Biochem Lec Full Combined

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[BIOCHEM] LEC 1- BASIC CONCEPTS OF METABOLISM

Dr. Maria Aloiza Hadloc | 02/08/22
LECTURE OUTLINE 2. Anabolic (synthetic)

I. INTRODUCTION - forms complex end products from simple precursors
A. Two types of Metabolism Pathways - Example: synthesis of polysaccharide, glycogen,
1. Catabolism
from glucose.
2. Anabolism
II. METABOLISM MAP
III. CATABOLIC PATHWAYS
A. Three stages of Catabolic pathways
1. Hydrolysis of complex molecules
2. Conversion of building blocks to simple intermediates
3. Oxidation of Acetyl CoA
IV. ANABOLIC PATHWAYS
V. REGULATION OF METABOLISM
A. Intracellular
B. Intercellular
C. Second messenger systems
1. Adenylyl cyclase
a. GTP-dependent regulatory protein
2. Protein kinases
a. Dephosphorylation of Proteins
b. Hydrolysis of Camp
VI. REVIEW QUESTIONS
VII. REFERENCES
Fig.1. Catabolism vs. Anabolism

I. INTRODUCTION
• Enzymatic reactions are organized into multistep sequences
called pathways. II. METABOLIC MAP
o product of one reaction serves as the substrate of
the subsequent reaction. • To investigate metabolism, we need to examine its component
o different pathways can also intersect, forming an pathways.
integrated and purposeful network of chemical
Each pathway is composed of multienzyme sequences
-
reactions.
Each enzyme may exhibit important catalytic or
-
When these different pathways interact with each other, we regulatory features.
call them METABOLISM • Metabolic map
- Contains the important central pathways of energy

Metabolism - sum of all the chemical changes occurring in a
metabolism (refer to Figure 2)
cell, a tissue, or the body.
- useful in:
o tracing connections between pathways o
A. Two types of Metabolism Pathways visualizing the purposeful “movement” of
1. Catabolic (degradative) metabolic intermediates
- process of breaking down complex molecules o picturing the effect on the flow of intermediates
- Example: degradation of proteins to amino if a pathway is blocked by a drug or an inherited
acids, carbohydrates to monosaccharides and enzyme deficiency.
fats to glycerol and fatty acids.
- 2 Purposes:
o To release energy in the form of ATP
o To come up with building blocks for
the synthesis of another complex
molecule
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- Building blocks are further degraded to acetyl coenzyme A

(CoA) and a few other, simple molecules. Some energy is captured
as ATP
3. Oxidation of acetyl CoA:
- The tricarboxylic acid (TCA) cycle is the final common

pathway Oxidation of acetyl CoA generates large amounts of ATP
via oxidative phosphorylation as electrons flow from NADH and
FADH2 to oxygen
- Generates larger amount of ATP
IV. ANABOLIC PATHWAYS
• Combine small molecules, such as amino acids, to form

complex molecules, such as proteins.
• Require energy which is provided by the breakdown of ATP to
adenosine diphosphate (ADP) and inorganic phosphate (Pi).
• Involve chemical reductions in which the reducing power is
most frequently provided by the electron donor NADPH
Fig.2. Metabolic Map
III. CATABOLIC PATHWAYS
• Catabolic reactions capture chemical energy in the form of

Adenosine Triphosphate (ATP)
• Catabolism also allows molecules to be converted into
building blocks for synthesis of complex molecules.
A. Three stages of Catabolism

Fig.3. Catabolism vs. Anabolism Reactions
1. Hydrolysis of complex molecules:
-Complex molecules are broken down into component

building blocks.
Example: V. REGULATION OF METABOLISM
Proteins >>> amino acids
• Individual cells do not function in isolation but are part of a
Poly - saccharides >> monosaccharides
community of interacting tissues.
Fats (triacylglycerols) >>> free fatty acids and glycerol
• A sophisticated communication system has evolved to
2. Conversion of building blocks to simple intermediates: coordinate the functions of the body.
• Regulatory signals that inform an individual cell of the
metabolic state of the body as a whole include hormones,
neurotransmitters, and the availability of nutrients.

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• Catabolism and anabolism pathways must be regulated.

This is by means of regulatory signals - hormones and
neurotransmitters
- Nonregulation → excessive catabolism = ↑ energy,
no building blocks
- excessive anabolism = require ATP, less energy to
form/synthesize complex molecule synthesis
- Example:
o Acromegaly - caused by excessive
production of growth hormone-releasing
hormone (GHRH).
o In healthy individual, growth hormone is
counteracted by somatostatin.
Fig.6. Intracellular communication
The ligand binds to the receptor causing change in receptor’s

shape and activity allowing signal transmission and eventually
producing response inside the cell.
B.Communication between cells (Intercellular)

• Signalling between cells provides for long-range integration
of metabolism, and usually results in a response that is
slower than is seen with signals that originate within the
cell.
• Communication between cells can be mediated by surface-
to-surface contact and by formation of gap junctions
• For energy metabolism, the most important route of
communication is chemical signalling between cells by
bloodborne hormones or by neurotransmitters.
Fig.4. Some commonly used mechanisms for transmission of

regulatory signals between cells.
3 Ways to Regulate Metabolism
A. Signals from within the cell (Intracellular)

• Metabolic pathways respond to regulatory signals that
arise within the cell.
• For example, the rate of a pathway may be influenced by Fig.7. Intercellular communication
the substrates, product inhibition, or alterations in the
Sending cell produces ions or hormones which will serve as
levels of allosteric activators or inhibitors.
ligands to the receptors of the target cell. These ligands cause
• Intracellular signals elicit rapid responses, and are change in shape/activity to the receptor causing transmission of
important for the moment-to-moment regulation of signal inside the cell eventually producing a response.
metabolism.

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C. Second messenger systems
• Hormones or neurotransmitters – signals
- Receptors - signal detectors
• Many receptors signal their recognition of a bound ligand
by initiating a series of reactions >> intracellular response.
• “Second messenger”
- intervene between the original messenger (the
neurotransmitter or hormone) and the ultimate effect
on the cell
- Examples:
o Calcium/phosphatidylinositol system
o Adenylyl cyclase system
Fig.9. Structure of a typical I G protein-coupled receptor

(GPCR) of the plasma membrane.
a. GTP- dependent regulatory protein
• The effect of the activated, occupied GPCR on second

messenger formation is not direct but is mediated by
specialised trimeric proteins (α, β, γ subunits) of the cell
membrane.
o G proteins
- bind guanosine nucleotides (GTP and GDP), form
Fig.8. Second Messenger systems a link in the chain of communication between
the receptor and adenylyl cyclase.
1. Adenylyl cyclase - In the inactive form of a G protein, the α-subunit
• The recognition of a chemical signal by some membrane is bound to GDP
receptors, such as the β- and α2-adrenergic receptors, • Binding of ligands causes a conformational change in the
triggers either an increase or a decrease in the activity of receptor, triggering replacement of GDP with GTP.
adenylyl cyclase (adenylate cyclase). o The GTP-bound form of the α subunit dissociates from
o A membrane-bound enzyme that converts ATP to the βγ subunits and moves to adenylyl cyclase which is
3',5'-adenosine monophosphate (also called cyclic thereby activated.
AMP or cAMP).
• Many molecules of active Gα protein are formed by one
• Tissues that respond to more than one chemical signal activated receptor. A hormone or neurotransmitter has
must have several different receptors, each of which can the ability to stimulate or inhibit adenylyl cyclase depends
be linked to adenylyl cyclase. on the type of Gα protein that is linked to the receptor.
o G protein-coupled receptors (GPCR)
o extracellular ligand-binding region, seven
o Gs - stimulates adenylylcyclase
transmembrane helices, and an intracellular
domain that interacts with G proteins. o Gi - inhibits the enzyme
• Gα–GTP complex are short-lived because, Gα has an
inherent GTPase activity, resulting in the rapid hydrolysis
of GTP to GDP causing inactivation of Gα.

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• Protein kinase A can also phosphorylate proteins that bind

to DNA, causing changes in gene expression.
Fig.11. Actions of cAMP
2 Ways of Protein Kinase Regulation

o Protein phosphatases
- Cleave or remove the phosphate that was added to
protein substrate in order to stop the reaction
Fig.10.The recognition of chemical signals by certain membrane o Cyclic AMP phosphodiesterase
receptors triggers an increase (or decrease) in activity of adenylyl - hydrolysis of cAMP to 5’-AMP
cyclase.
a. Dephosphorylation of Proteins
o The phosphate groups added to proteins by protein
2. Protein kinases: kinases are removed by protein phosphatases—
enzymes that hydrolytically cleave phosphate esters.
• Next key link in the cAMP second messenger system: o This ensures that changes in protein activity induced
activation by cAMP of a family of enzymes called cAMP- by phosphorylation are not permanent.
independent protein kinases Protein kinase A
• Cyclic AMP activates protein kinase A by binding to its two b. Hydrolysis of cAMP
regulatory subunits, causing the release of active catalytic o 9cAMP is rapidly hydrolyzed to 5'-AMP by cAMP
subunits. phosphodiesterase, a family of enzymes that cleave
• The active subunits catalyse the transfer of phosphate the cyclic 3',5'-phosphodiester bond.
from ATP to specific serine or threonine residues of o Effects of neurotransmitter- or hormone-mediated
protein substrates. increases of cAMP are rapidly terminated if the
extracellular signal is removed.
• The phosphorylated proteins may act directly on the cell’s
ion channels, or, if enzymes, may become activated or
inhibited.

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• Phosphodiesterase is inhibited by methyl xanthine 4.__________Signal that provides long-range integration of

derivatives, such as Theophylline and Caffeine metabolism and usually a slow signal
5. _________Signal that elicits rapid responses and is important
o Caffeine (1,3,7-trimethylxanthine) for the moment-to-moment regulation of metabolism
- CNS stimulant
6. Which is true about Second Messenger Systems?
- Inhibit cAMP phosphodiesterase = ↑ cAMP =
promote activation of protein kinase A I. Hormones or neurotransmitters act as signals
o Theophylline II. Receptors acts as signal detectors
- drug which we give in patients with COPD or those VII. Many receptors signal their recognition of a bound
with emphysema secondary to chronic smoking - ligand by initiating a series of reactions.
used in order to inhibit Phosphodiesterase type III VIII. IV. Second messengers intervene between the
and IV which is responsible in breaking down cAMP original messenger and the ultimate effect on the
in smooth muscle resulting now to bronchodilation. cell.
a) All statements are true

b) Only statements I, II, III are true
c) Only statements I and II are true
d) Only statements I, II, IV are true
7. True or False: Gi stimulates adenylyl cyclase while the Gs

inhibit the enzyme
8. Regulatory signals that inform an individual cell of the

metabolic state of the body as a whole, except:
a) Hormones
b) Availability of nutrients
c) Neurotransmitters
d) None of the Above
Fig.12. Effects of Theophylline 9. Which stage of catabolism generates larger amount of ATP?
a) Stage 1
VI. REVIEW QUESTIONS b) Stage 2
c) Stage 3
1. Which is true about the catabolic pathway?
10. True or False. Catabolism is degradative while Anabolism is
a) Involve chemical reductions in which the reducing
synthetic.
power is most frequently provided by the electron
donor NADPH.
b) Combine small molecules, such as amino acids, to form
complex molecules. VII. REFERENCES
c) Capture chemical energy in the form of ATP.
d) Require energy which is provided by the breakdown of 1. HAdloc, M.A. (2022). Basic Concept of Metabolism PPT
ATP to ADP and inorganic phosphate. and discussion.
2. In what form do catabolic reactions capture chemical 2. Ferrier, D. (2017). Lippincott Illustrated Reviews:
energy? Biochemistry (7th ed.). Philadelphia, PA: Lippincott and
a) Protein Wilkins.
b) ATP
c) ADP
d) Oxygen
3. Three stages of catabolism except:
a) Oxidation of acetyl CoA. True.
b) Conversion of building blocks to simple intermediates. stimulates adenylyl cyclase Gi inhibits the enzyme8.D.9.C.10.
c) Hydrolysis of complex molecules. 1.C.2.B.3D.4.Intercellularsignal5.Intracellular signal6.A.7.False - Gs
d) None of the above.

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[BIOCHEM]-LC2-Metabolic Effects of Insulin and Glucagon
Dr. M. Hadloc | 02/08/22
OUTLINE
I. NTRODUCTION
II. INSULIN
A. Structure of Insulin
B. Synthesis of Insulin
III. STIMULATION OF INSULIN SECRETION
IV. INHIBITION OF INSULIN SECRETION
V. METABOLIC EFFECTS OF INSULIN
A. Effects on Carbohydrate Metabolism
B. Effects on Lipid Metabolism
C. Effects on Protein Synthesis
VI. MECHANISM OF INSULIN ACTION
A. Membrane Effects of Insulin
B. Receptor Regulation Figure 1. Mechanisms of communication between four major
C. Time Course of Insulin Actions tissues.
VII. GLUCAGON
A. Stimulation of Glucagon Secretion II. INSULIN
B. Metabolic Effects of Glucagon
C. Mechanism of Action of Glucagon • Polypeptide hormone produced by the β cells of the islets of
D. Stimulation and Inhibition of Glycogen Degradation Langerhans embedded in the endocrine portion of the pancreas.
VIII. HYPOGLYCEMIA o Islets of Langerhans make up only about 1–2% of the total
A. Symptoms of Hypoglycemia
cells of the pancreas.
B. Glucoregulatory Systems
IX. TYPES OF HYPOGLYCEMIA • The most important hormone coordinating the use of fuels by
X. ALCOHOL INTOXICATION tissues.
XI. APPENDIX • Its metabolic effects are anabolic, and synthetic favoring, for
XII. TEST YOUR KNOWLEDGE example, synthesis of glycogen, triacylglycerols, and protein.
XIII. REFERENCES
I. INTRODUCTION
• Four major organs that play a dominant role in fueling
metabolism:
1. Liver
2. Adipose Tissue
3. Muscle
4. Brain
These 4 organs need to be in communication with each other by
means of hormones (Insulin and Glucagon) and catecholamines
(Epinephrine and Norepinephrine), so that the body will know if it
needs to store energy or needs to make the stored energy
available. For example, during survival crises, severe injury, or
when stressed, instead of storing energy, the body will make way
to make the stored energy (for example, the glucagon) to release Figure 2. Islets of Langerhans.
glucose.
• Communication between tissues is mediated by the nervous
system, by the availability of circulating substrates, and by
variation of plasma hormones.
• Two peptide hormones that control the integration of energy
metabolism (control the 4 major organs):
1. Insulin
2. Glucagon
• Catecholamines that play supporting role:
1. Epinephrine
2. Norepinephrine
• These hormones allow the body to store energy during feeding,
or to make stored energy available during “survival crises,” such
as famine, severe injury, and “fight-or-flight” situations.
Figure 3. Insulin and Glucagon are made in the pancreas.
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Dr. M. Hadloc | 02/08/22
o Type II Diabetes Mellitus - you have enough insulin

A. STRUCTURE OF INSULIN available in the body (intact pancreatic cells) but the
• Insulin is composed of 51 amino acids arranged in two problem is inability of the peripheral tissues to use this
polypeptide chains, A and B, linked by two disulfide bridges insulin (the body is not reacting to insulin). But later on,
• The insulin molecule contains an intramolecular disulfide bridge when the Type 2 DM is already prolonged, there will be an
between amino acid residues of the A chain. inability of the pancreas to produce insulin.
• When used in humans for the treatment of diabetes, antibodies 2. Proinsulin sequentially cleaved to form the active hormone
to these foreign proteins develop. plus the Connecting or C-peptide
• Use of human recombinant insulin has eliminated this problem.
Figure 4. Structure of Insulin.
B. SYNTHESIS OF INSULIN
When the Islet of Langerhans synthesizes insulin, it doesn’t Figure 5. Formation of human insulin from Preproinsulin.
automatically release insulin itself.
• Biosynthesis of Insulin involves two inactive precursors:
Preproinsulin and Proinsulin.
1. Preproinsulin - will be cleaved to become Proinsulin
o 4 components:
▪ Signal sequence
▪ B chain
▪ A chain
▪ C peptide
o C peptide - is very important because it is essential for
proper insulin folding of the 2 chains in order to properly
synthesize the insulin.
o In preproinsulin, there’s still a signal sequence (color blue).
Eventually when it reaches the endoplasmic reticulum in
the islet of Langerhans, it will cleave the signal sequence,
now producing the proinsulin. Proinsulin on the other hand,
will be cleaved once it is on the Golgi apparatus finally
releasing the insulin that is composed of only the A and B
chain.
o What is the difference between C peptide compared to the
insulin? The C peptide has a longer half-life in the plasma,
but they are released from the pancreas, at the same time
with the insulin and also in about equal amount. Because C Figure 6. Intracellular movements of insulin and its precursors.
peptide has a longer half-life, it stays in the blood longer
The insulin is stored in the cytosol in granules, and if given the
compared to insulin. It can actually tell how much insulin
proper stimulus, they will actually be released by exocytosis.
the body is making, thus the reason of using C peptide also
as a diagnostic test for Type 1 DM, if the doctor needs to o Insulin has a plasma half-life of approximately 6 minutes.
know how much your insulin still makes or the ability of the
pancreatic cells to produce insulin. For Type 2 DM, the C III. STIMULATION OF INSULIN SECRETION
peptide is used to find out if it is already time to start • Insulin secretion by the β cells of the islets of Langerhans of the
insulin, because majority for Type 2 DM, we start with oral pancreas is closely coordinated with the release of glucagon by
antidiabetic medications and later on if the pancreatic cells the pancreatic alpha cells. Because if you only allow insulin to
are not able to produce insulin anymore, then insulin will be be taking its effect then most likely we will be having
needed. hypoglycemia, so it needs to be regulated also by the glucagon
o Type I Diabetes Mellitus – autoimmune disease (immune release.
cells attack the cells of the pancreas → pancreatic cells are
destroyed → the Islet of Langerhans are not able to
produce insulin).
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Dr. M. Hadloc | 02/08/22
3 stimulants for insulin secretion:

A. Glucose:
- β cells are the most important glucose-sensing cells in the
body
- β cells contain GLUT-2 transporters with glucokinase
activity and can phosphorylate glucose.
Take note: Liver and Pancreatic cells have GLUT-2 transporters.
- A carbohydrate-rich meal leads to a rise in blood glucose,
which is a signal for increased insulin secretion.
- It is the most important stimulus for insulin secretion.
B. Amino acids:
- Protein ingestion → transient rise in plasma amino acid
levels → immediate secretion of insulin.
C. Gastrointestinal hormones:
- Cholecystokinin and gastric-inhibitory polypeptide
(glucose-dependent insulinotropic peptide) increase insulin
secretion in response to oral glucose, and referred to as
Figure 7. Changes in blood levels of glucose, insulin, and glucagon
“incretins.”
after ingestion of a carbohydrate-rich meal.
- For example, you eat a piece of cake, this cake will pass
through the intestines and once it’s in the intestines, this
will trigger now the release of incretins from the small
intestine, the cholecystokinin and gastric-inhibitory
polypeptide acting now on pancreatic cells to produce
insulin.
- Take note: Only oral glucose will trigger the release of
gastrointestinal hormones.
- Incretins are released from the small intestine after
ingestion of food → rise in insulin levels.
- Same amount of glucose given orally induces a much
greater secretion of insulin than if given intravenously.
- Patients who are in the hospital, who are not able to take
food through their mouth have a higher risk to have
hyperglycemia, because of the inability to produce enough
insulin.
Example:
Figure 8. Glucose-dependent glucagon secretion from the alpha
For a patient diagnosed with intestinal obstruction, you
can’t feed the patient and unfortunately, cannot undergo cell.
surgery immediately so you hook the patient to a Total
• Glucose enters the GLUT-2 transporter of the β cells of
Parenteral Nutrition (fluid that is given to a patient
pancreas (yellow receptor) → glucose inside the cell will be
through an intravenous line) which contains all the
metabolized into ATP → ATP will sensitize the K-channels (blue
nutrients needed by the body, even though the patient
channels) and will cause this K-channels to close → causing
cannot take food by mouth. The problem is, there is an
depolarization of the plasma membrane → depolarization
inability for the body to produce gastrointestinal
causes Ca-channels (green) to open up and there will be influx
hormones because the food didn’t pass through the
of Calcium → influx of Calcium will cause the vesicles containing
gastrointestinal system. So, there will be a lot of
insulin to finally be released from the β cells via exocytosis.
nutrients/glucose in the blood but there is less insulin that
is produced, which is the reason for the high risk for • Mechanism of Action of anti-diabetic medications
hyperglycemia. (Sulfonylurea): it closes the ATP sensitized K-channels (blue
channel) → it phosphorylates the entire cell → causing
- If the patient is in Total Parenteral Nutrition and especially production of insulin.
diabetic, the doctor will continuously monitor the glucose
of the patient. If the glucose is elevated, for example, more IV. INHIBITION OF INSULIN SECRETION
than 200 or 300, then insulin will be given by means of
subcutaneous route. • Synthesis and release of insulin are decreased when there is a
scarcity of dietary fuels, and also during periods of stress,
mediated primarily by epinephrine—secreted by the adrenal
medulla in response to stress, trauma, or extreme exercise.
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Dr. M. Hadloc | 02/08/22
• Fasting - during fasting, we do not have enough carbohydrate • In the muscle and adipose, insulin increases the number of
in the body. We do not want insulin to be using the rest of the glucose transporters in the cell membrane by a specific or
glucose to produce glycogen because we need glucose at this special mechanism. Once glucose enters the pancreas and
moment. Insulin secretion will be halted. Glucagon will be releases insulin, the insulin will signal the muscle and adipose
acting during fasting. tissue to further increase glucose uptake in these 2 tissues. The
• During periods of stress (fever, infection, during surgery) - this muscle and adipose tissues will increase the numbers of
is the reason why we check the sugar level of patients who glucose transporters in the cell membrane, which is the GLUT-4
underwent surgery. It is usually high because of stress. transporters.
Epinephrine is the one responsible in inhibiting insulin secretion • IV administration of insulin causes an immediate decrease in the
during stress. We need glucose at the moment rather than concentration of blood glucose.
using glucose for storage. • In the liver, insulin decreases production of glucose through
• Release of epinephrine is controlled largely by the nervous inhibition of glycogenolysis and gluconeogenesis.
system.
o It causes a rapid mobilization of energy-yielding fuels,
including glucose from the liver (produced by
glycogenolysis or gluconeogenesis) and fatty acids from
adipose tissue. Because when you are sick or undergoing
extreme exercise, you want a lot of energy in the body by
means of glucose.
o In addition, epinephrine can override the normal glucose-
stimulated release of insulin.
• In emergency situations, the sympathetic nervous system
largely replaces the plasma glucose concentration as the
controlling influence over β-cell secretion.
Figure 10. Effects of insulin on glucose and lipid metabolism.
B. EFFECTS ON LIPID METABOLISM

Main Goal: to decrease glucose and fatty acids in the blood. The
adipose tissue responds within minutes to the administration of
insulin, it will cause a significant reduction in the release of fatty
acids.
Figure 9. Regulation of Insulin release from pancreatic β cells.
(Note: Gastrointestinal peptide hormones also stimulate insulin 1. Decreased Triacylglycerol degradation:
release.) - Insulin decreases the level of circulating free fatty acids by
• Glucose and amino acids cause the release of insulin while inhibiting the activity of hormone-sensitive lipase that
epinephrine inhibits the release of insulin. degrades triacylglycerol in adipose tissue.
- Insulin promotes the dephosphorylation and inactivation of
V. METABOLIC EFFECTS OF INSULIN the enzyme.
2. Increased triacylglycerol synthesis:
A. EFFECTS ON CARBOHYDRATE METABOLISM
- Since insulin is anabolic, it keeps energy for future use.
• Insulin is anabolic, it promotes glucose storage - Insulin increases the transport and metabolism of glucose
• Insulin will cause storage of carbohydrate. Most prominent in into adipocytes, providing the substrate glycerol 3-
three tissues: phosphate for triacylglycerol synthesis.
1. Liver
- Insulin also increases the lipoprotein lipase activity of
2. Muscle
adipose tissue by increasing the enzyme's synthesis, thus
3. Adipose
providing fatty acids for esterification.
• In the liver and muscle, insulin increases glycogen synthesis. - In liver, insulin promotes the conversion of glucose to
triacylglycerols.
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Dr. M. Hadloc | 02/08/22
C. EFFECTS ON PROTEIN SYNTHESIS A. MEMBRANE EFFECTS OF INSULIN

• In most tissues, insulin stimulates the entry of amino acids into
cells, and protein synthesis.
• Insulin stimulates protein synthesis through activation of
factors required for translation
VI. MECHANISM OF INSULIN ACTION
*See larger image in appendices

Figure 12. Insulin causes the recruitment of glucose transporters
(GLUTs) from intracellular stores in skeletal and cardiac muscle and
adipose tissue.
• Insulin causes the recruitment of glucose transporters (GLUTs)
from intracellular stores in skeletal and cardiac muscle and
adipose tissue.
B. RECEPTOR REGULATION
• Binding of insulin is followed by internalization of the
hormone–receptor complex.
• Once inside the cell, insulin is degraded in the lysosomes.
• The receptors may be degraded but most are recycled to the
cell surface.
C. TIME COURSE OF INSULIN ACTIONS
• The most immediate response of the binding of insulin is an
increase in glucose transport into adipocytes and skeletal
muscle cells that occurs within seconds of insulin binding to its
receptor.
*See larger image in appendices • Insulin-induced changes in enzymic activity will occur minutes
Figure 11. Mechanism of action of insulin. IRS = Insulin receptor to hours in many cell types reflect changes in the
substrate; P = phosphate; Tyr = tyrosine; S-S = disulfide bond. phosphorylation states of existing proteins.
• The pink and blue one is called insulin receptor. There are also • Insulin also initiates an increase in the amount of many
alpha and beta subunit of the receptor that is linked to the 2 enzymes, such as glucokinase, liver pyruvate kinase, acetyl CoA
tyrosine residues. carboxylase, and fatty acid synthase, which requires hours to
• Insulin receptor is synthesized as a polypeptide that is cleaved days.
into alpha and beta subunits, which are then assembled into a
tetramer by disulfide bonds.
• The hydrophobic domain in each beta subunit expands the
plasma membrane, the extracellular alpha subunit (pink)
contains the insulin binding site. Cytosolic domain of the beta
subunit is a tyrosine kinase which is activated by insulin
(orange).
• Once insulin binds to alpha subunit, this induces
conformational changes, that will eventually transduce into
beta subunit. Once it reaches the beta subunit, this will
promote auto phosphorylation of the tyrosine residues that are
attached to the beta subunit.
Figure 13. Characteristics of glucose transport in various tissues.
• The autophosphorylation will now initiate cascade of
(Note: Many tissues have insulin insensitive systems for glucose
responses, one of which is the phosphorylation of Insulin
transport. For example, hepatocytes, erythrocytes, and cells of the
Receptor Substrate (IRS). The phosphorylated IRS will interact
nervous system, intestinal mucosa, renal tubules, and cornea do
now with other signaling molecules through specific domains,
not require insulin for glucose uptake.)
eventually activating multiple signaling pathways.
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Dr. M. Hadloc | 02/08/22
VII. GLUCAGON - In these situations, regardless of the concentration of

blood glucose, glucagon levels are elevated in anticipation
of increased glucose use. In contrast, insulin levels are
depressed.
Figure 14. Opposing actions of insulin and glucagon plus

epinephrine.
• Responsible during fasting state.
Figure 15. Regulation of glucagon release from pancreatic α cells.
• Polypeptide hormone secreted by the α cells of the pancreatic
(Note: Amino acids increase release of insulin and glucagon,
islets of Langerhans.
whereas glucose increases release of insulin only.)
• Along with epinephrine, cortisol, and growth hormone
(“counter-regulatory hormones”), counteracts or opposes B. METABOLIC EFFECTS OF GLUCAGON
many of the actions of insulin. 1. Effects on carbohydrate metabolism:
• Maintain blood glucose levels by activation of hepatic • Intravenous administration of glucagon leads to
glycogenolysis and gluconeogenesis. immediate rise in blood glucose from increase in the
o Glucagon wants a lot of glucose in the blood, especially breakdown of liver (not muscle) glycogen and an increase
during fight-or-flight activities. in gluconeogenesis.
• Composed of 29 amino acids arranged in a single polypeptide
2. Effects on lipid metabolism:
chain.
• Glucagon activates lipolysis in adipose. The free fatty
• Synthesized as precursor Preproglucagon that is converted to
glucagon through a series of selective proteolytic cleavages, acids released are taken up by liver and oxidized to acetyl
coenzyme A, used in ketone body synthesis.
similar to those described for insulin biosynthesis.
• Important with prolonged fasting where glycogen is
• Glucagon wants glucose to be present in the blood.
unavailable for gluconeogenesis or glycogenolysis,
A. STIMULATION OF GLUCAGON SECRETION therefore it relies on ketone body.
1. Low blood glucose (Hypoglycemia) 3. Effects on protein metabolism:
- A decrease in plasma glucose concentration is the • Glucagon increases uptake of amino acids by the liver,
primary stimulus resulting in increased availability of carbon skeletons for
- E.g. if you are undergoing an after-6 diet (you do not eat gluconeogenesis in order to have more glucose in the
anymore after 6 pm) or for those fasting in Ramadan, blood.
glucagon is the one responsible in preventing
C. MECHANISM OF ACTION OF GLUCAGON
hypoglycemia
2. Amino acids • Glucagon binds to high-affinity G protein-coupled receptors on
- Amino acids from a meal containing protein stimulate the the cell membrane of hepatocytes.
release of both glucagon and insulin. It’s an immediate • Glucagon receptors are not found on skeletal muscle.
counter regulatory mechanism. • Glucagon binding results in activation of adenylyl cyclase in the
- The glucagon will prevent hypoglycemia that will take plasma membrane → rise in cAMP (the “second messenger”),
effect as a result if you only allow insulin secretion to → activates cAMP-dependent protein kinase and increases the
occur after a protein meal. phosphorylation of specific enzymes or other proteins.
• This cascade results in the phosphorylation-mediated activation
3. Epinephrine
or inhibition of key regulatory enzymes involved in
- Elevated levels of epinephrine by the adrenal medulla, or
carbohydrate and lipid metabolism.
norepinephrine by sympathetic innervation of the
• An example of such a cascade in glycogen degradation is shown
pancreas, or both, stimulate release of glucagon.
on Figure 17. [Note: Glucagon also affects gene transcription.]
- During stress, trauma, or severe exercise, elevated
epinephrine can override the effect on the α cell of
circulating substrates.
BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
kinase molecules. This causes the production of many active

glycogen phosphorylase A molecule that can degrade glycogen.
• Because of the importance of maintaining blood glucose
levels, the synthesis and degradation of its glycogen
storage form are tightly regulated. In the liver,
glycogenesis accelerates during periods when the body
has been well fed, whereas glycogenolysis accelerates
during periods of fasting.
• In skeletal muscle, glycogenolysis occurs during active
exercise, and glycogenesis begins as soon as the muscle is
again at rest.
• Regulation of glycogen synthesis and degradation is
accomplished on two levels:
1. Glycogen synthase and glycogen phosphorylase are
hormonally regulated to meet the needs of the body
as a whole.
2. The pathways of glycogen synthesis and degradation
are allosterically controlled to meet the needs of a
particular tissue.

Figure 17. Stimulation and inhibition of glycogen degradation.
VIII. HYPOGLYCEMIA
Figure 16. Mechanism of action of glucagon.
(Note: For clarity, G-protein activation of adenylyl cyclase has been
omitted.) R = regulatory subunit; C = catalytic subunit; cAMP =
cyclic adenosine monophosphate; ADP = adenosine diphosphate;
P = phosphate.
D. STIMULATION AND INHIBITION OF GLYCOGEN
DEGRADATION
Summary of the regulation of glycogen degradation:
• The cascade of reactions results in glycogenolysis. The large • The central nervous system has an absolute requirement for
number of sequential steps serves to amplify the effect of the continuous supply of blood-borne glucose in order to serve as a
hormonal signal, that is, a few hormone molecules binding to fuel for energy metabolism.
their receptors result in a number of protein kinase A molecules • Severe hyperglycemia can cause cerebral dysfunction.
being activated that can each activate many phosphorylase Prolonged hyperglycemia can cause brain death. Therefore, the
body has multiple mechanisms in order to correct

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
hypoglycemia. The most important hormone that combats A. SYMPTOMS OF HYPOGLYCEMIA

hypoglycemia are glucagon and epinephrine. The symptoms of hypoglycemia can be divided into two
categories:
Hypoglycemia Diagnosis:
1) Central Nervous System (CNS) symptoms, including 1) Adrenergic symptoms
confusion, tremor, aberrant behavior, or coma • Anxiety, palpitation, tremor, and sweating—mediated by
2) Simultaneous blood glucose level ≤ 40 mg/dl epinephrine release regulated by the hypothalamus in
a. < 40 mg/dl is severe hypoglycemia response to hypoglycemia
b. Blood glucose < 70 mg/dl is considered a level 1 • Usually occur when blood glucose levels fall abruptly or
hypoglycemia when hypoglycemia is only acute or sudden.
3) Symptoms being resolved within minutes following the 2) Neuroglycopenic symptoms
administration of glucose. When the patient is unresponsive • Neuroglycopenia—the impaired delivery of glucose to the
or unconscious, IV line must be inserted, and D 50-50 vials (IV brain → impairment of brain function, causing headache,
sugar) will be administered. The patient should be awakened confusion, slurred speech, seizures, coma, and death.
after 5 mins of administration. After seeing the response of o Common with patients who are bed-bound, those
the patient then you can diagnose that the patient has who rely on other people to feed them because they
hypoglycemia. have high risk for prolonged hypoglycemia
• Hypoglycemia is an emergency case because the brain requires • Result from a gradual decline in blood glucose, often to
continuous supply of glucose. levels < 40 mg/dl, or from prolonged hypoglycemia and
o Transient hypoglycemia – effect will be mild cerebral prolonged fasting.
dysfunction • The slow decline in glucose deprives the CNS of fuel, but
o Prolonged hypoglycemia – may cause comatose or even fails to trigger an adequate epinephrine response.
death
B. GLUCOREGULATORY SYSTEMS
• Two overlapping glucose-regulating systems that are activated
by hypoglycemia:
a. Islets of Langerhans, which release glucagon
b. Receptors in the hypothalamus which trigger secretion of
epinephrine (mediated by ANS) and release of
adrenocorticotropic hormone (ACTH) and growth
hormone by the anterior pituitary
1. Glucagon and epinephrine:
- Hypoglycemia is combatted by decreased release of
insulin and increased secretion of glucagon, epinephrine,
cortisol, and growth hormone
- Glucagon and epinephrine are most important in the
acute, short-term regulation of blood glucose levels.
- Glucagon stimulates hepatic glycogenolysis and
gluconeogenesis.
- Epinephrine promotes glycogenolysis and lipolysis,
*See larger image in appendices inhibits insulin secretion, and inhibits the insulin-
Figure 18. A. Actions of some of the glucoregulatory hormones in mediated uptake of glucose by peripheral tissues.
response to low blood glucose. B. Glycemic thresholds for the - Epinephrine assumes a critical role when glucagon
various responses to hypoglycemia. + = weak stimulation; ++ = secretion is deficient, for example, in the late stages of
moderate stimulation; +++ = strong stimulation; 0 = no effect. type 1 DM.
[Note: Normal fasted blood glucose is 70–99 mg/100 ml.] 2. Cortisol and growth hormone:
ACTH = adrenocorticotropic hormone - Less important in the short-term maintenance of blood
• The figure above show that the body has multiple overlapping glucose concentrations.
mechanisms to prevent or correct hypoglycemia. The most - They however, play a role in the long-term management
of glucose metabolism.
important hormone changes in combating hypoglycemia are
elevated glucagon and epinephrine, plus a combined effect for
diminished release of insulin.

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
IX. TYPES OF HYPOGLYCEMIA 2. Postprandial Hypoglycemia:

1. Insulin-Induced Hypoglycemia: - Second most common form of hypoglycemia
- Hypoglycemia occurs frequently in patients with diabetes - Caused by exaggerated insulin release following a meal,
receiving insulin treatment prompting transient hypoglycemia with mild adrenergic
o e.g., You have a patient who has DM for almost 15 symptoms.
years and he is on insulin treatment approximately o e.g., You went for a buffet. You didn’t eat breakfast;
14 units of insulin, but patient is strict in his diet. He you didn’t eat lunch because you are anticipating
doesn’t want to eat a lot because he is afraid of your buffet. You ate a lot on the buffet. Later on,
hyperglycemia. He is very compliant with insulin, after 2 hours of eating, you already feel mild
and does not eat more than a half cup of rice. tremors, you feel tired, and have mild palpitations.
Because he tries to achieve control of his glucose This is secondary to or marked release of glucose
levels, hypoglycemia may occur. transporters in the skeletal muscles and adipose
- If the patient is conscious but starts to have dizziness, tissue because of exaggerated insulin release
starts to have mild tremors, you can treat mild causing transient hypoglycemia.
hypoglycemia is treated by oral administration of - Plasma glucose level returns to normal even if the patient
carbohydrate or glucose containing food. (This is reason is not fed.
for drinking of soft drinks to diabetic patients who have - Only treatment required: frequent small meals (every 2-3
hypoglycemia.) hours) rather than the usual three large meals.
- More commonly, patients are unconscious or have lost - At risk: people who fast almost the whole day but eat a
the ability to coordinate swallowing. lot at once after → reactive hypoglycemia secondary to
o e.g., For unconscious patients with glucose level of exaggerated release of insulin → every glucose in the
30 mg/dl, we hook D50-50 vials which contain glucose body will enter the cell → no more glucose left in blood →
that is administered intravenously. Minutes after weakness after eating.
administration the patient will wake up.
- Glucagon subcutaneously or intramuscular (IM) is the
3. Fasting Hypoglycemia:
treatment of choice
- Low blood glucose during fasting is rare since glucagon
will make way to promote glycogenolysis and
gluconeogenesis.
- Fasting hypoglycemia, which tends to produce
neuroglycopenia symptoms, may result from a reduction
in the rate of glucose production by hepatic
glycogenolysis or gluconeogenesis.
- This is very rare if you have a working liver.
- Often seen in patients with hepatocellular damage or
adrenal insufficiency, or in fasting individuals who have
consumed large quantities of ethanol
- This is common for patients with liver cirrhosis or chronic
alcohol intoxication because of the liver’s inability to
produce glucose by glycogenolysis or gluconeogenesis.
- Alternately, fasting hypoglycemia may be the result of an
increased rate of glucose use by the peripheral tissues
due to overproduction of insulin by rare pancreatic
tumors.
- If left untreated, patient may lose consciousness and
experience convulsions and coma.
Figure 20. Reversal of insulin-induced hypoglycemia by

administration of subcutaneous glucagon.

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
4. Hypoglycemia and Alcohol Intoxication: X. ALCOHOL INTOXICATION

Figure 21. A. Normal gluconeogenesis in the absence of ethanol Figure 22. Effects of chronic alcohol consumption on liver
consumption. B. Inhibition of gluconeogenesis resulting from morphology.
hepatic metabolism of ethanol.
• May precipitate hypoglycemia in individuals with depleted
• Alcohol is metabolized in the liver by two oxidation reaction. stores of liver glycogen.
• Ethanol is first converted to acetaldehyde by alcohol o E.g., You went to Vikings for 3 hours before drinking with
hydrogenase. It will be subsequently oxidized to acetate by your barkada. Your liver glycogen is depleted because of
aldehyde dehydrogenase. And in its reaction, NAD+ will be food consumption prior to drinking. Hypoglycemia can
transformed to NADH resulting to large amounts of NADH. happen.
• This abundance of NADH will now favor the reduction of • Hypoglycemia can produce behaviors of alcohol intoxication—
pyruvate to lactate and oxaloacetate to malate. agitation, impaired judgement, and combativeness because you
• Both the pyruvate and oxaloacetate are very important do not have stores of liver glycogen.
because both are intermediates in the synthesis of glucose by • Alcohol consumption in vulnerable individuals for
gluconeogenesis. hypoglycemia—fasted or have engaged in prolonged,
• Eventually, because of load pyruvate and oxaloacetate, there strenuous exercise—can produce hypoglycemia that will
will be decreased synthesis of glucose, thus alcohol contribute to the behavioral effects of alcohol.
consumption causes low blood sugar causing light-headedness • Chronic alcohol consumption can also result in fatty liver due to
and fatigue. increased synthesis of TAGs as a result of decreased fatty acid
• Patients present with hypoglycemia after intake of alcohol oxidation due to a fall in the NAD+/NADH ratio, and increased
especially to those who are depleted with glycogen at that lipogenesis due to increased availability of fatty acids and of
moment. glyceraldehyde 3-phosphate.
o For example, you drink alcohol after a strenuous exercise. • With continued alcohol consumption, alcoholic fatty liver can
Since you underwent exercise → the glycogen stores in progress first to alcoholic hepatitis, and then to alcoholic
the body were used → there is not enough glycogen in cirrhosis.
the body. • Alcohol consumption can increase the risk of hypoglycemia in
o Binge drinking alcohol after exercise → not enough patients using insulin. Therefore, patients in intensive insulin
glycogen stores in the body and because of the effect of treatment are counseled about the risk of hypoglycemia hours
the elevated NADH → there will be decreased glucose in after alcohol has been consumed.
the body → high risk for alcohol intoxication
hypoglycemia eventually presenting with agitation or
combativeness.
o But for a normal individual taking alcohol who did not
exercise → they still have enough glycogen in the body →
the body can still use this glycogen for gluconeogenesis →
may not present with hypoglycemia immediately.
o It is better to have heavy meals or pulutan when drinking
alcohol.
• Remember: DO NOT fast, exercise or engage in prolonged
fasting before drinking alcohol.

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Dr. M. Hadloc | 02/08/22
XI. APPENDIX
Figure 11. Mechanism of action of insulin. IRS = Insulin receptor substrate; P = phosphate; Tyr = tyrosine; S-S = disulfide bond.

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
Figure 12. Insulin causes the recruitment of glucose transporters (GLUTs) from intracellular stores in skeletal and cardiac muscle and adipose
tissue.
Figure 17. Stimulation and inhibition of glycogen degradation.

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
Figure 18. A. Actions of some of the glucoregulatory hormones in response to low blood glucose. B. Glycemic thresholds for the various
responses to hypoglycemia. + = weak stimulation; ++ = moderate stimulation; +++ = strong stimulation; 0 = no effect. [Note: Normal fasted
blood glucose is 70–99 mg/100 ml.] ACTH = adrenocorticotropic hormone
Figure 21. A. Normal gluconeogenesis in the absence of ethanol consumption. B. Inhibition of gluconeogenesis resulting from hepatic
metabolism of ethanol.

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
KEY CONCEPT MAP

BIOCHEMISTRY
Dr. M. Hadloc | 02/08/22
XII. TEST YOUR KNOWLEDGE 8. Which of the following statement/s is/are true?
1. Which of the following is an antagonist of insulin?
A. Glucose I. Blood insulin levels increase as a meal is digested, following
B. Amino Acids the rise in blood glucose levels.
C. Norepinephrine II. The uptake of dietary glucose by tissues (particularly liver,
D. Lysine adipose, and muscle) causes blood glucose to increase.
III. During fasting, insulin levels decrease and glucagon levels
2. Which of the following is a gastrointestinal agonist of insulin? increase. These hormonal changes promote glycogenolysis
A. Aldosterone and gluconeogenesis in the liver so that blood glucose levels
B. Lysine are maintained.
C. Norepinephrine IV. During exercise, blood glucose levels are maintained by
D. Cholecystokinin essentially the same mechanisms that are used during fasting.
E. Somatostatin
A. All statements are true
3. A 39-year-old woman is brought to the emergency room B. Only statements I, II, III are true
complaining of dizziness. She recalls getting up early that morning C. Only statements I, III, IV are true
to do as much shopping as possible and had skipped breakfast.
She drank a cup of coffee for lunch and had nothing to eat during 9. Increased expression of the genes for glucose synthase as
the day. She met with friends at 8 p.m. and had a drink at the bar. stimulated by insulin is an example of:
She soon became weak and dizzy and was transported to the A. Rapid short-term control
hospital. Following examination, the patient was given orange B. Long term coarse control
juice and immediately felt better. The patient has: C. Both
A. decreased glucagon D. Neither
B. blood glucose greater than 70 mg/dl
C. decreased hepatic glycogen 10. Type of hypoglycemia caused by exaggerated insulin release
D. presence of insulinoma following a meal, prompting transient hypoglycemia with mild
adrenergic symptoms:
4. An abnormal increase in this hormone is a potent stimulus for A. Insulin-induced hypoglycemia
the 4 key enzymes of gluconeogenesis: B. Postprandial hypoglycemia
A. Epinephrine C. Fasting hypoglycemia
B. Cortisol D. Hypoglycemia and alcohol intoxication
C. Glucagon
D. Growth hormone XIII. REFERENCES
• Hadloc, M.A. (2021). Metabolic Effects of Insulin and Glucagon
5. What is an allosteric activator of phosphofructokinase-1? [PPT].
A. fructose 1,6-bisphosphatase • Ferrier, D. (2011). Lippincott Illustrated Reviews: Biochemistry
B. Hexokinase (5th ed). Philadelphia, PA: Wolters Kluwer.
C. fructose 2,6-bisphosphate
D. Glucokinase ANSWER KEY: C D C B C B A C B B
6. Activation of cAMP-dependent Protein Kinase A causing

phosphorylation of glycogen synthase and glycogen
phosphorylase will result to which of the following?
A. Increased glycogenesis and inhibition of glycogenolysis
B. Increased glycogenolysis and inhibition of glycogenesis
C. Inhibition of both glycogenesis and glycogenolysis
D. Activation of both glycogenesis and glycogenolysis
7. What is the half-life of insulin?

A. 3-8 minutes
B. 1.5-2 minutes
C. 2.5 minutes
D. 10.5 minutes
E. 8-12.5 minutes

BIOCHEMISTRY
[BIOCHEM]-LC2.2-METABOLISM IN THE WELL-FED STATE
Dr. M.A. Hadloc-Tiu | 02/08/22
OUTLINE II. ENZYMATIC CHANGES IN THE WELL-FED STATE

I. OVERVIEW OF THE WELL-FED STATE
II. ENZYMATIC CHANGES IN THE WELL-FED STATE
The flow of intermediates through metabolic pathways is controlled by
A. Allosteric Effects three mechanisms:
B. Regulation of Enzymes by Covalent Modification A. ALLOSTERIC EFFECTS
C. Induction and Repression of Enzyme Synthesis • Allosteric changes usually involve rate-determining reactions.
III. LIVER: THE NUTRIENT DISTRIBUTION CENTER • Glycolysis in the liver is stimulated following a meal by an
IV. LIVER: CARBOHYDRATE METABOLISM
increase in fructose 2,6-bisphosphate—an allosteric activator of
V. LIVER: FAT METABOLISM
VI. LIVER: AMINO ACID METABOLISM phospho fructo kinase-1.
VII. ADIPOSE TISSUE: ENERGY STORAGE DEPOT • In contrast, gluconeogenesis is inhibited by fructose 2,6-
VIII. ADIPOSE TISSUE: CARBOHYDRATE METABOLISM bisphosphate, an allosteric inhibitor of fructose 1,6-
IX. ADIPOSE TISSUE: FAT METABOLISM bisphosphatase.
X. RESTING SKELETAL MUSCLE
XI. RESTING SKELETAL MUSCLE: CARBOHYDRATE METABOLISM B. REGULATION OF ENZYMES BY COVALENT MODIFICATION
XII. RESTING SKELETAL MUSCLE: FAT METABOLISM
XIII. RESTING SKELETAL MUSCLE: AMINO ACID METABOLISM • Many enzymes are regulated by the addition or removal of
XIV. BRAIN
phosphate groups from specific serine, threonine, or tyrosine
XV. BRAIN: CARBOHYDRATE METABOLISM
XVI. BRAIN: FAT METABOLISM residues.
XVII. REFERENCES • In the absorptive state, most enzymes regulated by these
XVIII. TEST YOUR KNOWLEDGE covalent modifications are in the dephosphorylated form and
are active.
• Three exceptions are glycogen phosphorylase kinase, glycogen
I. OVERVIEW OF THE WELL-FED STATE phosphorylase, and hormone-sensitive lipase of adipose tissue,
which are inactive in their dephosphorylated form.
C. INDUCTION AND REPRESSION OF ENZYME SYNTHESIS
• Increased (induction of) or decreased (repression of) enzyme

synthesis leads to changes in the total population of active sites
• Enzymes subject to the regulation of synthesis are those that are
needed at only 1 stage of development or under selected
physiologic conditions.
• in the fed state, elevated insulin >> increase in the synthesis of
acetyl coenzyme A (CoA) carboxylase and 3-hydroxy-3-
methylglutaryl- coenzyme A (HMG-CoA) reductase
The absorptive (well-fed) state is the 2- to 4-hour period after ingestion

of a normal meal. There will be transient increases in plasma glucose,
amino acids, and triacylglycerols (TAG). Islet tissue of the pancreas will
cause increased secretion of insulin and a decreased release of
glucagon. The elevated insulin in comparison with the glucagon and the
ready availability of circulating substrates will make the absorptive
state an anabolic period characterized by increased synthesis of TAG
and glycogen to replenish fuel stores as well as the enhanced synthesis
of protein. During this absorptive period, virtually all tissues use
glucose as a fuel.

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III. LIVER: THE NUTRIENT DISTRIBUTION CENTER 2) Increased glycogen synthesis:

• Liver: one of the most important organs in the body aside
from the brain and the heart as metabolism is going on here The conversion of glucose 6-phosphate to glycogen is favored by the
continuously. activation of glycogen synthase—both by dephosphorylation and by
• The liver process and distribute dietary nutrients because the the increased availability of glucose 6-phosphate
venous drainage of the gut and pancreas passes through the
hepatic portal vein before entry into the general circulation. 3) Increased activity of the hexose monophosphate pathway (HMP):
• After a meal, the liver is bathed in blood containing absorbed The increased availability of glucose 6-phosphate in the well-fed state,
nutrients and elevated levels of insulin secreted by the combined with the active use of NADPH in hepatic lipogenesis, stimulates
pancreas. the HMP.
• During the absorptive period, the liver takes up
carbohydrates, lipids, and most amino acids. 4) Increased glycolysis:
• These nutrients are then metabolized, stored, or routed to
A cytoplasmic pathway breaks down glucose into 2-3 carbon compounds
other tissues. Thus, the liver smooths out potentially broad
and generates energy.
fluctuations in the availability of nutrients for the peripheral
- In the liver, glycolytic metabolism of glucose is significant only during the
tissues.
absorptive period following a carbohydrate-rich meal. The conversion of
• Two blood supplies of the liver: glucose to acetyl CoA is stimulated by the elevated insulin to glucagon
o Hepatic portal vein: 75% ratio that results in increased activity of pyruvate kinase
o Hepatic artery: 25%
- Pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl CoA,
is active (dephosphorylated) because pyruvate inhibits PDH kinase
- Acetyl CoA is used as a building block for FA synthesis or provides energy
by oxidation in the (TCA) cycle.
5) Decreased gluconeogenesis:
Production of sugars (glucose) for catabolic reactions from non-
carbohydrate precursors.
Pyruvate carboxylase, which catalyzes the first step in gluconeogenesis, is
largely inactive due to low levels of acetyl CoA—an allosteric effector
essential for enzyme activity
IV. LIVER: CARBOHYDRATE METABOLISM

The liver is normally a glucose-producing rather than a glucose-using
organ especially during fight-or-flight (trauma, stress). However, after
a meal containing carbohydrates, the liver becomes a net consumer of
glucose, retaining roughly 60g of every 10 g of glucose presented by
the portal system. This increased use is not a result of stimulated
glucose transport into the hepatocyte (remember: only the muscles
and adipose tissues are the ones that increase the glucose transport
into these cells) rather, hepatic glucose metabolism is increased by the
following mechanisms.
Hepatic glucose metabolism is increased by the following

mechanisms:
1) Increased phosphorylation of glucose:
Elevated glucose within hepatocytes allows glucokinase to

phosphorylate glucose to glucose 6-phosphate.

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1. Increased amino acid degradation: In the absorptive period, more
amino acids are present than the liver can use in the synthesis of proteins.
The surplus amino acids are not stored but are either released into the
blood for all tissues to use in protein synthesis or are deaminated, with
the resulting carbon skeletons being degraded by the liver to pyruvate,
V. LIVER: FAT METABOLISM
acetyl CoA, or TCA cycle intermediates. These metabolites can be oxidized
Well-fed state: Anabolic state
for energy or used in fatty acid synthesis. The liver has a limited capacity
1. Increased fatty acid synthesis: to degrade the branched-chain amino acids leucine, isoleucine, and valine.
They pass through the liver essentially unchanged and are preferentially
• Liver is the primary tissue for de novo synthesis of fatty acids metabolized in muscle.
• This pathway occurs in the absorptive period, when dietary
caloric intake exceeds energy expenditure by the body. 2. Increased protein synthesis
• Fatty acid synthesis is favored by the availability of substrates
(acetyl CoA and NADPH derived from the metabolism of VII. ADIPOSE TISSUE: ENERGY STORAGE DEPOT
glucose) and by the activation of acetyl CoA carboxylase, both • Adipose tissue is second only to the liver in its ability to distribute
by dephosphorylation and by the presence of its allosteric fuel molecules.
activator, citrate. • In a 70-kg man, adipose tissue weighs approximately 14 kg, or
• This enzyme catalyzes the formation of malonyl CoA from about half as much as the total muscle mass.
acetyl CoA— rate-limiting for FA • In obese individuals’ adipose tissue can constitute up to 70% of
body weight.
2. Increased TAG synthesis: • Nearly the entire volume of each adipocyte can be occupied by
a droplet of TAG
• TAG synthesis is favored because fatty acyl CoA is available
both from de novo synthesis from acetyl CoA and from
hydrolysis of the TAG component of chylomicron remnants
removed from the blood by hepatocytes
• Glycerol 3-phosphate, the backbone for TAG synthesis, is
provided by the glycolytic metabolism of glucose
• Liver packages TAG into VLDL particles that are secreted into
the blood for use by extrahepatic tissues particularly adipose
and muscle tissue
VI. LIVER: AMINO ACID METABOLISM
VIII. ADIPOSE TISSUE: CARBOHYDRATE METABOLISM

In the liver, there are 5 different ways for the metabolism of
carbohydrates while in adipose tissue, there are only 3.
1) Increased glucose transport:
• Only the skeletal muscle and adipose tissues can increase

glucose transport by increasing the number of glucose
transporters in the cell membrane.
• Glucose transport by GLUT-4 into adipocytes is sensitive to
insulin concentration in blood.
• Circulating insulin levels are elevated in the absorptive state,
resulting in an influx of glucose into adipocytes.
2) Increased glycolysis:
• The increased intracellular availability of glucose results in an

enhanced rate of glycolysis
• Glycolysis serves a synthetic function by supplying glycerol
phosphate for TAG synthesis.
3) Increased activity in the HMP:

BIOCHEMISTRY
• Adipose tissue can metabolize glucose by means of the HMP,

thereby producing NADPH, which is essential for fat synthesis.
IX. ADIPOSE TISSUE: FAT METABOLISM

1) Increased synthesis of fatty acids:
X. RESTING SKELETAL MUSCLE
• De novo synthesis of fatty acids from acetyl CoA in adipose
tissue is low in humans, except when refeeding previously • The energy metabolism of skeletal muscle is unique in being able
fasted individual. to respond to changes in the demand for ATP that accompanies
• Most of the fatty acids added to the lipid stores of adipocytes muscle contraction.
are provided by dietary fat. • At rest: 30% of body’s oxygen consumption
• During vigorous exercise: up to 90% of total oxygen
2) Increased TAG synthesis:
consumption.
• After consumption of a lipid-containing meal, hydrolysis of • This illustrates the fact that skeletal muscle, despite its potential
the TAGs (from the intestine) and VLDL (from the liver) for transient periods of anaerobic glycolysis, is an oxidative
provides adipose tissue with fatty acids. tissue.
• Fatty acids are released from lipoproteins by lipoprotein • Heart muscle differs from skeletal muscle in three important
lipase, adipose, and muscle. ways:
• In the fed state, elevated levels of glucose and insulin favor 1) the heart is continuously active, whereas skeletal muscle
storage of TAG, all carbons are supplied by glucose. contracts intermittently on demand;
2) the heart has a completely aerobic metabolism;
3) Decreased TAG degradation: 3) the heart contains negligible energy stores, such as glycogen
or lipid.
• Elevated insulin favors the dephosphorylated (inactive)form
of hormone-sensitive lipase.
• TAG degradation is thus inhibited in the fed state.
XI. RESTING SKELETAL MUSCLE: CARBOHYDRATE

METABOLISM
1) Increased glucose transport:
• Increase in glucose and insulin after a carbohydrate-rich meal >>
increase in glucose transport into muscle cells by GLUT-4
• Glucose is phosphorylated to glucose 6- phosphate by
hexokinase and metabolized to provide the energy needs of the
cells.
2) Increased glycogen synthesis:
• Increased insulin to glucagon ratio and availability of
glucose 6- phosphate favor glycogen synthesis, particularly
if glycogen stores have been depleted as a result of exercise.

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XIV. BRAIN
• Although contributing only 2% of the adult weight, the brain
accounts for 20% of the basal oxygen consumption of the body
at rest.
• Because the brain is vital to the proper functioning of all organs
of the body, special priority is given to its fuel needs.
XII. RESTING SKELETAL MUSCLE:
• To provide energy, substrates must be able to cross the blood-
FAT METABOLISM
brain barrier.
Fat metabolism
• Normally, glucose serves as the primary fuel for the brain.
• Fatty acids are released from chylomicrons and VLDL by • If blood glucose levels fall below approximately 40 mg/100 ml,
the action of lipoprotein lipase the cerebral function is impaired.
• Fatty acids are of secondary importance as a fuel for • Even in a short time, severe and potentially irreversible brain
muscle during the fed state, in which glucose is the damage may occur.
primary source of energy • During a fast, ketone bodies play a significant role as a fuel for
XIII. RESTING SKELETAL MUSCLE: the brain, reducing its dependence on glucose
AMINO ACID METABOLISM
1. Increased protein synthesis:
• A spurt in amino acid uptake and protein synthesis occurs in
the absorptive period after a protein-containing meal.
• Synthesis replaces proteins degraded since previous meal
2. Increased uptake of branched-chain Amino Acids
• Muscle is the principal site for degradation of branched-chain

amino acids as it contains the required transaminase.
• Branched-chain amino acids, Leucine, Isoleucine, and
Valine, escape metabolism by the liver, are taken up by the
muscle and used for protein synthesis and as sources of
energy.

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XV: BRAIN: CARBOHYDRATE METABOLISM

• In the fed state, the brain uses glucose exclusively as a fuel,
completely oxidizing approximately 140 g/day to CO2 and
H2O.
• The brain contains no significant stores of glycogen and is,
therefore, completely dependent on the availability of blood
glucose.
• For people who study, it is not advisable to go on a very strict
diet.
XVIII. TEST YOUR KNOWLEDGE
1. In Carbohydrate metabolism, the conversion of glucose 6-phosphate to

glycogen is favored by the activation of__________.
a) glycogen synthase
b) carboxylase
c) acetyl CoA
2. Nearly the entire volume of each adipocyte can be occupied by a droplet

XVI: BRAIN: FAT METABOLISM
of ____________.
• The brain has no significant stores of TAG, and the fatty acids
a) TAG
circulating in the blood make little contribution to energy
b) protein
production because fatty acids bound to albumin do not
c) cholesterol esters
efficiently cross the blood-brain barrier.
XVII: REFERENCES 3. In adipose tissue, The increased intracellular availability of glucose

results in an enhanced rate of
a) decarboxylation
• Doctor Hadloc-Tiu’s PowerPoint presentation. b) glycolysis
• Harvey, R.A. & Ferrier, D. R. (2011). Lippincott’s Illustrated c) gluconeogenesis
Reviews: Biochemistry Fifth Edition. Philadelphia: Lippincott
Williams & Wilkins, a Wolters Kluwer business 4. In Carbohydrate metabolism, Elevated glucose within hepatocytes
allows ___________ to phosphorylate glucose to glucose 6-phosphate.
a) glucokinase
b) phosphatase
c) carboxylase
5. The conversion of glucose to acetyl CoA is stimulated by the elevated

a) Glucose to insulin ratio
b) insulin to glucose ratio
c) insulin to glucagon ratio
d) glucagon to insulin ratio

BIOCHEMISTRY
6. __________ is used as a building block for FA synthesis, or provides
energy by oxidation in the (TCA) cycle.
a) Acetyl CoA
b) glucose 6 phosphate
c) glucose
d) Fumarate
7. __________is the primary tissue for de novo synthesis of fatty acids

a) pancreas
b) liver
c) heart
d) brain
8. In a well-fed state what is the primary and secondary source of

energy respectively?
a) glucose; Fatty acids
b) fatty acids; glucose
c) glycogen; protein
d) protein; glycogen
9. If blood glucose levels fall below approximately ___ cerebral

functions are impaired.
a) 20 mg/100 ml
b) 30 mg/ 100 ml
c) 40 mg/ 100 ml
d) 50 mg/100 ml
10. The brain contains significant stores of glycogen and is, therefore,
completely dependent on the availability of blood glucose. True or
False?
ANSWER KEY: 1.) A 2.) A 3.) B 4.) A 5.) C 6.) A 7.) B 8.) A 9.) C 10.) FALSE

BIOCHEMISTRY
[BIOCHEM]-LEC 3-METABOLISM DURING FASTING AND IN
DIABETES MELLITUS
OUTLINE
I. METABOLISM DURING FASTING

A. Fasting
B. Fuel Stores
II. ENZYMATIC CHANGES IN FASTING
III. CARBOHYDRATE METABOLISM IN THE LIVER
IV. FAT METABOLISM IN THE LIVER
V. ADIPOSE METABOLISM IN FASTING
A. Carbohydrate Metabolism
B. Fat Metabolism
VI. RESTING SKELETAL MUSCLE IN FASTING
A. Carbohydrate Metabolism
B. Lipid Metabolism
C. Protein Metabolism
VII. BRAIN IN FASTING
VIII. KIDNEY IN LONG TERM FASTING II. ENZYMATIC CHANGES IN FASTING
IX. METABOLISM IN DIABETES MELLITUS
A. Type I Diabetes
B. Type II Diabetes ● Flow of intermediates through the pathways of energy
X. TEST YOURSELF metabolism is controlled by four mechanisms:
XI. APPENDIX 1.) the availability of substrates
2.) allosteric regulation of enzymes
3.) covalent modification of
I. METABOLISM DURING FASTING enzymes; and
A. Fasting 4.) induction-repression of enzyme synthesis.
- 2 to 4 hrs after a normal meal is considered fasting.
- Q: In the absence of food, what happens? In fasting, substrates are not provided, tissues for example,
- A: Levels of plasma glucose, amino acid and during lipolysis with release of fatty acids and glycerol from
triacylglycerols triggering a decrease in insulin your triacylglycerol in adipose tissue and proteolysis with
secretion and a increase in glucagon release. In release of amino acids from the muscle.
fasting, there will be decreased insulin and
circulating substrates, making fasting period a ● Most of the enzymes in the fasted state are
catabolic period. In the Catabolic period, there will phosphorylated and active.
be degradation of energy sources such as Three exceptions
triacylglycerol, glycogen and protein. 1. Glycogen phosphorylase
- Decline in insulin secretion and an increase in 2. Glycogen phosphorylase kinase, and
glucagon release. 3. Hormone – sensitive lipase of adipose tissue
- Exchange of substrates among liver, adipose tissue, These 3 are inactive in a de-phosphorylated state.
muscle, and brain guided by two priorities: ● Substrate are from breakdown of stores and/or tissues
1.) To maintain adequate plasma levels of glucose III. CARBOHYDRATE METABOLISM IN THE LIVER
to sustain energy metabolism of the brain, RBCs,
and other glucose- requiring tissues; and
2.) To mobilize fatty acids from adipose tissue, and - Note: The primary role of the liver in energy
the synthesis and release of ketone bodies from the metabolism during fasting is to maintain blood
liver, to supply energy to all other tissues. glucose in your body. Liver synthesizes glucose and
it distributes it to the different organs of the body.
B. Fuel Stores The liver first uses glycogen degradation (glucose
Note: stores) and after degradation, the next is
1. There is an enormous caloric source available in the gluconeogenesis in order to maintain blood glucose
form of triacylglycerol or fat compared with those levels and to sustain energy metabolism of the brain
contained in glycogen. and other glucose requiring tissues in the fasted
2. Although protein is listed as an energy source, only state.
about 1/3 of the body’s protein can be used for
energy production without compromising the vital
functions.

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DIABETES MELLITUS
1. Increased glycogen degradation: 2. Increased gluconeogenesis:

● Increased glucagon to insulin ratio causes a rapid After degradation of the glucagon, the synthesis of
mobilization of liver glycogen stores (contain about glucose and its release into the blood are vital hepatic
80g of glycogen in the fed state) due to functions during fasting. The carbon skeletons that you need
phosphorylation (activation) of glycogen for gluconeogenesis are derived primarily from:
phosphorylase. 1. Glucogenic amino acids and lactate from the
muscles
● Liver glycogen is nearly exhausted after 10-18 hours 2. Glycerol from the adipose tissue.
of fasting. After 10 to 18 hours of fasting, there will ● Gluconeogenesis is important in maintaining blood
be a hepatic glycogenolysis considered transient glucose both in overnight and prolonged fasting.
response to the 18hrs fasting. ● Begins 4-6 hours after the last meal and becomes fully
- During the brief well-fed period, the food that you active as stores of liver glycogen are depleted.
ate is the major source of blood glucose. Several
hours later, blood glucose levels have declined
IV. FAT METABOLISM IN THE LIVER
sufficiently to cause increased secretion of your
glucagon.
1.) Increased fatty acid oxidation:
● Oxidation of FA from TAG hydrolysis in adipose tissue
is the major source of energy in hepatic tissue during
fasting.
● The fall in malonyl CoA due to phosphorylation
removes the brake of carnitine palmitoyl transferase-
1 (CPT-1), allowing beta-oxidation. CPT-1 initiates B-
oxidation during fasting.
2.) Increased synthesis of ketone bodies

- Liver is unique and it is generous because it
synthesizes and releases ketone bodies primarily 3-
hydroxybutyrate to be used by peripheral tissues but take
note, not by the liver itself.
● Ketogenesis is favored when concentration of acetyl
CoA, from fatty acid metabolism, exceeds the
oxidative capacity of the TCA cycle.
● Ketogenesis starts during the first days of fasting.
- The availability of circulating ketone bodies can be
used for fuel when the blood is high. This reduces
the need for gluconeogenesis from amino acids
carbon skeleton, thus preserving essential protein.
Diagram: Glycogen degradation on the superior portion of V. ADIPOSE TISSUE IN FASTING

the diagram as part of the overall metabolic response of the
liver during fasting.
A. Carbohydrate metabolism
- Glucose transport by insulin-sensitive GLUT-4 into
the adipocytes are depressed >>> decreased in fatty
acid and TAG synthesis.
- GLUT-4 transporters are both found in adipose
tissue and skeletal muscle. When you have insulin
in the blood, this triggers more GLUT-4 transporters
to go into the cell membrane and allows more
glucose to enter into the cell. Hence, glucose
transport by insulin sensitive GLUT-4 transporters
into the adipocytes are depressed due to low levels
--Appendices for clearer photo-- of circulating insulin during the fasting state. This

BIOCHEMISTRY
DIABETES MELLITUS
leads to a decrease in fatty acid and triacylglycerol the plasma membrane, subsequent glucose
synthesis. metabolism is depressed because of low insulin.
-
B. Fat metabolism A. Carbohydrate metabolism
1. Increased degradation of TAG: ● Glucose transport into skeletal muscle cells via
- Activation of lipase and hydrolysis of stored TAG are insulin-sensitive GLUT-4 proteins and subsequent
enhanced by the elevated catecholamines glucose metabolism are depressed because of low
epinephrine and, particularly, norepinephrine. levels of circulating insulin.
There will be increased degradation of
triacylglycerol, increased release of fatty acid and B. Lipid metabolism
decrease uptake of fatty acid. ● During the first 2 weeks of fasting, muscle uses fatty
acids from adipose tissue and ketone bodies from
2. Increased release of fatty acids: the liver as fuels
● Fatty acids from hydrolysis of stored TAG are ● After about 3 weeks of fasting, muscle decreases its
primarily released into the blood. Bound to use of ketone bodies and oxidizes fatty acids almost
albumin, transported to tissues for use as fuel. exclusively. This leads to an already elevated level
of circulating ketone bodies. Note that the
3. Decreased uptake of fatty acids: increased use of ketone bodies by the brain as a
● Lipoprotein lipase activity of adipose tissue is low. result of increased concentration in the blood is
Consequently, circulating TAG of lipoproteins is not correlated with a decreased use of these
available to adipose tissue. compounds by the muscle.
C. Protein metabolism
● During fasting, muscle proteolysis happens,
initiated by the fall in insulin and sustained by the
rise in glucocorticoids. Remember that you only
have insulin receptors not glucagon receptors, thus
proteolysis is initiated by the fall of insulin,
sustained by the rise in glucocorticoids.
● During the first few days of fasting, there is a rapid
breakdown of muscle protein, in order to provide
amino acids used by the liver for gluconeogenesis.
● Alanine and glutamine are the most important
gluconeogenic amino acids released from muscle.
● By several weeks of fasting, the rate of muscle
proteolysis decreases paralleling a decline in the
need for glucose as a fuel for the brain, which has
begun using ketone bodies as a source of energy.
VI. RESTING SKELETAL MUSCLE IN FASTING
- Note: There is a difference between resting and

exercising muscle.
- Resting muscle uses fatty acid as its major fuel
source. In contrast, exercising muscle initially will
use glycogen stores as a source of energy.
- During intense exercise, glucose 6 phosphate
derived from glycogen is converted to lactate by
anaerobic glycolysis. As this glycogen reserves are
depleted, the free fatty acid provided by the
mobilization of triacylglycerol from adipose tissue
becomes the dominant energy source.
- For carbohydrate metabolism in the resting skeletal
muscle, glucose transport into the muscles via your - Major metabolic pathways in skeletal muscle during
GLUT-4 transporters which are insulin sensitive. In starvation. [Note: The numbers in the circles, which
appear both in the figure and in the corresponding

BIOCHEMISTRY
DIABETES MELLITUS
citation in the text, indicate important pathways for

fat or protein metabolism.]
VII. BRAIN IN FASTING
● During the first days of fasting, the brain continues

to use glucose exclusively as a fuel. Blood glucose is
maintained for the brain by hepatic
gluconeogenesis from glucogenic precursors such
as amino acid from proteolysis and glycerol from
lipolysis.
VIII. KIDNEY IN LONG TERM FASTING
- Kidney does not wait for sources of energy to arrive,

rather it will make way in providing the pool of
glucose in the blood.
● As fasting continues into early starvation and

beyond, the kidney plays very important roles:
1) Kidney expresses the enzymes of
gluconeogenesis, including glucose 6-phosphatase
2) In late fasting, about 50% of gluconeogenesis
even occurs here.
Note that a portion of this glucose is used by the
● In prolonged fasting (>2–3 weeks), plasma ketone
kidney itself.
bodies reach significantly elevated levels, and
3) Kidney also provides compensation for the
replace glucose as the primary fuel for the brain.
acidosis that accompanies the increased production of
Thus, reduces the need for protein catabolism for
ketone bodies.
gluconeogenesis.
● The glutamine released from the muscle’s
● Ketone bodies spare glucose and muscle protein.
metabolism of branched-chain amino acids is taken
You can't allow proteolysis to happen, because this
up by the kidney and acted upon by renal
will cause cachexia and muscle wasting. The
glutaminase and glutamate dehydrogenase,
metabolic changes during fasting ensures that all
producing α-ketoglutarate used as a substrate for
the tissues have an adequate supply of fuel
gluconeogenesis, plus ammonia (NH3).
molecules.
● NH3 picks up H+ from ketone body dissociation, and
is excreted in the urine as NH4 + decreases the acid
load in the body.
- In long term fasting, there is a switch from nitrogen
disposal in the form of urea to disposal in the form
of ammonia, this is the reason why patients with
kidney diseases, they are high risk for acidosis
during fasting and patients with moderate to severe
chronic disease (ESRD) or those who are going with
dialysis, they are discouraged from fasting.

BIOCHEMISTRY
DIABETES MELLITUS
Comparison of Type I and Type II Diabetes

TYPE I DM TYPE II DM
Age of onset Childhood or At age 35,

puberty, symptoms develop
symptoms gradually
develop rapidly
Nutritional Frequently Obesity usually

status at time undernourished present
of disease
onset
Prevalence 10% 90%
Genetic Moderate Very strong

predisposition
Defect or B cells are Insulin resistance

deficiency destroyed, combined with
eliminating inability of B cells to
production of produce appropriate
insulin quantities of insulin
Ketosis Common Rare

--Appendices for clearer photo--
Plasma insulin Low to absent High in early in
IX. METABOLISM IN DIABETES MELLITUS
disease; low in
disease of long
Diabetes Mellitus duration
● Elevation of fasting blood glucose caused by either
a relative or absolute deficiency in insulin. It is the Acute Ketoacidosis Hyperosmolar state
leading cause of adult blindness, amputation, renal complications
failure, heart attack, nerve damage and strokes.
● In the Philippines, 1 in 14 Filipino adults lives with Response to Unresponsive Responsive
diabetes. In 2019, the international Diabetes oral
Federation showed that about 4 million of the total hypoglycemic
63 million Filipino adult population have diabetes. It drugs
has a 6.3% prevalence of diabetes in adults.
Treatment Insulin is always Diet, exercise, oral
necessary hypoglycemic drugs;
insulin may or may
not be necessary.
Reduction of risk
factors (smoking
cessation, blood
pressure control,
treatment of
dyslipidemia) is
essential to therapy.
- In type I diabetes, the majority or all of the beta cells

are destroyed, eliminating the ability of pancreas to

BIOCHEMISTRY
DIABETES MELLITUS
produce insulin while on type II, the problem is

insulin resistance.
- Type I diabetes are unresponsive to oral
hypoglycemic drugs so as soon as they are
diagnosed with type I diabetes, they are already
started on insulin therapy, while on Type II, they are
given hypoglycemic drugs. If blood glucose is
elevated, patients will also start with insulin
therapy.
A. TYPE I DIABETES
Prevalence: 10%
● absolute deficiency of insulin by auto -immune
attack on the β cells of the pancreas.
● gradual depletion of the β-cell population.
● There is an autoimmune destruction because islets
of Langerhans become infiltrated with activated T
lymphocytes, leading to insulitis.
● symptoms appear abruptly when 80–90% of the β
cells have been destroyed.
● Insulin therapy is required in order to restore
metabolic control and prevent life threatening
ketoacidosis.
● β Cell destruction requires both a stimulus from the
environment (such as a viral infection) and a genetic
determinant that allows the β cells to be recognized
as “nonself.”.
● Among monozygotic or identical twins, if one sibling
develops Type I DM, the other twin has only a 30 to
- Note that FPG of 100 to 125mg/dl is an impaired
50% chance of developing the disease. These
fasting glucose. Fasting in blood glucose means
contracts with Type II DM in which the genetic
there is no caloric intake for at least 8 hours.
disease is stronger and all or both the monozygotic
- When uncertain, for example a patient has a FBG of
twins will develop.
160 mg/dl, however the patient does not present
with any symptoms, then test for:
Diagnosis of Type I Diabetes
Circulating islet-cell antibodies
● Onset: childhood or puberty
● Symptom onset: sudden
triggered by stress or illness
● Primary Symptoms:
-polyuria (frequent urination)
-polydipsia (excessive thirst), and
-polyphagia (excessive hunger)
● Accompanying symptoms:
-Fatigue
-weight loss
-Weakness
● Confirmation/ Ancillary test:
FBG: ≥ 126 mg/dl
Accompanied by ketoacidosis

BIOCHEMISTRY
DIABETES MELLITUS
Metabolic Changes • Note: Acetyl coenzyme-A from beta-oxidation gives

the substrate for ketogenesis.
• Diabetic ketoacidosis occurs in 25–40%, and may
recur if the patient becomes ill or noncompliant
with therapy. DKA is treated by replacing fluid and
electrolytes, and administering short-acting insulin
to gradually correct hyperglycemia.
• DKA is one of the most common complications of
type I diabetes. Considered a life-threatening
complication.
• S/S: most common symptoms of DKA are vomiting,
abdominal pain, increased urination plus
encephalopathy symptoms like confusion and
sometimes loss of consciousness.
• Patients with DKA have fruity breath because of
acidosis. The onset of symptoms is usually very
rapid, and DKA most commonly occurs in those who
know that they already have Diabetes but actually it
may also be the first presentation in someone who
has not been living known to be diabetic.
• DKA may be diagnosed when the combination of
hyperglycemia, so you test for the sugar of the
Figure 7-2: Shows the metabolic abnormalities of type I patient (the blood glucose), most of the time it is
Diabetes Mellitus that result from the deficiency of insulin >250-300. Ketones in the blood are also present, so
which profoundly affects the metabolism in the liver, muscle, when you are suspecting for DKA, you request for:
and adipose tissue. 1.Blood glucose
--Appendices for clearer photo— 2. Urine ketones – there should be the presence of
ketones in the urine.
Metabolic changes in Type I Diabetes 3. Arterial blood gas – which eventually shows
1. Hyperglycemia and ketoacidosis: acidosis in the blood.
● Elevated levels of blood glucose & ketones are
hallmarks of untreated type I Diabetes. 2. Hypertriacylglycerolemia
● Hyperglycemia is caused by increased hepatic ● Not all fatty acids flooding the liver be disposed
production of glucose, and diminished through oxidation or ketone body synthesis.
peripheral utilization ● Excess fatty acids are converted to triacylglycerol,
packaged and secreted in very-low-density
Remember that in type I Diabetes, hyperglycemia is caused lipoproteins (VLDL) thus type I diabetes is also a risk
by: for atherosclerosis and cardiovascular diseases.
1. Increased hepatic production of glucose – ● Chylomicrons are synthesized from dietary lipids by
because your body and liver cannot sense that there the intestinal mucosal cells following a meal.
is so much glucose in the blood since there is no ● Because lipoprotein degradation by lipoprotein
insulin available. lipase in the capillary beds of muscle and adipose
2. Diminished peripheral utilization – in the tissue is low in DM, plasma chylomicron and VLDL
muscle and adipose tissues, you have insulin- levels are elevated in the blood, resulting now in
sensitive GLUT-4 receptors that allow further influx hypertriacylglycerolemia, thus the high risk of
of glucose into the cell thus decreasing blood stroke or myocardial infarction among patients with
glucose. Since the body cannot identify the high type I diabetes. So it is very important to not only
amount of glucose already present in the blood, the monitor the blood sugar of patients with type I
body continuously makes way to provide us with diabetes but also need to monitor their lipid profile,
energy sources. check for both triacylglycerol and LDL.
• Ketosis results from increased mobilization of fatty
acids from adipose tissue, with accelerated hepatic
fatty acid β-oxidation and synthesis of 3-
hydroxybutyrate and acetoacetate.

BIOCHEMISTRY
DIABETES MELLITUS
● INTENSIVE
○ more closely normalize blood glucose thru
more frequent monitoring or more
frequent injection of insulin.
○ more injections of insulin—three or more
times a day.
○ Mean blood glucose: 150 mg/dl
○ HbA1C approximately 7%
• Normal mean blood glucose is approximately 100

mg/dl and HbA1C is 6% or less
• Patients show a 50% or more reduction in the long-
term microvascular complications of diabetes—
retinopathy, nephropathy, and neuropathy
This is the very reason why patients with type I DM
are advised to also consult an ophthalmologist in
order to check for changes in their retina already.
For patients with type I DM, as soon as they are
diagnosed with type I DM, you immediately advise
--Appendices for clearer photo-- them to check for a consultation with their
Treatment Of Type I Diabetes ophthalmologist because it took years for type I DM
• Individuals with type I DM must rely on exogenous to occur but symptoms came in abruptly. While in
insulin injected subcutaneously to control patients with type II DM, we advise the patients to
hyperglycemia and ketoacidosis. We cannot treat check for changes in their retina through their
them anymore with oral medications, you ophthalmologist 5 years after they are diagnosed.
immediately start with insulin therapy which is
given subcutaneously. Hypoglycemia in Type I Diabetes
• Hypoglycemia by excess insulin is the most common
● STANDARD complication of insulin therapy (> 90%)
○ Typically consist of one or two daily • High with intensive treatment regimens
injections of recombinant human insulin. The frequency of hypoglycemic episodes can
○ Mean blood glucose of patients who are in actually cause comatose, or seizures particularly
standard treatment: 225–275 mg/dl those with intensive treatment regimen.
○ Hemoglobin A1C (HbA1C): 8–9% of total • In normal individuals hypoglycemia triggers a
hemoglobin compensatory secretion of glucagon and
○ HbA1C is proportional to the average epinephrine (counter-regulatory hormones) which
blood glucose concentration over the promote hepatic production of glucose. So for
previous 3 months. normal patients, when you have hypoglycemia,
your liver will immediately trigger this
compensatory secretion.
• Type I diabetics, however, also develop a deficiency
of glucagon secretion, which occurs early in the
disease and is almost universally present 4 years
after diagnosis.
• Patients thus rely on epinephrine secretion to
prevent severe hypoglycemia. However, as the
disease progresses, they show diabetic autonomic
neuropathy and impaired ability to secrete
epinephrine in response to hypoglycemia.
• The combined deficiency of glucagon and
epinephrine secretion creates a condition
sometimes called “hypoglycemia unawareness.”
Thus patients with long-standing diabetes are
particularly very vulnerable to hypoglycemia.

BIOCHEMISTRY
DIABETES MELLITUS
• Hypoglycemia can also be caused by strenuous

exercise. Exercise promotes glucose uptake into B. TYPE II DIABETES
muscle and decreases the need for exogenous • Type 2 DM is the most common form and it afflicts
insulin. Patients now with diabetes are advised to approximately 90% of the diabetic population.
check their blood glucose level before or after • Develops gradually without obvious symptoms. The
intensive exercise to prevent or abort disease is often detected by a routine screening test.
hypoglycemia. • However, many individuals have symptoms of polyuria
• The reason why we do not allow the patients to and polydipsia of several weeks duration. Polyphagia is
have a very intensive therapy or why we allow the less common. But many are unaware that the symptoms
patients to at least just have a blood glucose level of of polyuria or polydipsia are actually significant.
around 150mg/dL and not achieve immediately a Polyphagia may be present but it is less common to type
normal blood glucose of <100mg/dL is because of II DM.
hypoglycemia. • Combination of insulin resistance and dysfunctional β
• One of the therapeutic goals in cases of diabetes is cells
to decrease blood glucose level in an effort to • Do not require insulin to sustain life, although eventually
minimize the development of complications (i.e. will be required to control hyperglycemia and keep
neuropathy, nephropathy, retinopathy) however, HbA1c below 7% in 90% of patients.
appropriate dosage of insulin is very difficult to • Metabolic alterations are milder because the secretion
achieve. of insulin in type II DM, although not adequate and does
not restrain ketogenesis.
Contraindications for Tight control
● Children
○ hypoglycemia may adversely affect brain
development
● Elderly people
○ hypoglycemia can cause strokes and heart
attacks
● Tight control is most worthwhile for healthy people who
can expect to live at least ten more years.
● Major goal of tight control is to prevent complications
many years later.
Diagnosis of Type II Diabetes

• Onset: usually after age 35
• Symptom onset: gradual does not involve viruses nor
autoimmune antibodies. The diagnosis is based most
commonly on the presence of hyperglycemia (fasting
blood glucose concentration of > 126mg/dL with
symptoms.

BIOCHEMISTRY
DIABETES MELLITUS
● Primary Symptoms: Insulin Resistance

○ polyuria (frequent urination)
○ polydipsia (excessive thirst), and
○ polyphagia (excessive hunger)
● Confirmation/ Ancillary test:
○ FBG: ≥ 126 mg/dl
Insulin Resistance
- Decrease ability of the target tissues (liver, adipose,
muscle) for them to respond properly to normal or
elevated concentrations of insulin. For example,
insulin resistance is characterized by uncontrolled
hepatic glucose production and decreased glucose
uptake by muscles and adipose tissue. Figure 11-1: time course for the development of
Insulin Resistance and Obesity hyperglycemia and the loss of beta cell function.
○ Obesity is the most common cause of insulin --Appendices for clearer photo—
resistance. Although most people with obesity
and insulin resistance do not automatically Insulin resistance and type 2 diabetes
become diabetic. ● Insulin resistance alone will not lead to type 2
○ In the absence of a defect in β-cell function, diabetes.
nondiabetic, obese individuals can compensate ● Type 2 diabetes develops in insulin-resistant
for insulin resistance with elevated levels of individuals who also show impaired β-cell function.
insulin in order to prevent DM.
○ Insulin secretion is two to three times higher in ● People at risk to develop Type 2 diabetes
obese subjects than it is in lean individuals as a ○ Elderly
compensatory mechanism to their insulin ○ Obese
resistance. ○ physically inactive
○ 3–5% of pregnant women who develop
GDM (Gestational Diabetes Mellitus)
● If you experience or come up with a patient who has
history of GDM, you have to monitor them by
requesting FBS years later especially if
overweight/obese or if they have a first-degree
relative with type II DM.
● Causes of insulin resistance

○ Insulin resistance increases with weight
gain and, conversely, diminishes with
weight loss. A very reason why diet and
exercise are both highly advised and are
included in primary treatment therapy is
that weight loss causes insulin resistance
to be improved.
○ Fat accumulation is important in the
development of insulin resistance.
○ Adipose tissue is also a secretory organ.
○ Substances produced by adipocytes,
Leptin and Adiponectin may contribute to
the development of insulin resistance.
○ In addition, the elevated levels of free
fatty acids (FFA) in obesity have also been
implicated in insulin resistance.
Dysfunctional B cells
• Initially, the pancreas retains β-cell capacity >>
insulin levels vary from above normal to
below normal.

BIOCHEMISTRY
DIABETES MELLITUS
• With time, years later, the β cell becomes chylomicron and VLDL levels are elevated resulting in
increasingly dysfunctional and fails to secrete hyperTAG
enough insulin to correct the prevailing • Low HDL levels are also associated with type 2 diabetes.
hyperglycemia. • Just like in type I DM, you also need to monitor the lipid
For example, insulin levels are high in typical obese type profile of the patient. Check for TAG, LDL & HDL.
II DM patients, but not as high as in similarly obese
individual who are non-diabetic. Treatment of Type II DM
• The natural progression of the disease results in a Goals:
declining ability to control hyperglycemia with ● Maintain blood glucose concentrations within
endogenous secretion of insulin. normal
• The deterioration of beta cell function may be ● Prevent development of long-term complications.
accelerated by the toxic effect of sustained ● The American Diabetes Association generally
hyperglycemia and elevated free FA. recommend the following target for blood sugar
levels:
80-130mg/dL – if FBS
<180mg/dL – postprandial or 2 hours after meal
● Weight reduction
● Exercise
● Medical Nutrition therapy
--Appendices for clearer photo-- ● Hypoglycemic agents or insulin therapy
● In addition to medication, you have to advise your
Metabolic changes in Type II DM patients to lessen the intake of sugary foods or at
least remove that useless food (i.e. soft drinks or
sweetened juice, fruit juice). Advise the patient to
take 1 cup of rice per day because once the rice is
metabolized it will eventually lead to elevated sugar
in the body.
● If the patient is not able to control his/her blood
glucose level by diet or weight reduction, you must
start with oral medications such as metformin,
glimepiride, gliclazide, vildagliptin etc. and later on
if years already have passed and the patient is still
not able to control his/her blood glucose level, you
may start the patient with insulin therapy already.
Your goal here is to control the blood glucose level
--Appendices for clearer photo-- between 80-130mg/dL for FBS in order to prevent
your complication.
1. Hyperglycemia
• Hyperglycemia is caused by increased hepatic
production of glucose, combined with diminished
peripheral use.
• Ketosis is usually minimal or absent (in type II DM)
because the presence of Insulin diminishes hepatic
ketogenesis
• Note that Metformin, the first-line agent/treatment for
type II DM, inhibits hepatic gluconeogenesis, thus is
considered a very effective treatment for type II DM.
2. Dyslipidemia:
• In the liver, fatty acids are converted to triacylglycerols,
which are packaged and secreted in VLDL.
• Chylomicrons are synthesized from dietary lipids by the
intestinal mucosal cells following a meal
• Lipoprotein degradation catalyzed by lipoprotein lipase
in adipose tissue is low in diabetics, >>> plasma

BIOCHEMISTRY
DIABETES MELLITUS
The long-standing elevation of blood glucose is associated

with the chronic complications of diabetes: ● So we advise the patient to have intensive
1. Premature atherosclerosis including cardiovascular treatment among patients with DM. They have to
disease and stroke. monitor their blood sugar level to prevent
2. Diabetic retinopathy hypoglycemia to attain <130mg/dL sugar level to
3. Nephropathy prevent complications.
● Diabetes is the no. 1 cause of end-stage
renal disease. So the majority of patients ● For HbA1c, you want the patient to have less than 7
undergoing dialysis, about 60% of them, HbA1c. HbA1C is a better test to know if the patient
have DM. This is the very reason why you with diabetes is compliant with the medication and
really need to control the sugar level of the responsive to the therapy.
patient to <130mg/dL.
4. Neuropathy
● The reason why a lot of patients have
amputations. Not only nerve cells are
affected but also arteries in lower
extremities due to atherosclerosis. So you
must check on the presence of
hyperpigmentation on lower extremities,
the reason here is what we call your
peripheral arterial occlusive disease,
there is occlusion on the arteries of the
lower extremities, decrease supply of
blood in the lower extremities causing
hyperpigmentation and non healing
wounds. Because of neuropathy, the
patient is unaware that they have a
nonhealing wound, and because of
atherosclerosis there is an inability for
these wounds to heal on their own, thus
leading now to amputation.
Chronic Effects and Prevention of DM Effect of body weight and exercise on the development of
type 2 diabetes.
X. TEST YOURSELF
INCREASE / DECREASE
1-2. Fasting is ____ in insulin secretion and an ____ in

glucagon release.
3. ____ glucagon to insulin ratio causes a rapid mobilization

of liver glycogen stores (contains about 80g of glycogen in the fed
state) due to phosphorylation (activation) of glycogen
phosphorylase.
4. In lipid metabolism, after about 3 weeks of fasting, muscle ____

its use of ketone bodies and oxidizes fatty acids almost exclusively.
This leads to an already increased level of circulating ketone bodies.
5. In fat metabolism, activation of lipase and hydrolysis of stored TAG

are enhanced by the elevated catecholamines epinephrine and,
particularly, norepinephrine. There will be _________ degradation

BIOCHEMISTRY
DIABETES MELLITUS
of triacylglycerol, increased release of fatty acid and _______ uptake

of fatty acid.
6. One of the most common complications of type I diabetes.

Considered a life-threatening complication. Patients with this
complication may present with fruity breath.
a. Lactic acidosis
b. Diabetic ketoacidosis
c. Hypoglycemia
d. Ketosis
7. Individuals with type I DM must rely on exogenous insulin to

control hyperglycemia and ketoacidosis. What is the most common
route of administration for insulin?
a. Intramuscular
b. Intradermal
c. Intravenous
d. Subcutaneous
8. What condition will arise for combined deficiency of glucagon and

epinephrine secretion?
a. Diabetes insipidus
b. hypoglycemia unawareness
c. Diabetic neuropathy
d. Addison’s Disease
9. Occlusion on the arteries of the lower extremities will lead to

decreased supply of blood in the lower extremities causing
hyperpigmentation and non healing wounds. This condition is
commonly referred to as?
a. peripheral arterial occlusive disease
b. peripheral vascular disease
c. coronary artery disease
d. varicose veins
10. Metformin is the first-line agent/treatment for type II DM,

inhibiting hepatic gluconeogenesis. Which of the following is the
most prominent side effect of metformin?
a. hypoglycemia
b. lactic acidosis
c. nausea and vomiting
d. diabetes ketoacidosis
ANSWER:
1. DECREASE
2. INCREASE
3. INCREASE because after 10 to 18 hours of fasting, there will be a
hepatic glycogenolysis considered transient response to the 18hrs
fasting.
4. DECREASES, during the first 2 weeks of fasting, muscle uses fatty
acids from adipose tissue and ketone bodies from the liver as fuels.
After about 3 weeks of fasting, muscle decreases its use of ketone
bodies and oxidizes fatty acids almost exclusively
5. INCREASE, DECREASE
6. B
7. D
8. B
9. A
10. B

BIOCHEMISTRY
DIABETES MELLITUS
XI. APPENDIX

BIOCHEMISTRY
DIABETES MELLITUS

BIOCHEMISTRY
DIABETES MELLITUS

BIOCHEMISTRY
DIABETES MELLITUS

For Your Eyes Only
VITAMINS and MINERALS
OVERVIEW
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A. Vitamins
- organic compounds that human tissues cannot synthesize  supplied by the diet
- small amounts are required (insufficient in mass to be suppliers of carbon or nitrogen or to act as energy
sources)
- below required level intake  deficiency symptoms appear
- time of onset of symptoms is a function of
- size
- daily flux of body reserves
- excessive vitamin A and D consumption  accumulation of toxic quantities
1. Required by the Body
- to perform specific cellular functions
- for normal growth and development
2. Functions
- prevent acute deficiency diseases (ex: scurvy, beri-beri)
- maintenance of optimal health
- chronic disease prevention
- daily intake of vitamin E (100 IU or 300% RDA) significantly reduces the risk of
coronary artery disease (CAD)
B. Sources of Vitamins
1. Foods
2. Synthesized by intestinal microorganisms
C. Classification
1. Water-Soluble Vitamins
- B vitamins
- folic acid
- niacin
- pantothenic acid
- biotin
- vitamin C
2. Fat-Soluble Vitamins
- vitamin
-A
-D
-E
-K
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D. Cofactors
1. Vitamins and Derivatives
- often serve as precursors of coenzymes for the enzymes of intermediary metabolism
a. Vitamin K
- the only fat-soluble vitamins which has a coenzyme function
2. Holoenzymes
- active enzymes that have the necessary cofactor bound to apoenzymes
- ex: pyruvate carboxylase
- needs biotin to be active
3. Prosthetic Groups
- cofactors that remain tightly bound to the enzymes and do not dissociate from it
4. Minerals
- may also serve as cofactors
- confer upon the enzyme a property that it does not possess in their absence
- particularly transition metals
- zinc
- iron
- copper
- may - play direct role in catalysis
- serve as redox reagent
- form complexes with substrates
E. Causes of Deficiency
1. Inadequate Dietary Intake
2. Inadequate Absorption
a. Biliary Obstruction
- lack of bile  decreased fat-soluble vitamin absorption
b. Regional Ileitis
c. Tropical Sprue
d. Celiac Disease (Nontropical Sprue)
- induced by wheat gluten in the diet of susceptible persons
e. Pernicious Anemia
- due to lack of intrinsic factor
3. Inadequate Use
a. Lack of transport protein
b. Failure to synthesize the active form that is ingested in the inactive precursor form
4. Increased Requirements
- increased caloric requirement  increased vitamin requirement
a. Growth
b. Pregnancy
c. Lactation
d. Wound Healing and Convalescence
5. Increased Excretion
- ex: renal malfunction
6. Drug-Induced
a. Antibiotic Therapy
 decreased microbial population
b. Isoniazid
- treatment for tuberculosis
- antagonist of pyridoxal phosphate derived from pyridoxine (vitamin B6)
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VITAMIN SUPPLEMENTS
A. Traditional Role of Vitamin Supplements
1. For Patients at Risk for Nutritional Deficiencies
- individuals consuming modified diets
- fad diets
- weight reduction regiments
- strict vegetarian diets
2. Some Normal Physiologic Conditions
- pregnancy
- lactation
3. Pharmacologic Amounts in Patients With
- malabsorption syndromes
- non-nutritional diseases (pharmacologic use of niacin in patients with hyperlipidemia)
B. Vitamin Supplementation in the General Population
- increased vitamin intake may be achieved by dietary modification
- ex: 3-5 daily servings of vegetables
2-4 portions of fruit
- the general population is unaccustomed to such diet  vitamin supplementation
WATER-SOLUBLE VITAMINS
- many are precursors of coenzymes for the enzymes of intermediary metabolism
- not toxic
- stored amount in the body are usually small
- when ingested in excess  readily excreted in the urine  must be continually supplied in the diet
A. Thiamine (Vitamin B1)
1. Structures
a. Thiamine Pyrophosphate (TPP)
- biologically active form

- formed from the transfer of pyrophosphate group from ATP to thiamine
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i. Formed in the
- liver
- lesser extent
- muscle
- brain
- heart
- reticulocytes
ii. Coenzyme in the
iia. Transketolation reactions found in the pentose phosphate pathway
iib. Conversion of pyruvate to acetyl-CoA
iic. Conversion of -ketoglutarate to succinyl-CoA
iid. Creation of branched-chain amino acids leucine, isoleucine, and valine
2. Distribution
- richest source
- pork
- organ meats
- lean meats
- yeast
- eggs
- green vegetables
- whole grain cereals
- nuts
- legumes
- outer layer of seeds
- whole wheat bread
- white bread prepared from milled grain is low in thiamine
- no significant supply from intestinal microorganisms
3. Nutritional Requirements
- RDA - males - 1.5 mg/day
- females
- 1.1 mg/day
4. Reactions
i. Oxidative Decarboxylation (Dehydrogenase Reactions) of Pyruvate (Carbohydrate
Metabolism) and -Ketoglutarate
- role in energy metabolism of most cells particularly important in nervous tissues
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ii. Transketolation
4. Clinical Indications
a. Thiamine Deficiency
i. Results to
- decreases the amount of acetyl-CoA available to enter the TCA cycle,
increases the amount of pyruvate available for anaerobic oxidation 
increased lactic acid production  decreased ATP production 
impaired cellular function
- decreased transketolase activity
ii. Effects
- cardiovascular and neurologic lesions
- emotional disturbances
iii. Clinically Signs
- muscle cramps
- paresthesias
- irritability
- beriberi (wet or dry)
iv. Diagnosis
- diagnosed by an increase in RBC transketolase activity
observed upon addition of thiamine pyrophosphate
b. Beri-beri
- severe thiamine-deficiency syndrome
- in areas where polished rice is the major component of the diet
i. Signs of Infantile Form
- tachycardia
- vomiting
- convulsions
- death if untreated
- rapid onset in nursing infants of thiamine deficient mothers
ii. Signs of Adult Form
- dry skin
- irritability
- disorderly thinking
- progressive paralysis
iii. “Dry” Beriberi
- diet chronically contains slightly less than the requirement
- symptoms
- peripheral neuropathy (motor and sensory neuropathy)
- extremities of greatest use most affected
- fatigue
- impaired capacity to work
- occurs with little physical exertion and decreased caloric intake
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iv. “Wet” Beriberi
- when deficiency is more severe
- manifestations
- neuropathy
- heart failure which may be high output
- includes a triad of
- peripheral vasodilation
- biventricular failure
- edema
- often brought on by physical exertion and increased carbohydrate intake
c. Wernicke's Encephalopathy
i. Triad - ophthalmoplegia (and nystagmus)
- truncal ataxia
- confusion
ii. Untreated Encephalopathy
- progresses to Korsakoff syndrome
d. Wernicke-Korsakoff Syndrome
i. Causes
- dietary thiamine deficiency in chronic alcoholism
- impaired intestinal absorption
ii. Manifestations
- apathy
- impaired short-term memory
- confabulation
- grossly normal cognition
B. Riboflavin (Vitamin B2)
1. Structures
a. Biologically Active Forms
- flavin mononucleotide (FMN)
- flavin adenine dinucleotide (FAD)
- formed from the transfer of AMP moiety from ATP to FMN
- capable of reversibly accepting 2 hydrogen atoms  FMNH2 or FADH2
- bound tightly (sometimes covalently) to flavoenzymes that catalyze oxidation or reduction of a
substrate
2. Distribution
- good sources
- milk
- eggs
- liver
- green-leafy vegetables
- kidneys
- limited supply from intestinal microorganisms
- readily destroyed by UV component of sunlight
- does not appear to be related to caloric requirements or to muscular activity
a. RDA - 1.7 mg/day males
- 1.3 mg/day females
b. Need - is related to protein use
- increased during
- growth
- pregnancy
- lactation
- wound healing
- convalescence
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c. Urinary Excretion
- affected by alterations in nitrogen balance
- positive nitrogen balance
- decrease in urinary riboflavin
4. Metabolism
- riboflavin  intestinal mucosal cells  flavin-adenine mononucleotide (FMN)  liver 
flavin-adenine dinucleotide (FAD)  excreted as riboflavin
5. Functions of Riboflavin
- precursor of the coenzymes FMN and FAD
- maintenance of tissues
- mucosal
- epithelial
- ocular
6. Deficiency
- results to ariboflavinosis
- lead to deficiencies in FMN and FAD
- not associated with a major human disease
- frequently accompanies other vitamin deficiencies
a. Manifestations
i. Lesions of the
- lips
- mouth
- skin
- genitalia
ii. Conditions
- angular stomatitis
- cheilosis (fissuring at the corners of the mouth)
- glossitis (tongue appearing smooth and purplish)
- corneal vascularization
- seborrheic dermatitis
- weakness
- anemia (glutathione reductase depends on riboflavin  red blood cell lysis 
anemia)
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C. Niacin (Nicotinic Acid, Vitamin B3)
1. Structure
a. Biologically Active Coenzyme Forms
- nicotinamide adenine dinucleotide (NAD+)
- nicotinamide adenine dinucleotide phosphate (NADP+)
b. Niacin
- substituted pyridine derivative
c. Nicotinamide
- derivative of nicotinic acid (nicotinic acid amide)
- contains amide instead of carboxyl group
- occurs in the diet
- readily deaminated in the body  nutritionally equivalent to nicotinic acid
i. Niacin and Nicotinamide
- equally effective in supplying human needs
- RDA - 19 mg/day males
- 15 mg/day females
- tryptophan is a precursor
- 60 mg niacin = 1 mg nicotinic acid
3. Distribution
a. Sources
- unrefined and enriched grains and cereals
- lean meats
- milk
- meats
- yeast
- liver
- legumes
b. Other Source
- tryptophan metabolism
- only in conditions of abundance of the amino acid (after the needs for protein
synthesis and energy production have been met)
- 60 mg of tryptophan yield 1 mg of nicotinic acid
- nicotinic acid derived from tryptophan catabolism  provide 10% of
pyridine nucleotide cofactor pool (remainder coming from ingested
nicotinic acid or nicotinamide)
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4. Metabolism
5. Functions
a. Synthesis of the Biologically Active Forms
- nicotinamide adenine dinucleotide (NAD+)
- phosphorylated derivative nicotinamide adenine dinucleotide phosphate (NADP+)
b. Coenzymes in Oxidation-Reduction Reactions
- undergo reduction of the pyridine ring accepting a hydride ion (hydrogen atom plus 1
electron)
c. Reduced Forms
- NADH
- NADPH
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a. Pellagra
- niacin deficiency
i. Causes
ia. Dietary Niacin Deficiency
ib. Isoniazid Use
ic. Hartnup Disease
- genetic defect in tryptophan membrane transport  intestinal
malabsorption and poor renal resorption
- simple replacement therapy is insufficient
id. Malignant Carcinoid Syndrome
- tryptophan is used for excessive 5-hydroxybyptamine (5-HT)
production and is less available for NAD synthesis 
pellagra that is nonresponsive to therapy
ii. Involves
- skin
- GIT
- CNS
iii. Manifestations
- dermatitis
- diarrhea from chronic intestinal mucosal inflammation
- dementia
- death if untreated
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iv. Non-Treatment
- can be fatal
v. Treatment
- dietary replacement of both tryptophan and niacin
b. Hyperlipidemia Treatment
- circulating free fatty acids  liver  TAG synthesis  VLDL production  LDL
- LDL - cholesterol-rich lipoprotein
- derived from VLDL in the plasma
- at doses of 1.5 g/day (100x the RDA)  strongly inhibits lipolysis in adipose tissue
(primary producer of circulating free fatty acids)  decreased hepatic TAG
synthesis  decreased plasma TAG (in VLDL)
decreased cholesterol (in VLDL and LDL)
 treatment of type IIb hyperlipoproteinemia (both VLDL and LDL
are elevated)
D. Biotin
1. Structure
- synthesized by intestinal microorganisms in large quantities  dietary source probably not
necessary
- amount of excreted biotin is 5x greater than the dietary intake
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3. Distribution
- present in almost all foods particularly
- liver
- kidneys
- milk
- egg yolk
- vegetables
4. Metabolism
a. Biotin
- as a prosthetic group
- covalently attached to the enzyme
- structure not modified with the attachment
b. Biotin Holocarboxylase Synthetase
- covalently links the free carboxyl group of biotin to a specific lysine residue of the
enzyme
c. Biotinidase
- catalyzes the removal of biotin from enzymes during protein turnover  recycling of
biotin
5. Functions
- coenzyme in carboxylation reactions
- carrier of activated carbon dioxide
- covalently bound to -amino groups of lysine residues of biotin-dependent enzymes
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6. Deficiency
- wide distribution  rare occurrence
a. Deficiency due to inadequate dietary intake with following accompanying factors
i. Antibiotics
- inhibit growth of intestinal bacteria  elimination of biotin source
ii. Avidin
- glycoprotein
- tightly binds biotin  prevents intestinal absorption
- in raw egg-white
- when introduced as source of protein (20 eggs/day) induces symptoms
of biotin deficiency
- seborrheic dermatitis
- glossitis
- loss of appetite
- nausea
- anorexia
- muscular pain
b. Multiple Carboxylase Deficiency
- defect in the enzyme biotin holocarboxylase synthetase  prevents attachment of
biotin to biotin-dependent enzymes
E. Pantothenic Acid (Vitamin B5)
1. Structure
- no RDA established
- requirements probably met at intake of 5-10 mg/day
3. Distribution
- widely distributed
- important sources
- eggs
- liver
- yeasts
- synthesized by some intestinal bacteria
4. Function
- component of coenzyme A
- component of fatty acid synthase
Coenzyme A
- functions in the transfer of acyl groups
- contains a thiol group that carries acyl compounds as activated thiol esters
- ex: succinyl CoA
fatty acyl CoA
acetyl CoA
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5. Metabolism
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6. Deficiency
- rare
- deficiency is not well characterized in humans
- experimentally induced deficiency
- nausea
- fatigue
- burning cramps in the limbs
- paresthesias or dysesthesias
- gastrointestinal distress
F. Folic Acid (Folate)
- key role in 1-carbon metabolism
- essential in the biosynthesis of
- purines
- pyrimidine thymine
- probably the most common deficient vitamin particularly among
- pregnant women
- alcoholics
1. Structure
- pteridine ring attached to p-aminobenzoic acid (PABA)
- conjugated with 1 or more glutamic acid residues
- humans cannot
- synthesize PABA
- attach the 1st glutamic acid
- tetrahydrofolic acid (THF)
- biologically active form
- produced by 2-step reaction by dihydrofolate (DHF) reductase
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a. DHF Reductase
- competitively inhibited by methotrexate
i. Methotrexate
- folic acid analogue
- used to effect the remission of acute leukemia in children
b. Sulfanilamide and its Derivatives
- structural analogues of PABA
- drugs that competitively inhibit folic acid synthesis  decrease the synthesis of
nucleotides needed for DNA and RNA replication
i. Sulfa Drugs
- do not affect human DNA or RNA synthesis because mammalian cells cannot
synthesize folic acid
2. Functions
a. THF - receives 1-carbon fragment from donors such as
- serine
- glycine
- histidine
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- transfers 1-carbon fragment to intermediates in the synthesis of

- amino acids
- purines and purine nucleotides
- deoxythymidylate
- thymidine
- characteristic pyrimidine of DNA
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3. Distribution
- green leafy vegetables
- liver
- lima beans
- whole grain cereals
- yeast
- fruits
- synthesis by intestinal bacteria
- the vitamin is easily destroyed by cooking
a. RDA - 200 g/day males
- 180 g/day females
b. Increased Requirements
- pregnancy
- lactation
5. Metabolism
a. Folate Enterohepatic Cycle
- dietary folate (available as polyglutamates)  hydrolysis and reduction from
enzymes on mucosal cell membranes  monoglutamate forms  folate
methylation by duodenal mucosal dihydrofolate reductase  absorption by
jejunal enterocytes  join plasma-binding proteins  liver  secreted in bile
 duodenum  repeat of cycle
b. Total Body Stores
- 12-15 mg
- lasts for 4-6 months
a. Folate Deficiency
i. Characteristics
- growth failure
- megaloblastosis of the bone marrow
- megaloblastic or macrocytic anemia (folic acid deficiency  deficient THF
derivatives  diminished DNA synthesis in erythropoietic stem cells
 anemia)
- gastrointestinal disturbances
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ii. Causes
iia. Increased Demand
- pregnancy
- lactation
- hemolytic anemias
iib. Malabsorptive Diseases
- malabsorptive diseases that affect the jejunum
- celiac sprue
- biliary disease
- alter the folate enterohepatic cycle
- drug treatment that are DHF reductase inhibitors (methotrexate)
iic. Inadequate Dietary Intake
- alcoholics
- persons who do not consume a lot of raw vegetables
iid. Liver Dysfunction
- alcoholic cirrhotics
- interfere with the enterohepatic cycle
- may interfere with production of plasma-binding proteins
iie. Medications
iiei. Methotrexate and Trimethoprim
- inhibit dihydrofolate reductase and decrease absorption of
dietary folate
iieii. Phenytoin
- can also interfere with absorption
iii. Folic Acid Deficiency
 decreased purine and pyrimidine synthesis  megaloblastic anemia 
affected cells unable to make DNA  cannot divide
iv. Treatment
iva. Folic Acid Supplementation
- pregnancy
- lactation
- alcoholism
ivb. Folinic Acid (Leucovorin)
- to protect normal cells from the methotrexate toxic effects
b. Nutritional Anemias
- classified according to the size of the RBCs or mean corpuscular volume (MCV)
- caused by inadequate intake of 1 or more essential nutrients
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i. Microcytic Anemia
- lack or iron (most common form)
ii. Macrocytic or Megaloblastic Anemia
- from cell deficiency of
- folic acid
- vitamin B12
- causes accumulation of large, immature red cell precursors
(megaloblasts) in the bone marrow
c. Folate and Neural Tube Defects in the Fetus

- early fetal development of the neural tube is critically dependent on folic acid presence
i. All Childbearing Women
- should consume 0.4 mg of folic acid/day  reduce risk of having pregnancy
affected by
- spina bifida
- anencephaly
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ii. 1st Weeks of Fetal Life

- many women are not yet aware of the pregnancy
- folate-dependent development occurs  adequate folate nutrition must occur
iii. Supplementation
- before conception
- during the 1st trimester
- avoid complicating diagnosis the diagnosis of vitamin B12 deficiency  folic acid
intake should not exceed 1 mg/day
G. Cobalamin (Vitamin B12)
1. Structure of Cobalamin and its Coenzyme Forms
a. Corrin Ring System
- 2 pyrrole rings linked directly (as differentiated from porphyrins which pyrrole rings
are linked through methylene bridge)
b. Cobalt
- held in the center of the corrin ring by 4 coordination bonds from the nitrogens of the
pyrrole groups
- other coordination bonds in commercial preparations in the form of cyanocobalamin
- with the nitrogen of 5,6-dimethylbenzimidazole
- with cyanide
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c. Coenzyme Forms of Cobalamin
i. 5’-Deoxyadenosylcobalamine
- cyanide is replaced with 5’-deoxyadenosine forming carbon-cobalt bond
ii. Methylcobalamin
- cyanide is replaced by a methyl group
2. Distribution
a. Cobalamin
- is synthesized only by microorganisms
- not present in plants
- obtained
- as preformed vitamins from natural bacterial flora
- eating food derived from other animals
- appreciable amounts in
- liver
- kidney
- whole milk
- eggs
- oysters
- fresh shrimp
- pork
- chicken
- RDA - 2 g/day males and females
- need is increased during pregnancy
4. Metabolism
- dietary vitamin B12  bind to intrinsic factor  cobalamin-intrinsic factor complex  bind to
surface receptors of mucosal cells in the ileum  general circulation  bind with
vitamin B12-binding proteins (globulin)  methylcobalamin and
5-deoxyadenosylcobalamin, (liver, bone marrow cell, reticulocytes)
- no significant catabolism
- small amounts excreted in the bile  most reabsorbed in the ileum  enterohepatic circulation
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5. Folate Trap Hypothesis
a. Effects of Deficiency are More Pronounced in Rapidly Dividing Cells
- bone marrow erythropoietic tissue
- intestinal mucosal cells
 need N5, N10-methylene and N10-formyl forms of THF required for the synthesis of
nucleotides for DNA replication
b. Vitamin B12 Deficiency
- N5-methyl form of THF is not efficiently used
- methylated form cannot be converted directly to other forms of THF  folate trapped in
the N5-methyl form  N5-methyl form accumulate
- levels of other forms decrease
 deficiency of THF forms needed in purine and thymine synthesis  symptoms of
megaloblastic anemia
6. Functions
a. B12 Dependent Enzymes
i. Methylmalonyl-CoA Mutase
- in the catabolism of
- isoleucine
- valine
- converts propionyl-CoA to methylmalonyl CoA and then to succinyl-CoA
- cobalamin deficiency  accumulation of abnormal fatty acids 
incorporated into cell membranes (including those of the
nervous system  neurologic manifestations)
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ii. Leucine Aminomutase

iii. Methionine Synthase (Homocysteine : H4-Folate Methyltransferase)
b. Role in Hemopoiesis
- related to folate metabolism
- methylcobalamin deficiency  deficiency of folate coenzyme pool
c. Maintenance of
- myelin sheath
- epithelial cells
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- significant amounts stored in the body (4-5 mg)  may take several years to manifest
symptoms of vitamin B12 deficiency
a. Vitamin B12 Deficiency
- rarely due to absence of the vitamin
- more commonly due to vitamin malabsorption
- long-term strict vegetarian diet  vitamin B12 deficiency
i. Dietary Deficiency
- rare (liver stores large quantities)
ii. Pernicious Anemia
- common cause of megaloblastic anemia (mean corpuscular volume [MCV] >
100 fL)
- most commonly due to autoimmune destruction of gastric parietal cells 
decreased production of glycoprotein intrinsic factor (accompanied by
achlorhydria and atrophic gastritis)
- dietary vitamin B12  bind to intrinsic factor  cobalamin-intrinsic factor
complex  bind to surface receptors of mucosal cells in the ileum 
general circulation  bind with vitamin B12 -binding proteins
(globulin)  methylcobalamin, 5-deoxyadenosylcobalamin (liver, bone
marrow cell, reticulocytes)
iii. Gastrectomy (and similarly Gastric Bypass Surgery)
- disrupts secretion of intrinsic factor by gastric parietal cells  decreased
absorption of vitamin B12
iv. Infectious Causes
iva. Helicobacter pylori
- causes chronic gastritis
ivb. Diphyllobothrium latum
- the fish tapeworm competes for vitamin B12 absorption in the
intestine
v. Blind Loop Syndrome
- bacterial overgrowth also competes for vitamin B12 absorption in the
intestine
vi. Structural Abnormalities of the Terminal Ileum
- Crohn disease
- surgical resection
- can cause decreased absorption of vitamin B12
vii. Pancreatic Insufficiency
- leads to a decrease in enzymes necessary to break down the R protein-
cobalamin complex  preventing cobalamin from binding with
intrinsic factor
- cobalamin release from the animal protein  bound by R protein
(haptocorrin)  stable R protein-cobalamin complex in the
low pH  R protein-cobalamin complex and secreted intrinsic
factor move into the duodenum  R protein-cobalamin
complex broken down by pancreatic enzymes (pancreatic
enzymes degrade R protein)  allows intrinsic factor to
bind vitamin B12
viii. Transcobalamin II Deficiency
- prevents cobalamin from entering systemic circulation
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b. Consequences of Vitamin B12 Deficiency

i. Megaloblastic Anemia
- elevated MCV (> 110 fL)
- hypersegmented neutrophils (lobe count~ 4)
ii. Bone Marrow Megaloblastosis

iii. Degeneration of Axis Cylinders of Spinal Cord Neurons
iv. Lesions of Mucous Surfaces
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v. Glossitis
vi. Methylmalonic Aciduria
vii. Neuropsychiatric Symptoms
viii. CNS Symptoms
- may occur in the absence of anemia
- irreversible
- pathogenesis is different from those described for megaloblastic anemia
viiia. Neuropathy
- characterized by defective myelin formation and consequent
subacute degeneration of the posterior and lateral spinal
columns  - symmetrical paresthesias and ataxia
- loss of proprioception and vibration senses
- in severe cases
- spasticity
- clonus
- paraplegia
- fecal and urinary incontinence
- mechanism
- (may be that) vitamin B12 deficiency  decreased folate 
decreased methionine levels  impaired myelin
production
c. Treatment
- intramuscular injection of cyanocobalamin
- high dose oral vitamin B12
- continued throughout the lives of patients suffering from pernicious anemia
d. Folic Acid Administration
- if given even alone reverses the hematologic abnormality
- masks vitamin B12 deficiency  severe neurologic dysfunctions and pathology
 megaloblastic anemia should not be treated with folic acid alone (should be the
combination of folate and vitamin B 12)
8. Case: Vitamin B12 deficiency
a. Presentation
A 6 month-old male infant was admitted to the hospital in a coma. The infant had been
normal at birth, but over the past 2 months his condition had deteriorated as he became
lethargic and unable to control his head. His weight was below average, and his head
circumference was very small (in the 3rd percentile of his age). Urinalysis revealed high
methylmalonic acid and compounds related to methionine metabolism. Blood
analysis revealed vitamin B12 levels were 20 pg/mL (normal value = 150-1000 pg/mL).
The infant’s mother indicated that she was a strict vegetarian who had eaten no animal
products, including milk and eggs, for the past 8 years, and had taken no vitamin
supplements. The infant was being exclusively breast-fed. Analysis of the mother’s milk
revealed vitamin B12 levels of 75 pg/mL (normal value = 1000-3000 pg/mL).
b. Diagnosis and Treatment
- infant’s condition
- vitamin B12 deficiency
- administered vitamin B12 at 1 mg/day for 4 days  condition improved dramatically
 discharged after 14 days
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c. Discussion
- vitamin B12
- is obtained in the diet from animal products
- not found in plants
- deficiency is rare
- conserved and stored in the liver
- 8 years of strict vegetarian diet  mother’s vitamin B12 stores decreased  insufficient
breastmilk vitamin B12 content
- compounds in the infant’s urine are metabolites normally processed by vitamin B12-
dependent enzymes
- methylmalonyl CoA mutase
- homocysteine methyltransferase
H. Pyridoxine (Vitamin B6)
- collective term for
a. Pyridoxine
- occurs primarily in plants
b. Pyridoxal
- found in foods obtained from animals
c. Pyridoxamine
- found in foods obtained from animals
- all 3 can function as vitamin B6
- derivatives of pyridine
- differ only in the nature of the functional group attached to the ring
- serve as precursors of the biologically active cofactor pyridoxal phosphate (PLP)
1. Structures
2. Nutritional Requirement
a. RDA - 2 mg/day males
- 1.6 mg/day females
b. Increased Requirements
- pregnancy
- lactation
- high protein intake
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3. Distribution
- wheat
- corn
- egg yolk
- liver
- fish
- nuts
- whole-grain cereals
- muscle meats
- some from intestinal bacterial synthesis
4. Metabolism
- nonphosphorylated forms (pyridoxine, pyridoxal, pyridoxamine)  absorbed in the upper
intestinal tract  converted to phosphate esters by pyridoxal kinase (phosphorylation)
using ATP (brain, liver, kidneys)
- extensive metabolism  pyridoxic acid as major metabolite formed in the liver
- excreted in the urine
- vitamin storage
- brain
- liver
- muscle
- half is bound to muscle glycogen phosphorylase
5. Functions
a. PLP - coenzyme for a large number of enzymes that use amino acids as substrates
- form covalent Schiff-base intermediates between PLP aldehyde portion and -amino
group of amino acids
- found in glycogen phosphorylase  as general acid-base catalyst in the conversion of
glycogen  glucose 1-phosphate
Reaction Type Example

Transamination Oxaloacetate + Glutamate  Aspartate + -ketoglutarate
Deamination Serine  Pyruvate + NH3
Decarboxylation Histidine  Histamine + CO2
Condensation Glycine + Succinyl CoA  -aminolevulinic acid
Aldol cleavages
Racemizations
Elimination and replacement
reactions at the beta and
gamma carbons
6. Deficiency
a. Isoniazid (INH)
- isonicotinic acid hydrazide
- drug used to treat tuberculosis
- forms an inactive derivative with pyridoxal phosphate  pyridoxine deficiency 
dietary pyridoxine supplementation should be an adjunct in INH treatment
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b. Dietary Pyridoxine Deficiencies
- rare
- observed in
- newborn infants fed with formulas low in vitamin B6
- women taking oral contraceptives
- alcoholics
c. Biochemical Indicators
- decreased blood PLP levels
- decreased RBC transaminase activity
- increased urinary excretion of xanthurenic acid following tryptophan load
- decreased urinary pyridoxic acid excretion
- increased urinary oxalic acid excretion
- increased urinary cystathionine excretion
d. Clinical Symptoms
- skin and mucosal lesions
- sideroblastic anemia
- neuronal dysfunction including convulsions
- personality changes
7. Pyridoxine Toxicity
- intake of >2 gm/day  neurologic symptoms
- when vitamin is discontinued  substantial improvement but no complete recovery
I. L-Ascorbic Acid (Vitamin C)
1. Structure
2. Nutritional requirements
- RDA - 60 mg/day males and females
3. Distribution
- citrus fruits and other juices
- strawberries
- cantaloupes
- raw or minimally cooked green vegetables
- potatoes (particularly the skin)
- tomatoes
4. Metabolism
- not metabolized
- functions as cofactor in its form
5. Functions
- ascorbic acid
- active form
- main function
- reducing agent in several different reactions
- coenzyme in hydroxylation of prolyl and lysyl residues during collagen biosynthesis by the
fibroblasts  maintenance of normal connective tissue
wound healing
- required in the synthesis of
- epinephrine
- norepinephrine
- antioxidant  protective agent against free radical damage
- aids dietary iron absorption from the intestines
- “does not prevent colds”
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a. Deficiency
i. Scurvy
- disease characterized by
- sore
- spongy gums
- loose teeth
- fragile blood vessels
- swollen joints
- anemia (often present)
- deficiency of collagen hydroxylation  defective connective tissue

 defective wound healing and bone formation
lessening of blood vessel integrity  hemorrhagic diathesis
b. Chronic Disease Prevention
i. Antioxidants
- vitamin C
- vitamin E
- -carotene
- supplementation  decreased incidence of chronic disease
- coronary heart disease
- cancers
- inactivate toxic oxygen free radicals
ii. Reactive Oxygen Radicals
iia. By-Product Of
- normal metabolism
- exposure to
- sunlight
- ozone
- tobacco smoke
- other environmental pollutants
iib. Damaging to
- lipid membranes
- proteins
- cellular DNA
iic. Role in the Development of
- heart disease
- lung disease
- cancer
- aging
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7. Toxicity
- no observed acute toxicity
a. Dehydroascorbic Acid
- oxidized form of ascorbic acid
- toxic
- high vitamin C doses could favor accumulation of dehydroascorbic acid
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FAT-SOLUBLE VITAMINS
- vitamins are released  absorbed  transported with the fat of the diet
- not readily excreted in the urine
- significant quantities are stored
- adipose tissue
- liver
- excessive vitamins A and D consumption  accumulation of toxic quantities
A. Vitamin A (Retinol)
1. Vitamin A Structure
a Vitamin A
- collective term for several related biologically active molecules
b. Retinoids
- family of molecules that are related to retinol
- includes both natural and synthetic forms of vitamin A that may or may not show
vitamin A activity
i. Essential for
- vision
- reproduction
- growth
- epithelial tissue maintenance
c. Carotenoids
- provitamins
i. -Carotene
- contained by plant foods
- oxidatively metabolized by intestinal mucosal cells  2 retinal molecules
- conversion in human is inefficient  vitamin A activity of -carotene is about
1/6 that of retinol
- found in green vegetables
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d. Retinol
- primary alcohol
- contains -ionone ring with an unsaturated side chain
- found in animal tissues as a retinyl ester with long-chain fatty acids
- used by the body
e. Retinal
- aldehyde derived from retinol oxidation
- can easily be interconverted with retinol
- used by the body
f. Retinoic Acid
- dietary retinol  oxidized to retinal  oxidized to retinoic acid
- cannot be reduced in the body  cannot give rise to retinol or retinal
- mediates most of the actions of the retinoids except vision (depends on retinal)
- high affinity binding to specific receptor proteins in the nucleus of target tissues 
activated retinoic acid-receptor complex interact with nuclear chromatin 
stimulate retinoid-specific RNA synthesis  specific protein synthesis 
physiologic functions
2. Vitamin A Absorption and Transport

a. Transport to Liver
- dietary retinol esters  hydrolysis in the upper intestinal mucosa   transported to
the liver via chylomicrons  release of retinol and free fatty acids
i. Retinol
- derived from
- esters
- cleavage and reduction of carotenes
 reesterified to long chain fatty acids (palmitate) in the intestinal mucosa 
secreted as a component of chylomicrons  lymphatic system 
hepatic uptake and storage (90%)
ii. Carotenoids
- may be directly absorbed (bile is required for micelle formation)
- most are cleaved to retinal  converted to retinol which is absorbed
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b. Release from Liver
- retinol released from the liver  transported by plasma retinol-binding protein (RBP)
and prealbumin  extrahepatic tissues  retinol RBP complex  attach to
specific receptors on the surface of cells of peripheral tissues  intracellular
retinol entry  cellular retinol-binding protein  retinol transported to
nuclear sites  action of vitamin in a manner analogous to steroid hormones
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3. Mechanism of Action of Vitamin A
- retinoid acid  high-affinity binding to specific receptor proteins in the nucleus of target
cells (ex: epithelial cells)  retinoic acid-receptor complex  activation  interaction
with nuclear chromatin  stimulation of retinoid-specific RNA synthesis 
production of specific proteins  mediate several physiologic functions
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a. Retinoids
- control keratin gene expression in most epithelial tissues
b. Specific Retinoic Acid-Receptor Proteins
- part of the superfamily of transcriptional regulators that include
- steroid
- thyroid hormones
- 1, 25-dihydroxycholecalciferol
4. Functions of Vitamin A
a. Role in Vision
- retinol  retina  esterified to a fatty acid (means of concentrating retinol within the
cell)  fatty acid esters hydrolysis  release of retinol  oxidized to retinal
(by specific NAD+-linked dehydrogenase)  form complexes with opsin in the
rods and cones  rhodopsin  visual cycle
i. Visual Cycle
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ia. Rhodopsin
- visual pigment of the rod cells of the retina
- consists of 11-cis retinal specifically bound to protein opsin
- light exposure  series photochemical isomerizations 
bleaching of visual pigment  release of all-trans retinal
and opsin  nerve impulse production  optic nerve  brain
- isomerization of all-trans retinal  11-cis retinal  spontaneous
combination with opsin  rhodopsin regeneration
** similar reactions are responsible for color vision in cone cells
b. Growth
i. Vitamin Deprivation
 taste bud keratinisation  appetite loss
 slow bone growth (fails to keep pace with growth of the nervous system) 
CNS damage
c. Reproduction
i. Retinol and Retinal
- essential for normal reproduction
- support spermatogenesis in the male
- prevent fetal resorption in the female
ii. Retinoic Acid
- inactive in maintaining
- reproduction
- visual cycle
- promotes growth and differentiation of epithelial cells
d. Differentiation and Proliferation of Epithelial Cells
- vitamin A is essential for
- normal differentiation of epithelial tissues
- respiratory tract
- skin
- cornea
- conjunctiva
- other tissues
- normal mucus secretion
5. Distribution
- good sources of preformed vitamin A (all-tans retinyl esters)
- liver
- kidney
- cream
- butter fat
- egg yolk
- fish oils
- meats
- dairy products
- eggs
- good dietary sources of carotenes (precursors of vitamin A)
- yellow and dark green vegetables
- fruits
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6. Requirement for Vitamin A
a. RDA for Adults
i. Males - 1000 retinol equivalents (RE)/day
ii. Females
- 800 RE/day
1 RE = 1 g of retinol
= 6 g of -carotene
= 12 g of other carotenoids
i. Retinol and Its Precursors (Retinyl Esters)
- used as dietary supplements
ii. Retinoic Acid
- useful in dermatology
a. Effects of Vitamin A Deficiency
- vision problems
- disorders of epithelial cell differentiation and proliferation
- impaired immune response
b. Symptoms
i. Headaches
ii. Skin changes
iii. Sore throat
iv. Alopecia
c. Progression of Visual Symptoms for Vitamin A Deficiency
i. Loss of Green Light Sensitivity
ii. Poor Adaptation to Dim Light
iii. Night Blindness
- loss of retinol in rod cells
- one of the earliest signs of vitamin A deficiency
- visual threshold is increased  difficult to see in dim light
- prolonged deficiency  irreversible loss in the number of visual cells
iv. Xerophthalmia
- squamous epithelial thickening
- due to severe vitamin A deficiency  pathologic conjunctival and corneal
dryness
- if no treatment  corneal ulceration  scarring (opaque)  blindness
iva. Bitot Spots
- squamous metaplasia
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ivb. Keratomalacia
- softening of the cornea
d. Respiratory Epithelial Metaplasia

- often common in cystic fibrosis due to failure of fat-soluble vitamin absorption
e. Frequent Respiratory Infections
- secondary to respiratory epithelial defects
f. Acne and Psoriasis
- effectively treated with retinoic acid or its derivatives
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i. Mild Cases of Acne (Darier’s Disease) and Aging

ia. Treatment
- topical application of
- tretinoin
- all trans retinoic acid
- very toxic for systemic administration
- treatment of skin aging
- benzoyl peroxide
- antibiotics
ib. Severe Recalcitrant Cystic Acne Unresponsive to Conventional
Therapies
- oral isotretinoin (13-cis retinoic acid)
g. Prevention of Chronic Disease
- high dietary -carotene  antioxidant and enhanced immune function  decreased
incidence of
- skin cancer
- cataracts
- macular degeneration
- -carotene is nontoxic even at high doses for extended periods
- clinical trials
a. -Carotene Supplementation
- did not decrease the incidence of lung cancer
- increased cancer in individuals who smoke
b. High-Dose -Carotene Supplementation
- increased death due to heart disease
h. Mucous Membranes
- abnormal formation of keratin
i. Bones - failure of bone remodelling  thick solid bones in the skull  increased
cerebrospinal fluid pressure
j. Reproductive System
i. Males - gonadal dysfunction
ii. Females
- miscarriage
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8. Toxicity
a. Hypervitaminosis A
- toxic syndrome due to excessive vitamin A intake
- should avoid intake of retinol >7.5 mg/day
i. Organ Systems Involved
ia. Skin - dry
- pruritic
ib. Liver
- enlarged
- can become cirrhotic
ic. Nervous System
- increased intracranial pressure can mimic that of brain tumor
b. Acute Toxicity
- can be caused from a large, single dose of vitamin A
- manifestations
- nausea
- vertigo
- blurry vision
c. Chronic Toxicity
- manifestations
- ataxia
- alopecia
- hyperlipidemia
- hepatotoxicity
d. As a Teratogenic Effect
- first trimester of pregnancy
- excess vitamin A can be very teratogenic (potential for causing congenital
malformations in the developing fetus)
- can lead to fetal loss
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i. Isotretinoin
- teratogenic
- absolutely contraindicated in women with childbearing potential
- prolonged treatment  hyperlipidemia, increased LDL/HDL ratio 
increased risk of cardiovascular disease
B. Vitamin D
- group of sterols that have hormone-like function
1. 1, 25-Dihydroxycholecalciferol (1,25-diOH D3)
- active molecule
- binds to intracellular receptor proteins  1,25-diOH D3-receptor complex  interaction with
DNA in the nucleus of target cells  stimulate gene expression
repress gene transcription
- most prominent actions
- regulate plasma levels of
- calcium
- phosphorous
2. Structure
3. Distribution
a. Diet
i. Ergocalciferol
- vitamin D2
- found in plants
- differ from the cholecalciferol by the presence of
- additional double bond
- methyl group
ii. Cholecalciferol
- vitamin D3
- found in animal tissues
b. Endogenous Vitamin Precursor
i. 7-Dehydrocholesterol
- intermediate in cholesterol synthesis
- converted to cholecalciferol in the dermis and epidermis in humans exposed
to sunlight
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ii. Preformed Vitamin D
- dietary requirement in individuals with limited sunlight exposure
4. Metabolism
a. 1,25-diOH D3 Formation
- vitamin D2 and D3 (inactive)  liver  hydroxylation at 25-position  25-
hydroxycholecalciferol (25-OH D3)  kidney  hydroxylation at 1-position 
1,25-diOH D3 (regulator of calcium metabolism)
i. 1,25-diOH D3
- most potent vitamin D metabolite
ii. 25-OH D3
- predominant form of vitamin D in the plasma
- major storage form
iii. 25-Hydroxycholecalciferol 1-Hydroxylase and 25-Hydroxylase
- employ
- cytochrome P450
- molecular oxygen
- NADPH
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b. 25-Hydroxycholecalciferol 1-Hydroxylase Regulation

- increased activity
- directly by low plasma phosphate
- indirectly by low plasma calcium  parathyroid hormone release
- insufficient dietary calcium  hypocalcemia  increased plasma 1,25-diOH
D3 ( decreased 1-hydroxylase activity)
5. Functions of Vitamin D
a. Maintain Adequate Calcium Plasma Levels by
- increasing intestinal calcium uptake
- minimizing renal calcium loss
- stimulating bone resorption
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b. Effect of Vitamin D on the Intestine

- 1,25-diOH D3  enters intestinal cell  binding with cytosolic receptor  1,25-diOH
D3-receptor complex  nucleus  interaction with cellular DNA  increased
synthesis of calcium-binding protein  enhanced calcium and phosphate
absorption
c. Effect of Vitamin D on Bone
- 1,25-diOH D3 + PTH  bone (important reservoir of calcium)  release of phosphate
and calcium  increased plasma calcium and phosphate
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6. Regulation
7. Distribution and Requirement

- occurs naturally in
- fatty fish
- liver
- egg yolk
- RDA - 5 g cholecalciferol/day
- 200 IU/day
- ultraviolet light  synthesized in the skin  required as accessory food factor when deprived of
sunlight
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a. Vitamin D Deficiency
 net bone demineralization  rickets in children
osteomalacia in adults
i. Causes
- insufficient exposure to daylight
- deficiencies in vitamin D consumption
ii. Occur Predominantly in
- infants
- elderly
iii. Characteristics
- continued bone collagen matrix formation, incomplete mineralization  soft,
pliable malformed bones
iv. Treatment
- vitamin D therapy
b. Rickets
i. Nutritional Rickets
ia. Characteristics
- signs of hypocalcemia
- bowing of the lower extremities
- poor dentition
ib. Treatment
- vitamin D therapy
ii. Renal Rickets (Renal Osteodystrophy)
- chronic renal failure  decreased ability to form the vitamin
- 1,25-diOH D3 (calcitriol) administration
- effective replacement therapy
iii. Vitamin D-Resistant Rickets
- vitamin D repletion does not treat the syndrome
- genetic abnormality may be present
iiia. Type I Vitamin D-Resistant Rickets
- genetic mutation of 1-hydroxylase
- treatment
- 1,25-dihydroxyvitamin D (bypass the conversion of 25-
hydroxy derivative in the kidney)
- if 1,25-dihydroxyvitamin D supplementation does not treat
the underlying problem  type II vitamin D-
resistant rickets
iiib. Type II Vitamin D-Resistant Rickets
- mutated 1,25-dihydroxyvitamin D receptor unresponsive to both
vitamin D and calcitriol
iv. X-Linked Rickets
- entirely due to renal phosphate wasting
- 1,25-0HD levels are elevated
c. Osteomalacia
- preexisting bone demineralization  increased susceptibility to fractures
- higher doses of 800 IU/day  reduce the incidence of osteoporotic fractures
i. Treatment
- vitamin D therapy
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d. Hypoparathyroidism  hypocalcemia and hypophosphatemia

- treatment
- vitamin D + parathyroid hormone
9. Toxicity
a. Vitamin D
- most toxic of all vitamins
- can be stored in the body
- slowly metabolized
b. High Doses (100,000 IU for weeks or months)
- loss of appetite
- thirst
- nausea
- stupor
- enhanced calcium absorption and bone resorption  hypercalcemia  calcium
deposition particularly the arteries and kidneys
c. Sarcoidosis
- can lead to excess vitamin D since pulmonary macrophages can produce calcitriol
d. Lymphoma
- can produce calcitriol
C. Vitamin K
- serve as coenzyme in the carboxylation of certain glutamic acid residues  post-
translational modification of various blood clotting factors
a. Several Forms
i. Phylloquinone (Vitamin K1)
- in plants
ii. Menaquinone (Vitamin K2)
- in intestinal bacterial flora
- found in mammalian liver
iii. Menadione
- synthetic derivative
- used for treatment
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1. Structure
2. Function
a. -Carboxyglutamate (Gla) Formation
i. Vitamin K
- required in the hepatic synthesis of
- prothrombin
- clotting factors
- II
- VII
- IX
-X
- required in the vitamin K-dependent carboxylation of glutamic acid
residues in the formation of clotting factors
ii. Requires
- O2
- CO2
- hydroquinone form of vitamin K
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iii. Sensitive to Inhibition by

iiia. Dicumarol
- anticoagulant naturally occurring in spoilt sweet clover
iiib. Warfarin
- synthetic vitamin K analogue
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b. Interaction of Prothrombin with Platelets
i. Gla Residues of Prothrombin
- good chelators of positively charged calcium ions
ii. Prothrombin-Calcium Complex
- bind to phospholipids essential for blood clotting on the surface of platelets
iii. Platelet Attachment
- increases the rate of proteolytic conversion of prothrombin to thrombin
c. Role of -Carboxyglutamate Residues in Other Proteins

- unknown
- Gla is also present in osteocalcin of bone
d. Cofactor in the Carboxylation Reaction
- requires oxygen
- found in
- cabbage
- cauliflower
- spinach
- egg yolk
- liver
- green leafy vegetables
- extensive synthesis by the bacteria in the gut
- RDA - 80 g/day males
- 65 g/day females
- recommended
- 70-140 g/day
- bile is required for absorption
a. Vitamin K Deficiency
- true deficiency is unusual
i. Antibiotic Therapy
 decreased bacterial population  decreased vitamin K production 
hypoprothrombinemia
- 2nd generation cephalosporins
- cefoperazone
- cefamandole
- moxalactam
- warfarin-like mechanism  hypoprothrombinemia
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ii. Symptoms
- similar to those of warfarin toxicity
- GI bleeding
- intracranial bleeding
- ecchymoses
- epistaxis
- hematuria
iii. Treatment
- vitamin K supplementation
b. Vitamin K Deficiency in the Newborn
- newborns  sterile intestines  cannot synthesize vitamin K
- breastmilk
- provides only 1/5 of daily requirement  require single intramuscular dose of
vitamin K  prevent hemorrhagic disease
5. Toxicity
- prolonged large dose administration  toxic effects on RBC membranes  hemolytic anemia
 jaundice in newborns
D. Vitamin E
- consists of 8 naturally occurring tocopherols
a. -Tocopherol
- most active
1. Structure

- rich sources
- liver
- eggs
- seed oil
- sunflower oil
- corn oil
- soybeans
- meats
- fruits
- vegetables
a. RDA - requirement increases as polyunsaturated fatty acid intake increases
- bile is required for absorption
- 60% of ingested tocopherols  excreted in the feces
i. -Tocopherol
- 10 mg for men
- 8 mg for women
ii. Vitamin E
- 15 IU
3. Absorption
- absorbed in the intestine  to the liver via chylomicrons
4. Metabolism
- unmetabolized in the body
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5. Functions
a. Antioxidant
- primary function
- prevention of nonenzymatic oxidation of cell components (polyunsaturated fatty acids)
by
- molecular oxygen
- free radicals
- works together with
- vitamin C
- -carotene
 delay onset of cataracts
b. Prevent free radical formation in polyunsaturated fatty acids by donating electrons
c. Participate with glutathione peroxidase in the removal of peroxides formed in the
polyunsaturated fatty acids
6. Deficiency
- almost entirely restricted to premature infants
a. Fat Malabsorption Diseases
- cystic fibrosis
- liver disease
- decrease the amount of vitamin E available
- usually associated with defective lipid absorption and transport
b. Signs - sensitivity of RBCs to peroxide  hemolysis
- appearance of abnormal cellular membranes
- creatinuria due to increased muscle breakdown
- peripheral neuropathy
- ophthalmoplegia
7. Vitamin E Excess
- can interfere with vitamin K metabolism
8. Vitamin E Supplementation
- not recommended for the prevention of chronic disease such as
- CHD
- cancer
a. -Tocopherol, -Carotene Cancer Prevention Study
- given high doses of vitamin
- lacked cardiovascular benefit
- increased incidence of stroke
9. Toxicity
- least toxic of the fat-soluble vitamins
- no toxicity observed at doses >300 mg/day
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SUMMARY
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MINERALS
- inorganic elements
- variety of functions
- cofactors in enzyme-catalyzed reactions
- acid-base balance regulation
- nerve conduction and muscle irritability
- structural elements in the body
- have adverse effects if ingested in excessive amounts
A. 2 Major Groups
- based upon extent to which they are required
1. Macrominerals
- essential for proper nutrition
- required in > 100 mg/day
- includes
- calcium
- chloride
- magnesium
- phosphorous
- potassium
- sodium
2. Microminerals (Trace Elements)
- required in < 100 mg/day
B. Classification
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C. Functions
D. Calcium and Phosphorous

- most abundant minerals in the body
1. Calcium
- exists in 2 forms that have different functions
a. Calcium Phosphate Crystals
- in the bones and teeth forming the cement (structural)
b. Unbound Ionic Form (Ca++)
- critical functions in
- muscle contractions
- nerve impulse transmission
- ion transport
- signal transmission across membranes
- hormone action
- blood clotting
- tightly regulated extracellular and intracellular concentrations
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2. Phosphorus
- required for the formation of
- ATP
- phosphorylated intermediates in metabolism
3. Parathyroid Hormone (PTH)
- decreased extracellular calcium level  PTH release
a. Renal Effects
- increases calcium absorption from the glomerular filtrate
- decreases phosphate absorption
b. Increases Synthesis of 1,25-Dihydroxycholecalciferol (Active Form of Vitamin D)
- stimulation of calcium release from the bones
- increased calcium transport from intestinal lumen to the blood
4. Calcitonin
- increased extracellular calcium level  specialized cells found among the thyroid follicular cells
 calcitonin secretion
- decreases plasma calcium levels by
- decreasing bone reabsorption
- increasing urinary calcium and phosphate loss
5. Osteoporosis
- leads to progressive loss of bone mass
- occurs in the elderly in both sexes (more pronounced in postmenopausal women)
- characterized by frequent bone fractures
a. Life-Style
- may influence calcium metabolism
- bone loss in immobilized or sedentary individuals
- increased bone mass with regular exercise
b. Therapy
i. Estrogen Replacement
- most effective prevention of postmenopausal bone loss
ii. Calcium Supplementation
- often given with vitamin D
- if the current calcium intake in pre- and postmenopausal women is 500 mg/day
- should be supplemented with additional 1000 mg
- obtained from
- milk and milk products
- calcium supplements
- calcium carbonate
- calcium gluconate
- calcium lactate
- excess calcium intake does not prevent or reverse bone loss due to inactivity or
estrogen insufficiency
For Your Eyes Only
E. Sodium, Potassium, and Chloride

1. Functions
- regulate pH
- maintain ECF and ICF osmolarity
- establish ion gradients across membranes
- maintain water balance
- neutralize positive and negative charges on proteins and other molecules
2. Diet and Hypertension
- increased sodium intake  increased osmolarity  increased blood volume  increased blood
pressure
3. Stroke and Potassium
- low sodium and high potassium diet is associated with the lowest blood pressure levels and
lower frequency of strokes
F. Iron - most found in
- heme component of hemeproteins (mostly hemoglobin and cytochromes)
- storage forms
- ferritin
- hemosiderin
- < 1% of total body iron found in
- plasma
- various iron-containing enzymes
1. Iron Deficiency
- leads to decreased hemoglobin synthesis  iron-deficiency anemia
For Your Eyes Only
a. Most Commonly Seen in
- premenopausal women (result of menstruation)
- men with undetected GI bleeding
b. Other Causes
- iron-poor diet
- impaired intestinal iron absorption
c. Treatment
i. Ferrous Salts
- ferrous sulfate
- ferrous gluconate
- ferrous fumarate
2. Iron Excess
- adult men should not use iron
- high iron tissue levels correlate with increased risk of myocardial infarction
- unbound inorganic iron can promote formation of reactive oxygen radicals (conversion of H2O2
to highly reactive hydroxyl radicals)  LDL oxidation
3. Forms of Dietary Iron
- availability of dietary iron depends on whether iron is present as
- heme iron (more rapidly absorbed)
- nonheme iron (less rapidly absorbed)
G. Magnesium
- activates many enzymes
- forms a complex with ATP
H. Zinc and Molybdenum
- required in very small quantities
I. Sulfur
- ingested principally in the amino acids cysteine and methionine
- found in connective tissue (cartilage, skin)
- functions in metabolism
- excreted in the urine as sulfate
For Your Eyes Only
For Your Eyes Only
J. Deficiencies
- rare
- symptoms are not well defined
1. Calcium
- inadequate intake may cause
a. Rickets
- in children
b. Osteoporosis
- in adults
2. Copper
a. Menke’s Disease
- due to inability to absorb copper from the intestines
3. Iodine
a. Goiter
- inadequate dietary intake  thyroid enlargement
b. Hypothyroidism
- due to chronic iodine deficiency  insufficient thyroid hormone (T3, T4) synthesis
- may cause cretinism in children
4. Iron - inadequate intake  microcytic anemia
- role of iron as a component of hemoglobin
5. Zinc - deficiency  associated with poor wound healing
6. Phosphorus
- results in bone loss along with weakness, anorexia, malaise, and pain
OUTLINE
I. THE IMMUNE RESPONSE
II. INNATE IMMUNITY
A. Physical Barrier
B. Chemical Barrier
C. Genetic Barrier
D. Biologic Barrier
III. ACQUIRED/ ADAPTIVE IMMUNITY
A. Humoral Immunity
1. Antibodies
2. Classical Pathway
B. Cell-mediated Immunity
1. Types of T – cells
C. Immunization
D. Passive Immunity Figure 2. Immunity compose of Innate and Adaptive immunity
IV. HYPERSENSITIVITY
A. Type I: Immediate
B. Type II. Antibody-mediated
C. Type III: Immune complex-mediated
II. THE INNATE IMMUNITY
D. Type IV: Cell-mediated
V. REVIEW QUESTIONS
VI. REFERENCES
VII. APPENDIX ● Non-specific, always present and available
● Also called natural, or native immunity
● Refers to the mechanisms that are ready to react to
I. THE IMMUNE RESPONSE infections even before they occur, and that have evolved to
specifically recognize and combat microbes
● First line of defence
● White blood cells (WBC) ● It is mediated by cells and molecules that recognize
- Huge role in the immune system products of microbes and dead cells and induce rapid
- Produced in the bone marrow protective host reactions
- Derived from multipotent cells called ● Functions in stages:
hematopoietic stem cells o 1. recognition of microbes and damaged cells
o 2. activation of various mechanisms
o elimination of the unwanted substances
A. PHYSICAL BARRIER
● Resistance of invasion through the skin (epithelial cells)
● Trapping of microorganisms by mucus in the respiratory
and GI tracts.
● Expulsion of inhaled pathogens (coughing and sneezing
reflexes, flushing by tears and saliva).
B. CHEMICAL BARRIER
● something that the body produces in order to protect
itself
Figure 1. Multipotential hematopoietic stem cell ● Sebaceous gland in the skin- lactic acid and fatty acids
The diagram shows how one multipotential hematopoietic ● Vaginal secretions- acidity
stem cell can become either thrombocyte, erythrocytes, ● Gastric cells- hydrochloric acid (pH= 2-3) and gastric
mast cells, myeloblast that will differentiate into basophil, enzymes
neutrophil, eosinophil or macrophage. They can also ● Lactoferrin- sequester iron, making it unavailable for the
become Natural killer cell, a T-cell, a B cell or a plasma cell. nutrition of the microorganisms
One HSC can have only one fate but it can have different ● COMPLEMENT SYSTEM (Classical, Alternative, Tip-over,
potentials MBL)
IMMUNITY
● The ability to resist almost all types of organisms or
C. GENETIC BARRIER
toxins ● Some people are more vulnerable to contract certain
● that tend to damage the tissues and organs diseases such as type 1 diabetes, etc.,
● “How the body protects itself”
● Classic definition: Protection from infectious pathogens
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D. BIOLOGIC BARRIER such as defensins, and lymphocytes located in the
epithelia combat microbes at these sites. If microbes do
● Release of Interferons that interfere with viral propagation breach epithelial boundaries, other defence
inside the cell (not viral entry) mechanisms are called in.
● Fever, most pathogens are mesophilic (thrive at temp ● Monocytes and neutrophils are phagocytes in the blood
35-37C) so increasing the body temperature has that can rapidly be recruited to any site of infection;
detrimental effects on Phagocytosis. monocytes that enter the tissues and mature are called
macrophages. All tissues contain resident macrophages,
the professional phagocytes of the body. These cells not
only sense the presence of microbes and other
offending agents, but also ingest (phagocytose) these
invaders and destroy them.
● Dendritic cells are a specialised cell population present
in epithelia, lymphoid organs, and most tissues. They
capture protein antigens and display peptides for
recognition by T lymphocytes. In addition to their
antigen presenting function, dendritic cells are
endowed with a rich collection of receptors that sense
microbes and cell damage and stimulate the secretion
of cytokines, mediators that play critical roles in
inflammation and antiviral defence. Thus, dendritic cells
are involved in the initiation of innate immune
Figure 3: When the bacteria or foreign body comes in contact responses, but, unlike macrophages, they are not key
with the receptor of the macrophage, they get engulfed participants in the destruction of microbes and other
producing a vesicle called phagosome that encloses the foreign offending agents.
body and the lysosome combines with the phagosome to form ● Natural killer cells provide early protection against many
phagolysosome. Inside the phagolysosome, the pathogen gets viruses and intracellular bacteria.
broken down and gets extruded from the cell through exocytosis. ● Several other cell types can sense and react to
microbes. These include mast cells, which are capable
of producing many mediators of inflammation and even
epithelial and endothelial cells.
● It has recently been recognized that cells with the
appearance of lymphocytes but with features more like
the cells of innate immunity may contribute to the early
defence against microbes.
● In addition to these cells, several soluble proteins play
important roles in innate immunity. The proteins of the
complement system, which were described in Chapter
3, are plasma proteins that are activated by microbes
using the alternative and lectin pathways in innate
immune responses; in adaptive immunity it is activated
by antibodies using the classical pathway. Other
circulating proteins of innate immunity are
mannose-binding lectin and C-reactive protein, both of
which coat microbes and promote phagocytosis. Lung
surfactant is also a component of innate immunity,
Figure 4: Sometimes phagocytosis doesn't just break the foreign providing protection against inhaled microbes.
body. Sometimes instead of just exocytosis of the different
particles, these particles attach to MHC II receptors of the cell Reactions of Innate Immunity
which offers it to the T-helper cell. ● The innate immune system provides host defense by
two main reactions.
a. Inflammation - Cytokines and products of
Components of Innate Immunity
complement activation, as well as other mediators,
● Epithelia of the skin and gastrointestinal and
are produced during innate immune reactions and
respiratory tracts provide mechanical barriers to the
trigger the vascular and cellular components of
entry of microbes from the external environment.
inflammation. The recruited leukocytes destroy
Epithelial cells also produce antimicrobial molecules
microbes and ingest and eliminate damaged cells.
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b. Antiviral defense - Type I interferons produced in
response to viruses act on infected and uninfected
cells and activate enzymes that degrade viral
nucleic acids and inhibit viral replication, inducing
what has been called an antiviral state.
● In addition to these defensive functions, innate immunity
provides the danger signals that stimulate the subsequent
more powerful adaptive immune response.
III. ACQUIRED IMMUNITY
ACQUIRED IMMUNITY
● Also called adaptive, or specific immunity
● Consists of mechanisms that are stimulated by (“adapt to”) Figure 5. The principal classes of lymphocytes and their
microbes and are capable of recognizing microbial and functions. B and T lymphocytes are cells of adaptive immunity
antimicrobial substances and natural killer (NK) cells are cells of innate immunity.
● Develops later, after exposure to microbes and other Several more classes of lymphocytes have been identified,
foreign substances, and is even more powerful than innate including NK-T cells and so-called innate lymphoid cells (ILCs);
immunity in combating infections the functions of these cells are not established.
● Acquired - develops weeks or months after the body is first
attacked by bacterium, virus or toxin.
- Antigen - unique and specific proteins or large
polysaccharides found in a toxin or organism that
initiate acquired immunity
- Lymphocytes
⮚ Lymphocytes and other cells involved in immune
responses are not fixed in particular tissues (as are
cells in most of the organs of the body) but
constantly circulate among lymphoid and other
tissues via the blood and the lymphatic circulation.
This feature promotes immune surveillance by
allowing lymphocytes to home to any site of
infection.
Figure 6. The principal mechanisms of innate immunity and
⮚ Mature lymphocytes that have not encountered adaptive immunity. NK cells, Natural killer cells.
the antigen for which they are specific are said to
be naive (immunologically inexperienced).
⮚ After they are activated by recognition of antigens
and other signals, lymphocytes differentiate into:
a. Effector cells - which perform the function
of eliminating microbes
Memory cells - which live in a state of
heightened awareness and are able to react
rapidly and strongly to combat the microbe in
case it returns
Figure 7. Common lymphoid progenitor tree
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doesn’t necessarily kill the antigen but it makes it
on-harmful for the body.
● Some of the B-cells become IgG expressing B-cells so
they produce IgG, another type of antibody. The
antibody attaches to the macrophage or phagocyte, so
being given instruction from IgG will now hunt for the
microbe and ingest it to kill it.
● Some of the IgG can also attach to a natural killer cell
and this natural killer cell will directly kill the microbe.
The attachment of the IgG to a phagocyte and natural
killer cell is through a receptor called Fc receptor.
● After the B-cell has proliferated, it differentiates and
some of them becomes High-affinity IgG-expressing
B-cell which produces IgG again which can activate the
complement pathway. The significance of this
Figure 8: Formation of antibodies and sensitized lymphocytes by a complement pathway is in killing the microbe and
lymph node in response to antigens. This figure also shows the inducing inflammation.
origin of thymic (T) and bursal (B) lymphocytes that respectively ● Lastly, the B-cell will also produce a memory B-cell, like
are responsible for the cell-mediated and humoral immune a historian, so when the body comes in contact again
processes with the same microbe, it doesn’t have to undergo a
long process already because the body is now able to
directly produce IgG and IgM because it already
encountered the same microbe from before.
A. HUMORAL IMMUNITY (B cells)
● Easy way to remember: HUMAN
BODY=HUMORAL—B CELL
● Protection against extracellular microbes and their
toxins
● Mediated by B (bone marrow–derived) lymphocytes
and their secreted products, antibodies (also called
immunoglobulins, Ig)
Figure 10. This is the reason why after the first introduction
of the antigen to the body, the body produces not much of
antibodies as primary response but when the body
encounters the same antigen for another time, it provides a
Figure 9. Humoral Immunity bigger secondary response and a higher antibody
concentration because it already knows that kind of antigen.
● A microbe contains an antigen, an ID that is specific to
certain microbes. ● B lymphocytes are the only cells in the body capable of
● When the microbe gets in touch with Naïve B-cell, the B producing antibody molecules, the mediators of
cell will recognize and produce antibodies that are humoral immunity. B lymphocytes develop from
specific to the microbe. precursors in the bone marrow. Mature B cells
● From there, the B-cell produces more copies of itself constitute 10% to 20% of the circulating peripheral
through the help of the T-helper cells. Once it lymphocyte population and are also present in
proliferates, now it differentiates. peripheral lymphoid tissues such as lymph nodes,
● Some of them becomes antibody-secreting plasma cell spleen, and mucosa-associated lymphoid tissues. B cells
that produce antibody called IgM, once this antibody is recognize antigen via the B-cell antigen receptor
produced, it attaches to the antigen or to the active site complex.
of the microbe/toxins and neutralises it, meaning it
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● Membrane-bound antibodies of the IgM and IgD
isotypes, present on the surface of all mature, naive B
cells, are the antigen-binding component of the B-cell
receptor complex.
● After stimulation by antigen and other signals, B Tells
develop into plasma cells, veritable protein factones for
antibodies. Antibody-secreting cells are also detected in
human peripheral blood; these are called plasmablasts.
1. ANTIBODIES
Figure 12. Classification of of Antibodies
How do antibodies work?

1. Direct Action
2. Complement system
1. Direct Action
● Agglutination or clumping
● Precipitation: antigen-antibody complex becomes so
large, it becomes insoluble
Figure 11. Prototype Structure of an Antibody ● Neutralisation: antibodies cover the toxic sites of
antigenic agents. Not necessarily killing the agent but
This is the prototype structure of an antibody. neutralising it making it non-harmful for the body.
Constant portion ● Lysis: antibodies directly attack the cellular
- consisting of heavy chains. membranes and cause rupture of the offending agent
- These heavy chains are connected by disulfide bonds
into variable portions.
Variable portion
- variable means it can change. Sometimes it can have
two antigen binding sites, or ten antigen binding sites
depending on the type of antibody.
Antigen-binding sites
- where the antigen or the ID of the microbe attaches
Five general classes of antibodies:

● IgG: 70-75%, small, can cross placenta. IgG of a mother
can be transferred through her blood to her unborn
baby.
Figure 13. Once the B- cell produces antibodies, they attach
- most common form of antibodies to the antigen and form clumps. So their potential to spread
● IgM: MASSIVE (10 binding sites), primary infection out in the body is lessened because they are tied up.
- kind of antibody produced during the first infection
- IgG- antibody during first infection and subsequent 2. Complement System
infection
● “complement” = “enhance”
● IgE: least, allergies (mast cells and recruitment of
● “Complement” is a term used to call a collection of 20
eosinophils)
proteins that are mostly enzyme precursors (inactive)
● IgA: mucosal surfaces (inhaled, ingested pathogens)
that have to be activated before they can perform their
● IgD: unclear function
job.
● Although the principal actors are just 11 proteins
namely: C1, C2, C3, C4, C5, C6, C7, C8, C9, B and D
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Complement (inactive) → must be activated to elicit a stronger
immune response → Classical Pathway, Alternative Pathway,
Lectin Pathway
2. CLASSICAL PATHWAY
Figure 15. Humoral immunity. Naive B lymphocytes recognize

antigens, and under the influence of TH cells and other stimuli
(not shown), the B cells are activated to proliferate and to
differentiate into antibody-secreting plasma cells. Some of the
Figure 14. Classical Pathway activated B cells undergo heavy-chain class switching and affinity
maturation, and some become long-lived memory cells.
Classical pathway - activating the complement system will result Antibodies of different heavy-chain classes (isotypes) perform
in different outcomes. Ultimately it results in lysis of cells. During different effector functions, shown on the right. Note that the
the process, it produces different effects such as : antibodies shown are IgG; these and IgM activate complement;
and the specialised functions of IgA (mucosal immunity) and IgE
(mast cell and eosinophil activation) are not shown.
1) Engulfing of bacteria by neutrophils and
Opsonization macrophages. Make the bacteria more
B. CELL-MEDIATED IMMUNITY (T-CELLS)
of Bacteria appetising to phagocytes
● Responsible for defense against intracellular microbes
● Mediated by T (thymus-derived) lymphocytes
2) Activate Release of histamine, heparin, etc, to ● T cells need to be: “introduced” to antigens via Major
mast cause increased blood flow and leak of Histocompatibility Complex (MHC) that is attached to an
cells and fluid and plasma into tissues that Antigen Presenting Cell (APC)
basophils help inactivate the antigenic agent ● Examples of APC: macrophages, B cells, dendritic cells (or
accessory cells)
3) Chemotaxis Attraction of neutrophils and a. Dendritic cells (sometimes called interdigitating
of macrophages to the area. dendritic cells) are the most important antigen-
white blood
presenting cells for initiating T-cell responses against
cells
protein antigens.
b. Several features of dendritic cells account for their key
4) Lysis of cells Formation of the MEMBRANE ATTACK role in antigen presentation.
COMPLEX that inserts itself into the lipid - First, these cells are located at the right place to
bilayer of cell membranes of offending capture antigens—under epithelia, the common site of
organisms and cause them to rupture. entry of microbes and foreign antigens, and in the
interstitium of all tissues, where antigens may be
produced. Immature dendritic cells within the
epidermis are called Langerhans cells.
- Second, dendritic cells express many receptors for
capturing and responding to microbes (and other
antigens), including TLRs and lectins.
- Third, in response to microbes, dendritic cells are
recruited to the T-cell zones of lymphoid organs, where
they are ideally located to present antigens to T cells.
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● The class II β2 domain has a binding site for CD4, and
- Fourth, dendritic cells express high levels of MHC and therefore, the class II-peptide complex is recognized by
other molecules needed for presenting antigens to and CD4+ T cells, which function as helper cells. In this
activating T cells. interaction, the CD4 molecule acts as the coreceptor.
c. Macrophages that have phagocytosed microbes Because CD4+ T cells can recognize antigens only in the
and protein antigens process the antigens and context of self class II molecules, they are referred to as
present peptide fragments to T cells. Thus, class II MHC-restricted.
macrophages function as antigen-presenting cells ● In contrast to class I molecules, class II MHC molecules
in T-cell activation. are mainly expressed on cells that present ingested
d. Macrophages are key effector cells in certain forms antigens and respond to T-cell help (macrophages, B
of cell-mediated immunity, the reaction that serves lymphocytes, and dendritic cells).
to eliminate intracellular microbes. In this type of
response, T cells activate macrophages and MHC molecules play several key roles in regulating T cell-
enhance their ability to kill ingested microbes. mediated immune responses.
e. Macrophages also participate in the effector phase ● First, because different antigenic peptides bind to
of humoral immunity. It efficiently phagocytose different MHC molecules, it follows that an individual
and destroy microbes that are opsonized (coated) mounts an immune response against a protein antigen
by IgG or C3b. only if he or she inherits the genes for those MHC
● The function of MHC molecules is to display peptide molecules that can bind peptides derived from the
fragments of protein antigens for recognition by antigen and present it to T cells. The consequences of
antigen- specific T cells. inheriting a given MHC (e.g., class II) gene depend on
the nature of the antigen bound by the class II
MHC I: cytotoxic T-cell molecule.
● Expressed on all nucleated cells and platelets. They are ● Second, by segregating cytoplasmic and internalised
heterodimers consisting of a polymorphic α, or heavy, antigens, MHC molecules ensure that the correct
chain (44-kD) linked noncovalently to a smaller (12-kD) immune response is mounted against different
nonpolymorphic protein called β2-microglobulin. The α microbes—CTL-mediated killing of cells harbouring
chains are encoded by three genes, designated HLA-A, cytoplasmic microbes, and helper T cell- mediated
HLA-B, and HLA- C, that lie close to one another in the antibody and macrophage activation to combat
MHC locus. The extracellular region of the α chain is extracellular microbes.
divided into three domains: α1, α2, and α3. The α1 and
α2 domains form a cleft, or groove, where peptides
bind.
● Display peptides that are derived from proteins, such as
viral and tumor antigens, that are located in the
cytoplasm and usually produced in the cell, and class
I–associated peptides are recognized by CD8+ T
lymphocytes.
MHC II: T-helper cell

● Encoded in a region called HLA-D, which has three
subregions: HLA-DP, HLA-DQ, and HLA-DR. Each class II
molecule is a heterodimer consisting of a noncovalently
associated α chain and β chain, both of which are
polymorphic. The extracellular portions of the α and β
chains both have two domains designated α1 and α2,
and β1 and β2.
● Similar to class I molecules, they have peptide- binding
clefts facing outward. This cleft is formed by an
interaction of the α1 and β1 domains, and it is in this Figure 16. This diagram shows an antigen-presenting cell that
portion that most class II alleles differ. Thus, as with has MHC protein where it offers a part of the broken down part
class I molecules, polymorphism of class II molecules of the pathogen for the T-cell to recognize.
are associated with differential binding of antigenic
peptides.
● Present antigens that are internalised into vesicles, and
are typically derived from extracellular microbes and
soluble proteins.
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Figure 18. Helper T lymphocyte
2. Cytotoxic T-cells
- Directly kills microorganisms or even the body’s own
cells.
- Surface protein is CD8+
- Perforins: perforates protein secreted by cytotoxic
T-cells that punch holes in the membrane of the
attacked cell.
- It attacks cancer cells, transplanted cells
Figure 17. Cell-mediated immunity. Dendritic cells (DCs) capture
microbial antigens from epithelia and tissues and transport the
antigens to lymph nodes. During this process, the DCs mature,
and express high levels of MHC molecules and costimulators.
Naive T cells recognize MHCassociated peptide antigens displayed
on DCs. The T cells are activated to proliferate and to differentiate
into effector and memory cells, which migrate to sites of infection
and serve various functions in cell-mediated immunity. CD4+
effector T cells of the TH1 subset recognize the antigens of
microbes ingested by phagocytes, and activate the phagocytes to
kill the microbes; other subsets of effector cells enhance leukocyte
recruitment and stimulate different types of immune responses.
CD8+ cytotoxic T lymphocytes (CTLs) kill infected cells harboring
microbes in the cytoplasm. Some activated T cells remain in the
lymphoid organs and help B cells to produce antibodies, and
some T cells differentiate into long-lived memory cells (not
shown). APC, Antigen-presenting cell.
TYPES OF T-CELLS
Figure 19. Cytotoxic T cell recognizes the antigen, attaches
1. T-helper cells itself, produces perforins that destroys integrity of cell
- 75%, lymphokines for proliferation of other T-cells and membrane of the attacked cell causing lysis. This is also
B-cells, inactivated by HIV. the way it attacks the transplanted cell. E.g. in kidney
transplant, lung transplant.
3. Regulatory T-cells
- “suppressor cell” like a referee
- Growth and proliferation of cytotoxic and regulatory
- Suppresses the functions of cytotoxic T-cells and
T-cells
T-helper cells to prevent excessive immune reaction
- IL-4, IL-5, IL-6: B-cell growth factors
and to prevent immune tolerance (attacks to the
body’s own tissues)
- Surface protein is CD4+
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Belgium)
Oxford Uni- Viral vector

AstraZeneca (UK)
Sputnik V (Russia) Viral vector
Sinovac (China) Attenuated virus
Viral vector- the virus is carried by another chemical or other

organism, but both of them are actually attenuated and they
still carry the antigen that can be recognized by the immune
system.
Attenuated virus- live virus but weakened significantly
D. PASSIVE IMMUNITY
● Blood donations- you also donate the antibodies and

Figure 20. An antigen is eaten by antigen presenting cells through activated T cells. This lasts in the recipient's blood for
MHC, then the antigen is offered to the T helper cells and T helper 2-3 weeks. If the recipient doesn’t have the active
cells produce lymphokines that stimulate B-cells to produce immunity, they lose the protection provided by the
different types of antibodies. Also, the lymphokine triggers the antibody and activated T cells that are found in the
differentiation of the cytotoxic T cells that can kill the bacteria at donor.
the same time suppressor T cell that regulate the activity of the T a. Infusion of antibodies, activated T-cells or both through
helper cells and the cytotoxic T cells. blood (lasts 2-3 weeks.
b. Mother to child through placenta (IgG)
C. IMMUNIZATION
● Passive introduction of antigen or pathogens to the body, it

is also by way of the active immunity that we get protected
IV. HYPERSENSITIVITY
● Injection of live organisms that have been attenuated/
(Over-reacting response)
weakened
- Example: smallpox, yellow fever, poliomyelitis, measles
● Implies an excessive or harmful reaction to antigen.
dead virus or virus that have been weakened
There are several important general features of
● Injection of dead organisms that are no longer capable of
hypersensitivity disorders:
causing disease but still have some chemical antigens
● Hypersensitivity reactions can be elicited by
- Example: typhoid, pertussis, diphtheria
exogenous environmental antigens (microbial and
● Injection of toxins/ proteins that have been treated with
non-microbial) or endogenous self-antigens.
chemicals so that their toxic nature has been destroyed but
● Hypersensitivity usually results from an imbalance
their antigens are still intact.
between the effector mechanisms of immune
- Example: tetanus, botulism
responses and the control mechanisms that serve
● Injection of mRNA or a viral vector of mRNA to give
to normally limit such responses.
instructions for our body to make antigens:
● The development of hypersensitivity diseases (both
- Example: COVID-19
allergic and autoimmune) is often associated with
the inheritance of particular susceptibility genes.
Vaccine Mechanism ● The mechanisms of tissue injury in hypersensitivity
Manufacturer reactions are the same as the effector mechanisms
of defense against infectious pathogens.
Pfizer- BioNTech mRNA
(US-Germany)
Moderna (US) mRNA
Johnson & Johnson- Viral vector

Janssen (US-
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ALLERGIES
● “Immune hypersensitivity”
● Dilation of blood vessels → redness
● Attraction of eosinophils & neutrophils to reactivate site
→ rash & itching
● Increased permeability of the capillaries with loss of
fluid into the tissues → secretions (lacrimation, runny
nose, diarrhea)
● Contraction of smooth muscles cells → difficulty of
breathing, diarrhea
Infection Allergic Reaction

Figure 21. If a person is allergic to pollen, and the pollen is
Antigen Allergen inhaled by the nose through your mucosal lining, it gets
recognized by B cell specifically, by T-helper2 B cell. T-helper
Antibody Reagin or sensitizing antibodies cell2 stimulates B cell, which also recognizes the pollen so that
the B cell becomes the plasma cell and produces Ig E antibody.
IgG, IgM: main antibodies IgE gets attached to the mast cell through the FceRI receptor
that are concerned in IgE then the mast cell releases vasoactive amines, lipid mediators
infection during the first few hours of exposure. Later on, around 4 hours
after the exposure it releases cytokines.
Neutrophil, macrophage Basophil, mast cells During the first few hours of exposure to these amines, lipid
mediators induce hypersensitive reactions that we commonly
Histamine, protease, eosinophil seen in our patients. The cytokines are the late phase reaction
chemotactic substance, or the different symptoms we observe several hours after initial
Lymphokines neutrophil chemotactic exposure to allergen. Later on, during repeat exposure to
substance, heparin, allergen (meaning there is another time the pollen come in
platelet-activating factor contact with the mucosal lining) since the body already
recognizes the allergen (pollen), it can directly bypass the
different steps from the B cell and plasma cell and it can
directly attach to the IgE (already attached to the mast cell).
Figure 22. Allergy symptoms

● Rash
● Lacrimation
● Runny nose
● Sneezing
● Itching
● Red eyes
○ Dilation of blood vessels 🡪 redness because dilation of
the blood vessels is part of the immune reaction
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smooth muscle spasm or glandular secretions. Evident
○ Attraction of eosinophils and neutrophils to reactive site within minutes after exposure to an antigen and
🡪 rash, itching because of the chemo reactive substances subside in a few hours.
released by the mast cells o Late-phase reaction - sets in 2 to 24 hours later
○ Increased permeability of the capillaries with loss of fluid without additional exposure to antigen and may last
into the tissues 🡪 secretions (lacrimation, runny nose, for several days. It is characterised by infiltration of
diarrhea). The allergen has contact with the mucosal tissues with eosinophils, neutrophils, basophils,
monocytes, and CD4+ T cells, as well as tissue
surface of the GI tract and produces the cascade of the
destruction, typically in the form of mucosal epithelial
allergic reaction. cell damage.
Contraction of smooth muscle cells 🡪 difficulty of breathing,
diarrhea
Clinical Syndrome Clinical and Pathological

A. TYPE I (IMMEDIATE)
Manifestations
Anaphylaxis(may be Fall in blood pressure (shock) cause by

caused by drugs, bee vascular dilation; airway obstruction
sting, food) due to laryngeal edema
Bronchial asthma Airway obstruction caused by

bronchial smooth muscle
hyperactivity; inflammation and tissue
injury caused by late-phase reaction
Allergic rhinitis, Increased mucus secretion;

sinusitis (hay fever) inflammation of upper airways,
sinusitis
Food allergies Increased peristalsis due to

contraction of intestinal muscles
Figure 23. Examples of Type 1 Hypersensitivity B. TYPE II (ANTIBODY-MEDIATED)

● Injury is caused by TH2 cells, IgE antibodies, and mast cells ● Secreted IgG and IgM antibodies injure cells by
and other leukocytes promoting their phagocytosis or lysis and injure tissues
o Mast cells release mediators that act on vessels and by inducing inflammation.
smooth muscle and proinflammatory cytokines that ○ Antibodies may also interfere with cellular
recruit inflammatory cells. functions and cause disease without tissue
● Rapid immunologic reaction occurring in a previously injury.
sensitized individual that is triggered by the binding of an ● IgG, IgM → binds to antigens → phagocytosis or lysis
antigen to IgE antibody on the surface of mast cells. of target cell by activated complement; recruitment of
● These reactions are often called allergy, and the antigens leukocytes
that elicit them are allergens. ● Antigens may be intrinsic to the host’s normal cell or
● Localized could be adsorbed from drug metabolites. Basically, it’s
o Urticaria/ Hives like the normal cascade of events during infection but
o Skin allergy, hives there’s involvement of the host’s normal cell.
o Allergic rhinitis and conjunctivitis
o Hay fever
o Bronchial asthma
o Allergic gastroenteritis (food allergy)
● Widespread
o Angiodema
o Anaphylaxis: severe, deadly because of widespread
vasodilation and loss of intravascular fluid and Figure 24. The opsonized cell has different antigens that present
contraction of bronchioles causing suffocation itself to the Fc receptor of the phagocytes. Because of this, the
● Many local type I hypersensitivity reactions have two whole cell gets eaten by the phagosome. This is seen in
well-defined phases: autoimmune hemolytic anemia, Immune Thrombocytopenic
o Immediate reaction - characterized by vasodilation, Purpura.
vascular leakage, and depending on the location,
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myocardial
Disease Target Antigen Mechanism Manifestati antigen
on
Myasthen Acetylcholine Antibody Muscle
Immune RBC Opsonisation & Hemolysis ia gravis receptor inhibits weakness,
Hemolytic membrane phagocytosis of Anemia acetylcholin paralysis
Anemia protein (Rh RBCs e binding,
antigen, I down
antigen) modulates
receptors
Immune Platelet Opsonisation & Bleeding
Thromboc membrane phagocytosis of Graves TSH receptor Antibody-m Hyperthyroidis
ytopenic proteins (GP platelet disease ediated m
Purpura IIb/ IIIa (hyperthy stimulation
integrin) roidism) of TSH
receptors
RBC and platelets in these situations contain different antigen. In
effect our body will eat your own blood cells and platelets Insulin-res Insulin Antibody Hyperglycemia
because they have these different antigens or allergens istant receptor Inhibits , ketoacidosis
recognized by your phagocytes. Opsonization → phagocytosis → diabetes bonding of
insulin
Hemolysis → anemia → thrombocytopenic purpura → Bleeding
(because there is a lack of platelets). Pernicious Intrinsic factor Neutralizati Abnormal
anemia of gastric on of myelopoiesis,
parietal cells intrinsic anemia
factor,
decreased
absorption
of vitamin
B12
Figure 25. Complement mediated inflammation: the one being
attacked are the normal cells.
Disease Target Antigen Mechanism Manifestation
Pemphigu RBC Opsonisatio Skin vesicles

s vulgaris membrane n& (bullae)
Figure 26. Antibody-mediated cellular dysfunction
protein (Rh phagocytosi
antigen, I s of RBCs
C. TYPE III HYPERSENSITIVITY (IMMUNE
antigen)
COMPLEX-MEDIATED)
Vasculitis Platelet Opsonisatio Vasculitis
caused by membrane n& ● IgG and IgM antibodies bind antigens usually in the
ANCA proteins (GP phagocytosi circulation, and the antigen-antibody complexes deposit
IIb/IIIa integrin s of RBCs in tissues and induce inflammation.
o The leukocytes that are recruited (neutrophils
Goodpast Noncollagenou Complemen Nephritis Lung and monocytes) produce tissue damage by
ure s protein (NC-I) t and Fc haemorrhage
release of lysosomal enzymes and generation
syndrome in basement receptor-me
membranes of diated of toxic free radicals.
kidney inflammatio ● Deposition of antigen-antibody complexes (not soluble)
glomeruli and n → complement activation → recruitment of leukocytes
lung alveoli → release of enzymes and other toxic molecules →
vasculitis, inflammation. Same mechanism as with
Acute Streptococcal Inflammatio Myocarditis infection (neutralization of antigens), but the problem
rheumatic cell wall n,
is, they get deposited in the endothelial cells of the
fever antigen; macrophage
antibody activation blood vessels causing inflammation of blood vessels
cross-reacts (vasculitis).
with ● Kidneys, joints, small blood vessels ← high filter areas
o Blood gets filtered under high pressure in
these areas. Because the immune complexes
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are not soluble, they get stuck in the filter in appear. Wherever complexes deposit the tissue damage
these areas. is similar. The mechanisms of inflammation and injury
were discussed above, in the discussion of
antibody-mediated injury. The resultant inflammatory
lesion is termed vasculitis if it occurs in blood vessels,
glomerulonephritis if it occurs in renal glomeruli,
arthritis if it occurs in the joints, and so on.
Disease Target Antigen Manifestation
Systemic Lupus Nuclear Antigen Nephritis, skin

Erythematosus lesions, arthritis,
others
Post-streptococc Streptococcal cell Nephritis

al wall antigens; may
glomerulonephr be “planted” in
itis glomerular
basement
membrane
Polyarteritis HBV antigens in Systemic vasculitis

nodosa some cases
Reactive Bacterial antigens Acute arthritis

arthritis (ex. Yersinia)
Serum sickness Various proteins Arthritis, vasculitis,

nephritis
Figure 27. Immune complex disease. The sequential phases in the Arthrus reaction Various proteins Cutaneous vasculitis
induction of systemic immune complex–mediated diseases (type
III hypersensitivity).
D. TYPE IV HYPERSENSITIVITY (T-CELL MEDIATED)

The pathogenesis of systemic immune complex disease can be ● Sensitized T lymphocytes (TH1 and TH17 cells and CTLs)
divided into three phases: are the cause of the tissue injury.
(1) Formation of immune complexes. The introduction of a o TH2 cells induce lesions that are part of
protein antigen triggers an immune response that immediate hypersensitivity reactions and are
results in the formation of antibodies, typically about a not considered a form of type IV
week after the injection of the protein. These hypersensitivity.
antibodies are secreted into the blood, where they ● Caused by inflammation resulting from cytokines
react with the antigen still present in the circulation and produced by CD4+ T cells and cell killing by CD8+ T cells.
form antigen-antibody complexes. ● CD4+ T cell–mediated hypersensitivity induced by
(2) Deposition of immune complexes. The circulating environmental and self-antigens is the cause of many
antigen-antibody complexes are deposited in various chronic inflammatory diseases, including autoimmune
tissues. In general, complexes that are of medium size, diseases.
formed in slight antigen excess, are the most ● CD8+ cells may also be involved in some of these
pathogenic. Organs where blood is filtered at high autoimmune diseases and may be the dominant
pressure to form other fluids, like urine and synovial effector cells in certain reactions, especially those that
fluid, are sites where immune complexes become follow viral infections.
concentrated and tend to deposit; hence, immune
complex disease often affects glomeruli and joints. CD4+ T Cell–Mediated Inflammation
(3) Inflammation and tissue injury. Once immune ● CD4+ → produces cytokines
complexes are deposited in the tissues, they initiate an ● The T cell attack our own cell in the body through the CD4
acute inflammatory reaction. During this phase that produce cytokines that induce inflammation and the
(approximately 10 days after antigen administration), inflammation itself cause tissue injury or they directly kill
clinical features such as fever, urticaria, joint pains
(arthralgias), lymph node enlargement, and proteinuria
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different cells of the body who presents the allergen or
antigen
● Cytokines produced by the T cells induce inflammation that

may be chronic and destructive.
● The prototype of T cell–mediated inflammation is
delayed-Type hypersensitivity (DTH), a tissue reaction to
antigens given to immune individuals. Figure 29. T cell- mediated cytolysis
● In this reaction, an antigen administered into the skin of a
previously immunized individual results in a detectable
cutaneous reaction within 24 to 48 hours (hence the term
V. REVIEW QUESTIONS
delayed, in contrast to immediate hypersensitivity).
1. What is the common progenitor of plasma cells?
● Both TH1 and TH17 cells contribute to organ-specific
a. lymphoid
diseases in which inflammation is a prominent aspect of
b. myeloid
the pathology. The inflammatory reaction associated with
c. hematopoietic
TH1 cells is dominated by activated macrophages, and that
d. osteoblastic
triggered by TH17 cells has a greater neutrophil
2. In innate immunity, what are these chemical barriers that
component.
make iron unavailable to microorganisms?
a. lactobacillus
b. lactoferrin
c. porferins
d. complement system
3. A highly acidic substance in the stomach that acts as a
chemical barrier in microorganisms in the food eaten and
aids for breaking down protein?
a. pepsin
b. amylase
c. HCl
Figure 28. Cytokine-mediated inflammation d. acetic acid
4. What do you call these unique and specific proteins that
act as identifiers of different organisms?
a. antigen
CD8+ T Cell–Mediated Cytotoxicity b. vaccine
● CD8+ → direct cell-cytotoxicity c. antibody
● In this type of T cell–mediated reaction, CD8+ CTLs kill d. B-cells
antigen-expressing target cells. 5. T-cells mature at?
● Tissue destruction by CTLs may be an important a. thymus gland
component of some T cell–mediated diseases, such as b. thyroid gland
c. parathyroid gland
type 1 diabetes.
d. bone marrow
● CTLs directed against cell surface histocompatibility
6. B-cells mature at?
antigens play an important role in graft rejection.
a. bone cells
● They also play a role in reactions against viruses. In a b. bone marrow
virus-infected cell, viral peptides are displayed by class I c. liver
MHC molecules and the complex is recognized by the d. thyroid gland
TCR of CD8+ T lymphocytes. 7. What kind of lymphocytes produce the antibodies?
● The killing of infected cells leads to the elimination of a. B-cells
the infection, but in some cases it is responsible for cell b. T-cells
damage that accompanies the infection (e.g., in viral c. mast cells
hepatitis). d. pathogens
● Tumor-associated antigens are also presented on the 8. Antibodies that serve as a marker for new onset
cell surface, and CTLs are involved in the host response infections?
to transformed cells. a. IgG
b. IgE
c. IgM
d. IgA
9. Antibodies that can cross the blood-placental barriers?

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a. IgG 20. A life-threatening manifestation of type 1 hypersensitivity
b. IgE reaction?
c. IgM a. anaphylaxis
d. IgA b. Coma
10. Which antibodies can be found in the mucosal surfaces? c. brain dead
a. IgG d. stroke
b. IgE 21. A drug administered to anaphylactic patients?
c. IgM a. water
d. IgA b. epinephrine
11. Antibodies that are produced in allergic reactions? c. dopamine
a. IgG d. serotonin
b. IgE 22. What type of hypersensitivity reaction wherein your immune
c. IgM cells phagocytized your own cells?
d. IgA a. type 1 hypersensitivity
12. What do you call the process of agglutination wherein b. type 2 hypersensitivity
formation of clumps occurs and it becomes insoluble in the c. type 3 hypersensitivity
blood? d. type 4 hypersensitivity
a. precipitation 23. What are the antigen presenting cells?
b. condensation a. neutrophils, eosinophils, basophils
c. opsonization b. RBC, WBC, platelets
d. neutralization c. dendritic cells, macrophages, B-cells
13. The process of making pathogens appetizing to neutrophils d. tumor cells, necrotic cells, toxic cells
and macrophages? 24. WBC, RBC and platelet precursors are derived from
a. precipitation multipotent cells called:
b. condensation a. Progenitor cells
c. opsonization b. Hematopoietic stem cell
d. neutralization c. Precursor cells
14. A protein used to present pathogens to a T-Helper cell? d. Stem cells
a. MHC I 25. TRUE or FALSE: On primary exposure to antigen, the body’s
b. MHC III response is slow and not many antibodies are produced, while on
c. MHC IV secondary exposure, a faster secondary response occurs and a
d. MHC II higher antibody concentration is produced.
15. A protein that is used by Killer T-cells in making holes in
pathogens’ cell membrane and causes lysis?
a. lactoferrin
b. perforins
1A.2B.3C.4A.5A.6B.7A.8C.9A.10D.11B.12A.13C.14D.15B.16A.17B.18C.19A.20
c. amylase A.21B.22B.23C.24A.25TRUE
d. pepsin
16. Lymphocytes responsible for organ rejection in organ
VI. REFERENCES
transplantation?
a. cytotoxic T-cells/ killer T-cells Andal, V. (2021). The Immune Response [PPT] and discussion
b. Tumor Necrosis factor
Lippincott Illustrated Biochemistry 6th ed Guyton and Hall
c. interleukin II
Textbook of Medical Physiology 14th ed
d. Helper T-cells
Robins Basic Pathology 9th ed.
17. What is the surface protein of the regulatory T-cells?
a. CD8+
b. CD4+
c. CD9+
d. CD6+
18. What type of hypersensitivity is lupus?
a. type 1 hypersensitivity
b. type 2 hypersensitivity
c. type 3 hypersensitivity
d. type 4 hypersensitivity
19. What type of hypersensitivity is a common allergy?
a. type 1 hypersensitivity
b. type 2 hypersensitivity
c. type 3 hypersensitivity
d. type 4 hypersensitivity
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VII. APPENDIX
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16
For Your Eyes Only
MOLECULAR BIOLOGY:
DNA STRUCTURE and FUNCTION
OVERVIEW
A. Nucleic Acids
1. Two Types
a. DNA
b. RNA
2. DNA and RNA
- store genetic information and make it available to the cell
a. Properties
i. Genetic information must be stored in a form that is stable and manageable in size
ii. Genetic information must be decoded by transcription and translation
iii. The information in DNA or RNA must be accessible to proteins and other nucleic
acids
iv. Replication, in the case of DNA, must be template-driven so that each daughter cell
receives the same genetic information
B. DNA
1. Present In
- chromosomes in the nucleus of eukaryotic organisms
- mitochondria
- chloroplasts in plants
2. Prokaryotic Cells
- lack nuclei
- have single chromosome
- may contain nonchromosomal DNA in the form of plasmids
3. Fertilized Eggs
- DNA encodes the information that directs the development of an organism
C. Functions of DNA
- the genetic information found in DNA is copied and transmitted to daughter cells through DNA
replication
For Your Eyes Only
D. Central Dogma of Molecular Biology
DNA STRUCTURE
A. DNA
- associated with various types of proteins (nucleoproteins) in eukaryotic cells
- present in the nucleus
1. Polydeoxyribonucleotide
- containing many monodeoxyribonucleotides linked by 3’, 5’-phosphodiester bonds
2. Double Stranded Molecules
- 2 strands wind around each other  double helix
- except of a few viruses containing single-stranded DNA
3. Protein-DNA Complex
- present in the nucleoids in prokaryotes
For Your Eyes Only
PRIMARY POLYMERIC STRUCTURE
A. Linkage of Nucleotides
1. 3’,5’-Phosphodiester Bonds
a. Sugar-Phosphate Linkages
- link nucleotides through a phosphate molecule between
- 3’-hydroxyl on the deoxypentose of a nucleotide
- 5’-hydroxyl on the deoxypentose of another nucleotide
- forms a symmetrical backbone
- 5’ end of 1 sugar always linked through a phosphate molecule to the 3’ end the
adjacent sugar
b. Polarity With Unattached Ends
- 5’-end
- 3’-end
c. Hydrolytic Cleavage By
i. Deoxyribonucleases
- for DNAs
ii. Ribonucleases
- for RNAs
iii. Endonucleases
- nucleases that cleave the nucleotide in the interior of the chain
iv. Exonucleases
- cleave the chain by removing individual nucleotides from 1 of the 2 ends
2. Bases - variable
- stick out from the backbone
- order of bases determines the coding or structural capacity of the nucleic acid
B. Notation
- DNA sequences are written in the 5’ to 3’ direction
For Your Eyes Only
DNA SECONDARY STRUCTURE
A. Double Helical (B Form) DNA
- right-handed helix
- 10 residues / 360o turn
- planes of the bases perpendicular to the axis of the helix
- in chromosomal DNA
- most common type of DNA helix
1. Two Antiparallel Strands Form a Right-handed Helix
a. Chains
- coiled around a common axis
- paired in an antiparallel manner
- 5’-end of 1 strand pairs with the 3’-end of the other
b. Spatial Relationship of the 2 Strands
- create grooves
- major (wide)
- minor (narrow)
i. Actinomycin D (Dactinomycin)
- anticancer drug
- cytotoxic effect by intercalating to the narrow groove  interfere with RNA
and DNA synthesis
For Your Eyes Only
2. Complementary Base Pairing
- bases of 1 strand pairs with base of the other strand
- 1 polynucleotide chain is a complement of the other strand
a. Stabilized By
i. Hydrogen Bonds
- 2 for A-T
- 3 for G-T
ii. Hydrophobic Interactions
- which tend to cause free base pairs to aggregate
iii. van der Waals Interactions (Stacking Interactions)
- between base pairs
 stabilize the double helix
For Your Eyes Only
b. Chargaff’s Rules
- resulted from the specific base pairing in DNA
- amount of adenine = amount of thymine
- amount of guanine = amount of cytosine
- total amount of purines = total amount of pyrimidines
3. Base Stacking
- hydrophilic deoxyribose-phosphate backbone is on the outside
- hydrophobic bases stacked in the inside of the molecule perpendicular to the axis of the helix
a. Thermodynamic Stability of the Double Helix Due To
- hydrogen bonding of base pairs
- van der Waals interactions of the stacked base pairs
4. Spiral Staircase
- each base pair is offset approximately 36 degrees from its neighboring base pairs  helix
appears like a spiral staircase
- there are 10 steps or base pairs for each complete turn of the helix
For Your Eyes Only
5. Dimensions
- 20 Ao (angstroms) wide
- 3.4 Ao between each stacked base pair  turn of helix (10 base pairs) is 34 Ao long
a. Grooves
- apparent from the outside of the helix
i. Major (Wide) Groove
- 22 Ao wide
ii. Minor (Narrow) Groove
- 12 Ao wide
- areas where many drugs and proteins make contact with the bases without opening of
the helix
- provide access for the binding of regulatory proteins to their specific recognition
sequences along the DNA chain
B. Alternate DNA Structural Forms

- in the aqueous cell environment
- DNA structure is dynamic  bend and adopt alternate structures
1. A Form
- wider and flatter
- produced by moderately dehydrating the B form
- more compact than B form
- right-handed helix
- 11 base pairs / turn
- turn occurring every 26 Ao
- planes of the base pairs are tilted 20o away from the perpendicular to the helical axis
- conformation found in
- DNA-RNA hybrids
- RNA-RNA double stranded regions
For Your Eyes Only
a. Major Groove
- deep
- narrow
b. Minor Groove
- shallow
- wide
2. Z Form
- left-handed
- 12 base pairs / turn
- turn occurring every 57 Ao
- deoxyribose-phosphate backbone “zigzags”  “Z” DNA
- can occur naturally in DNA regions having sequence of alternating purines and pyrimidines
- ex: poly GC
- if cytosine residues in a stretch of alternating guanines and cytosines are methylated  Z
form DNA can exist under physiologic conditions
- more elongated than B form
- forms in vitro in nucleic acids containing alternating purines and pyrimidines
a. Minor Groove
- deep
b. Major Groove
- not discernible
C. DNA Modification
- most common modification in eukaryotic DNA is the methylation of cytosines that precede guanosines
1. Frequency of Methylation
- modification occurs in less than 10% of all cytosines
- methylated cytosines vary among different tissue types
2. Function of Methylation
a. Methylcytosines in Genes
- strongly correlate with transcriptionally inactive genes
- in sequences of alternating C-G doublets favors the transition of DNA from B to Z
form
For Your Eyes Only
D. DNA Size
1. Different Organisms Contain Different Amounts of DNA
- the more DNA an organism contains, the greater its genetic potential
a. Yeast DNA
- largest chromosome contains a single DNA with 2,200,000 base pairs
b. Human DNA
- largest chromosome contains a single DNA with 240,000,000 base pairs
E. Separation of the 2 DNA Stands in the Double Helix
- when hydrogen bonds are disrupted
a. Occur In
- altered pH (treatment with alkali) of the DNA solution  ionization of the bases
- heated (increase in temperature) DNA solution
- no broken phosphodiester bond
b. Capability to Dissociate and Reassociate
- essential to the processes of
- replication
- transcription
1. Denaturation
- occurs when all the hydrogen bonds holding the 2 strands are broken  separation of the 2
strands  loss of DNA helical structure
a. Melting
- if the separation is due to increased temperature
For Your Eyes Only
b. Melting Temperature (Tm)

- temperature at which half of the helical structure is unwound or denatured
i. Factors Affecting the Tm
ia. Solvent
ib. Kind and Concentration of Ions in Solution
ic. pH
 heat, alkaline pH, chemicals (formamide, urea) are commonly used to
denature DNA
id. Mole Fraction of G-C Base Pairs

- A-T base pairs form 2 hydrogen bonds  DNA containing high
concentration of A-T  lower Tm
- G-C base pairs form 3 hydrogen bonds  DNA containing high
concentration of G-C  higher Tm
For Your Eyes Only
2. Renaturation (Reannealing)
- complementary strands can reform the double helix
CIRCULAR DNA MOLECULES

A. Eukaryotes
1. Chromosome in the Nucleus
- contains one long linear molecule of dsDNA (bound to a complex mixture of proteins to form
chromatin)
2. Closed Circular DNA
- in the mitochondria
B. Prokaryotes
- contains a single, double-stranded, supercoiled, circular chromosome (associated with histone-like
proteins and RNA that can condense the DNA to form a nucleoid)
For Your Eyes Only
1. Plasmids
- small, circular, extrachromosomal DNA
- carry genetic information
- undergoes replication that may or may not be synchronized to chromosomal division
- may carry genes that convey antibiotic resistance to the host bacterium
- may facilitate transfer of genetic information between bacteria
HIGHER ORDER (TERTIARY) DNA STRUCTURE

A. Supercoils
- circular DNA can be twisted into a compact supercoiled or superhelical form
- have higher free energy than nonsupercoiled (relaxed) DNA
1. Forms
a. Right-Handed (Positive)
- twisted in the same direction as of the right-handed helix of B-form
b. Left-Handed (Negative)
- twisted in the opposite direction
2. Negative Supercoils
- can - cause localized unwinding of the double helix
- exist as compact supercoil
- to adapt to torsional stress of supercoiling
3. Can Exist Naturally
- bacterial genomic DNA
- plasmid DNA
4. Purpose
- eukaryotic DNA  supercoiling (most are negative)  packaged into higher order chromatin
structures
For Your Eyes Only
B. Chromatin
a. During Mitosis
- DNA packaged in chromosomes
b. Chromosomes
- visible under light microscope
- 10,000 fold shortening of DNA from primary B-form
c. During Interphase
- DNA needs to be accessible to transcription and replication enzymes  packaged in
less dense structures (chromatin)
1. Histones
- small basic proteins
- bind to acidic DNA by noncovalent interactions  nucleosomes
- high lysine and arginine content  positively charged at physiologic pH  form
ionic bonds with the negatively charged DNA
- 11,000 - 21,000 MW
- major class of proteins associated with chromatin
- exist in mass equal to that of DNA in chromatin
- along with positively charged ions (Mg++)  help to neutralize large negative charge of the
DNA phosphate groups
- all are post-transcriptionally modified at various stages of the cell cycle
- phosphorylation
- methylation
- acetylation
a. 5 Classes
i. 4 Core Histones
- 2 copies
- H2A
- H2B
- H3
- H4
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ii. 1 Linker Histone
- H1
2. Nucleosomes
- resemble “string of beads”
- 7-fold shortening of linear B-form DNA
- basic packaging unit of chromatin
- occur once every 200 base pairs on the average
For Your Eyes Only
a. 10 nm Fiber
- repeating beads-on-a-string structure
b. Core Particles
- fundamental structural feature of nucleosomes
- formed by 2 molecules each of
- H2A
- H2B
- H3
- H4
- 4 core histones are subject to covalent modifications at the N-terminal ends

- reversible
- influence how tightly the histones bind to the DNA  affecting expression of
specific genes
i. Acetylation
- of histones H3 and H4
- associated with the activation or inactivation of gene
transcription
- of core histones
- associated with chromosomal assembly during DNA
replication
ii. Methylation
iii. ADP-Ribosylation (of Histones)
- associated with DNA repair
iv. Phosphorylation (of Histone H1)
- associated with the condensation of chromosomes during the
replication cycle
v. Covalent Linkage to Ubiquitin (H2A only)
- around the core is a DNA double helix segment wound nearly 2X  negatively
supertwisted helix
c. Linker DNA
- join neighboring nucleosomes
- averages 50 base pairs in length
d. Histone H1
- bind to linker DNA chain between the nucleosome beads
- most tissue-specific and species-specific of the histones
- protect much of the linker DNA from digestion by nucleases
- not found in the nucleosome core
- aid in nucleosome packing  more compact structures
For Your Eyes Only
e. Fate of Nucleosomes During DNA Replication

- highly structured and constrained chromatin must be relaxed  replication
- nucleosomes are displaced
- incomplete dissociation of nucleosome core from the DNA
- parental histones remain loosely associated with only 1 of the parental DNA strand
- new histone synthesis occurs simultaneously with DNA replication
- nucleosomes containing only newly synthesized histones associate with only 1 of the
new daughter helices
- parental histone octamers are conserved
3. Higher Levels of Organization
a. Polynucleosome (Nucleofilament)
- formed by nucleosomes joined by linker DNA (50 nucleotides in length)
- assumes a shape of a coil (30 nm fiber)
b. 30 nm Fiber
- organized into loops
- anchored by nuclear scaffold
- 40-fold shortening of B-form DNA
For Your Eyes Only
4. Higher Order Chromatin Structure

- forms of packaged DNA in most cells
- cells in interphase contain two types of chromatin - euchromatin, heterochromatin
a. Heterochromatin
- darkly stained portion of chromatin
- represent densely packaged DNA
- transcriptionally inactive
- do not appear to be unpackaged from their mitotic chromosome form
- found in mitosis as well as interphase
i. Constitutive Heterochromatin
- always condensed  inactive
- found - near the chromosomal centromeres
- at the chromosomal ends (telomeres)
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ii. Facultative Heterochromatin
- at times condensed  inactive
- at times uncondensed (appearing as euchromatin)  actively
transcribed
b. Euchromatin
- stain poorly
- not tightly packaged
- transcriptionally active
- generally corresponds to the nucleosomes (10-nm fibers) loosely associated
with each other (looped 30-nm fibers)
5. Nonhistone Chromatin Proteins
a. High Mobility Group (HMG) Proteins
- abundant
- fast-mobility upon electrophoresis
- required for cell viability
b. Scaffold Proteins
i. Topoisomerase II
- enzyme
- needed to relieve torsional stress in loops in DNA projecting from the
scaffold or nuclear matrix upon replication or during mitosis
ii. Other Scaffold Protein
- unknown function
For Your Eyes Only
For Your Eyes Only
ORGANIZATION of EUKARYOTIC GENOMIC DNA

1. Typical Human Cell
- 33,000,000,000 base pairs of DNA per haploid genome
- < 10% codes for a product
- 46 chromosomes
- total DNA approximately 1.74 meters in length
2. Eukaryotic DNA
- associated with tightly bound basic proteins (histones)
3. Histones
- serve to order the DNA into basic structural units (nucleosomes)
4. Nucleosomes
- resemble beads on a string
- arranged into more complex structures  organize and condense the long DNA molecule into
chromosomes that can be segregated during cell division
A. Three Major Classes of DNA
1. Nonrepetitive Sequences
- 60% of the DNA of eukaryotes consists of sequences that are found in only 1 or few copies in
the genome
- most protein-coding sequences
- genes that code for proteins are interspersed with middle repetitive sequences which are not
transcribed
a. Division of Genes for Most Proteins
- exons - expressed regions
- introns
- intervening regions that split up the exons
- transcribed with the exons but removed by splicing
2. Middle Repetitive Sequences
- sequences repeated anywhere from 10 to hundreds of times
- 30% of the DNA
- for DNA fingerprinting
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3. Highly Repetitive Sequences
- repeated from hundreds to hundreds of thousands of times
- 10% of DNA
B. Further Repetitive DNA Classification
1. Tandemly Repeated Genes
- rRNA genes
- tRNA genes
- histone genes
- tandem repeats of hundreds to thousands of copies separated by some non-coding DNA (spacer
sequences)
- humans
- 250 copies of genes for 45S pre-rRNA
- 2000 5S rRNA genes
- 1300 tRNA genes
- produce products that are needed abundantly in most cell types
2. Non-Coding Repetitive DNA
a. Simple Sequence Repeats
- short tandemly repeated sequences
- 5-200 base pairs
- overall length can reach 1,000,000 base pairs
- function is unknown
- located at the centromeres and telomeres  believed to play structural role
- hypervariable regions
- varies considerably between individuals  basis of a very specific
identification (DNA fingerprinting)
b. Interspersed Repeats
- random locations
- length vary
- between 150-300 base pairs (short interspersed repeats, SINES)
- between 1000-6000 base pairs (long interspersed repeats, LINES)
- many are mobile elements  move to different positions in the genomic DNA
- may arise from RNA (then converted to DNA) by the action of reverse transcriptase
- lack promoter and other sequences  lack expression  pseudogenes
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SUMMARY
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MOLECULAR BIOLOGY:
DNA REPLICATION
THE STRUCTURE of DNA REVEALS a MECHANISM for its REPLICATION

A. Double Helical Model
- suggests that the strands can
- separate
- act as template for the formation of a new complementary strand
1. Conservative Replication
- parental DNA strands remained together in 1 of the daughter cells
- the newly synthesized DNA strands went to the other daughter cell
2. Semiconservative Replication
- each daughter cell received 1 parental DNA strand and 1 newly synthesized complementary
strand
- parental strand was the template
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B. Meselson-Stahl Experiments
- demonstrated semiconservative replication
PROKARYOTIC REPLICATION
- much better understood than that of the eukaryotes
- basic requirements and components of replication are the same for prokaryotes and eukaryotes
a. Template-Directed Polymerases
- synthesize the complementary sequence with extraordinary fidelity
b. DNA Synthesis in Higher Organisms
- is less well understood
- involves the same types of mechanisms
A. Basic Requirements for DNA Synthesis
1. Substrates
- 4 deoxynucleotide triphosphates (dNTPs)
- deoxyadenosine triphosphate (dATP)
- deoxyguanosine triphosphate (dGTP)
- deoxycytidine triphosphate (dCTP)
- deoxythymidine triphosphate (dTTP)
- cleavage of the high energy phosphates bonds between the  and  positions provide
the energy for nucleotide addition
2. Template
- directs the addition of a complementary nucleotide
- in semiconservative replication, each of the parental DNA strands serve as templates
3. Primer
- prepares the template strand for the addition of nucleotides
- new nucleotides are added to the 3’-end of the primer  new synthesis is said to occur in a
5’  3’ direction
4. Enzyme
- DNA-dependent DNA polymerases
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B. Multiple DNA Polymerases with Multiple Enzymatic Activities
1. DNA Polymerase I (Pol I)

a. Function
- DNA replication
- repair of damaged DNA
- removes RNA primers via its 5′ → 3′ exonuclease activity
b. Structure
- single polypeptide
- 103,000 MW
c. Other Enzymatic Activities (Besides Polymerase Activity)
i. Proofreading
- recognizes mismatched nucleotides  halts polymerization  3’ to 5’
exonuclease removes mismatched nucleotide  polymerization
resumes
- assures high level replication fidelity
ii. Excision-Repair
- 5’  3’ exonuclease activity
- hydrolytically removes a DNA segment from the 5’-end of a strand of duplex
DNA
- 1-10 nucleotide segments removed at a time
- essential for primer removal in DNA replication
- essential for the repair of damaged DNA
iii. Nick-Translation
iiia. Nick
- break in a phosphodiester bond of 1 strand of DNA in a double
helix
- leaves a free
- 3’-hydroxyl end
- 5’-phosphate end
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iiib. Pol I
- function at nicks as
- exonuclease
- polymerase
- as the 5’-phosphate is removed or displaced  nick is moved or
translated in the direction of synthesis
2. DNA Polymerase II (Pol II)

- minor DNA polymerase in E. coli
a. Function
- some DNA repair processes
b. Structure
- single polypeptide
- 90,000 MW
c. Other Enzymatic Activities
- proofreading (3’  5’ exonuclease)
3. DNA Polymerase III (Pol III)
- primary DNA polymerase in cellular replication
a. Function
- catalyzes leading and lagging strand synthesis (5′ → 3′ polymerase activity)
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b. Structure
i. Pol III Holoenzyme
- active cellular form
- 10 subunit polypeptides
- 422,000 MW
c. Other Enzymatic Activities
- proofreading (3’  5’ exonuclease)
   i.  Subunit
- contains the DNA polymerase activity
ii.  Subunit
- contains the 3’  5’ exonuclease activity
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STEPS in PROKARYOTIC (ESCHERICHIA COLI) DNA SYNTHESIS
- classes of proteins involved in replication
A. Separation (Melting) of the Two Complementary Strands

1. Origin of Replication
- single unique nucleotide sequence
- where replication begins
2. Eukaryotes
- replication begins at multiple sites  mechanism for rapidly replicating the great length of
eukaryotic DNA molecules
- sites include a short sequence composed almost exclusively of A-T base pairs
(consensus sequence)
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B. Replication Fork Formation

- unwinding or separation of 2 strands  form a “V” where active synthesis occurs (replication fork)
1. Replication
- bi- or unidirectional (replication is bidirectional in most organisms)
- replication forks move away from the origin
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2. Proteins (Forming the Prepriming Complex) Required for DNA Strand Separation
- group of proteins that recognize replication origin and/or the replication fork
- direct the initiation of replication that occurs within oriC
i. OriC - single replication origin in E. coli
- sequence of about 240 base pairs
ii. Maintain the
- separation of the parental strands
- unwinding of the helix ahead of the replication fork
a. DnaA Protein
- required for proper initiation of replication at the origin
- binds to specific nucleotide sequences at the origin of replication  melting of short,
tandemly arranged AT-rich regions in the origin
i. Melting
- ATP-dependent
- results in strand separation  formation of localized regions of
ssDNA (single-stranded DNA)
b. Single-Stranded DNA-Binding (SSB) Proteins (Helix Destabilizing Proteins)
- tetrameric
- bind cooperatively
- binding of 1 molecule of SSB protein  easier for additional molecules of
SSB proteins to bind tightly to the DNA strand
- bind only to single-stranded DNA
- not enzymes
- shift the equilibrium between single- and double-stranded DNA in the single-stranded
direction
- prevents reannealing of the DNA in the area of replication origin
- enhance the activity of helicases
- protect the DNA from nucleases that cleave single-stranded DNA
- block formation of intrastrand duplexes of hairpins that can slow replication
- displaced from single-stranded DNA when DNA undergoes replication
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c. DNA Helicases
- enzyme
- bind to single-stranded DNA near the replication fork  move into neighboring double-
stranded region  force strands apart  double-helix unwinding  SSB
proteins bind  double helix reformation prevented  provision of single-
strand templates for replication
- derives energy from cleavage of high-energy phosphate bonds of nucleoside
triphosphates usually ATP
i. dnaB Protein
- hexameric
- principal helicase of E. coli
- component of the primosome
- further unwinds DNA in an ATP-dependent reaction
ii. Proteins With Helicase Activity
- a number have been isolated
- most play a role in
- DNA repair
- recombination
- bacteriophage replication
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3. Problem of Supercoils (Supertwists)
- as 2 strands are separated  entire chromosome ahead of the replication fork would either have
to - rotate
- accumulate positive supercoils (supertwists)  interferes with further double
helix unwinding
a. Topoisomerases
- alter DNA supercoiling by making transient single-strand breaks (Type I) or double-
strand breaks (Type II)
b. Type I DNA Topoisomerases

- reversibly cut a single strand of the double helix  transient “nick”  rotation of the
DNA helix on either side of the nick around the phosphodiester bond opposite
the nick  relief of supercoils
- store the energy from the phosphodiester bond they cleave ( do not require ATP) 
reuse the energy to reseal the strand
i. Activities
- nuclease (strand-cutting)
- ligase (strand-resealing)
ii. Types
iia. Type IA Topoisomerases
- relax only negatively supercoiled DNA (fewer helical turns than the
relaxed DNA) in E. coli by changing the linking number in
increments of one
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iib. Type IB Topoisomerases
- relax negative and positive supercoils by a controlled rotation
mechanism in eukaryotic cells
iii. Regulation of DNA Supercoiling by Type I Topoisomerases
iiia. Proposed Mechanisms of Action of Type I Topoisomerases
iiiai. Strand-Rotation Model
- type I topoisomerase binds to opposite strands of the DNA
double helix  topoisomerase nicks one strand and
remains bound to one of the nicked ends
- unbound end of the nicked strand is able to unwind by one
or more turns  religated to its parent strand
iiiaii. Strand-Passage Model
- type I topoisomerase binding to the DNA double helix 
melting of the two DNA strands  introduces a nick
into one strand, while remaining bound to each end
of the broken DNA strand
- broken strand is passed through the helix and religated
 net unwinding of the DNA
iiib. Camptothecins
- cancer chemotherapy
- inhibit the joining of the broken strand of DNA after strand passage
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c. Type II DNA Topoisomerases
- bind tightly to the DNA double helix
- make transient breaks in both strands  allow a 2nd stretch of DNA double-helix to
pass through the break  reseal transient break  relief of negative and
positive supercoils (ATP-dependent)
- required in both eukaryotes and prokaryotes for the separation of interlocked DNA
molecules following chromosomal replication
i. Regulation of DNA Supercoiling by Type II Topoisomerases

ia. The type II topoisomerase enzymes contain A', B', and ATPase domains. The
A' and B' domains engage the G-segment of the DNA double helix
ib. Interaction with the G-segment induces a conformational change in the type
II isomerase, causing it to “lock” around the DNA G-segment
ic. ATP binds to the ATPase domains of the topoisomerase. T-segment of the
DNA double helix enters and is “locked” into the B' domains
id. Once the enzyme is engaged with both DNA segments, the topoisomerase
cuts both strands of the G-segment DNA
ie. This double-stranded nick in the G-segment allows the T-segment to pass
through the G-segment to the opposite side of the topoisomerase.
if. The T-segment is released from the topoisomerase, and the G-segment nick
is religated. ATP is hydrolyzed to ADP, ADP dissociates from the
topoisomerase, and the cycle begins anew
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ii. DNA Gyrase

- type II topoisomerase in prokaryotes
- may introduce negative supercoils in resting circular DNA (negative
supercoils in eukaryotes result primarily from packaging DNA into
nucleosomes)
- facilitates future replication (negative supercoils neutralize positive
supercoils introduced during double helix opening)
- aids in the transient strand separation required during transcription
iia. 2 Subunits
- 1 that cleaves ATP for superhelices formation
- 1 that is required to remove superhelices
- does not require ATP
iii. Quinolone Antibiotics
iiia. Inhibit
- passage of the T-segment
- religation of the nicked G-segment by bacterial type II
topoisomerases
iiib. Therapeutic Concentrations
- promote topoisomerase subunit (domain) dissociation  double-
stranded breaks in the DNA  bacterial killing
iiic. Nalidixic Acid
- antibiotic
- inhibits the ability of DNA gyrase to remove superhelices
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iiic. Fluoroquinolones
- norfloxacin
- ciprofloxacin
- newer derivatives of nalidixic acid
- greater antibacterial activity
- broader activity spectrum
- bacteria do not develop resistance to them quickly
iv. Cancer Chemotherapeutic Agents
iva. Examples
- anthracyclines
- epipodophyllotoxins
- amsacrine
- etoposide
ivb. Inhibit
- passage of the T-segment
- religation of the nicked G-segment by human type II topoisomerases
ivc. Result
- double-stranded DNA breaks
- induce apoptosis of cancer cells
C. Basic Molecular Events in Replication Forks
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a. DNA Polymerase
- copying of DNA templates
- read the parental nucleotide sequence in the 3’  5’ direction
- synthesize the new strands in the 5’  3’ direction (antiparallel)
- 2 newly synthesized stretches of nucleotide chains grow in opposite directions
- 1 in the 5’3’ direction towards the replication fork
- 1 in the 5’3’ direction away from the replication fork
1. Leading Strand Synthesis
- continuous synthesis of one of the daughter strands in a 5’  3’ direction (direction of the
advancing replication fork)
- catalyzed by Pol III
2. Lagging Strand Synthesis
- synthesis of one of the daughter strands in the direction away from the replication fork
- synthesized discontinuously with small DNA fragments (Okazaki fragments) being copied
near the replication fork  fragments joined by DNA ligase single continuous strand
- DNA synthesis always occur in a 5’  3’ direction  discontinuous synthesis of the lagging
strand
- catalyzed by Pol III
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D. RNA Primer
1. DNA Polymerases
- cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded
template  require a primer
2. Primer
- short double-stranded region with a free hydroxyl group on the 3’-end of the shorter strand
- hydroxyl group serves as the 1st acceptor of a nucleotide by the action of DNA
polymerase
- free 3’-hydroxyl group is provided by a short stretch of RNA (4-12 nucleotides)
- complementary to the template strand
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3. Primase (DnaG Protein)

- specific RNA polymerase
- catalytic portion of the primosome that makes the RNA primer needed to initiate synthesis of the
okazaki fragments
- makes the primer that initiates leading strand synthesis at the origin
- synthesize the short stretches of RNA (10 nucleotide length) that is complementary and
antiparallel to the DNA template  hybrid duplex
- U in RNA pairs with A in DNA
- G pairs with C
- constantly synthesized at the replication fork on the lagging strand
- few are required on the leading strand
- building blocks are 5’-ribonucleoside triphosphates
- only 1 RNA sequence at the origin of replication is required on the leading strand
4. Primosome
a. Combination Of
- prepriming complex
- primase
i. Prepriming Complex
- formed prior to RNA primer synthesis
- group of several proteins
- binds to the single strand of DNA  displacing some of the single-stranded
DNA-binding proteins
- hexamer of
- helicase dnaB protein
- dnaG protein
- proteins
-n
- n’
- n”
-i
- displaces SSB in an ATP-dependent process
- initiates Okazaki fragment formation by moving along the lagging strand in the 5’  3’
direction
- periodically recognize specific nucleotide sequences that direct it to synthesize RNA primer in
the 5’3’ direction (antiparallel to the DNA template chain)
- moves in the opposite direction from the DNA synthesis of the lagging strand
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E. Chain Elongation
- prokaryotic and eukaryotic DNA polymerases
- add deoxyribonucleotides at the 3’-end  elongate a new DNA strand
- sequence of added nucleotides is dictated by the base sequence of the template strand
1. DNA Polymerase III
- catalyze DNA chain elongation
- uses the 3’-hydroxyl group of the RNA primer as the 1st acceptor of deoxyribonucleotide
- new strands grows in the 5’  3’ direction (antiparallel to the parental strand) 
5’  3’-polymerase activity
- all 4 deoxyribonucleoside triphosphates (5’- deoxyribonucleoside triphosphates) must be present

for DNA elongation to occur
- dATP
- dTTP
- dCTP
- dGTP
- if 1 of the 4 is depleted  DNA synthesis stops
- highly “processive” enzyme
- remains bound to the template strand as it moves along
- does not have to diffuse away and rebind before adding each new nucleotide
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2. DNA Synthesis Inhibition by Nucleoside Analogues

- when incorporated into the growing DNA chain  prevent further elongation  blocking DNA
replication  slowed division of rapidly dividing and growing cells and viruses
a. Nucleotide Analogue Formation
i. Modification in the Sugar Portion of the Nucleotide
- ex: removal of the hydroxyl group from the 3’-carbon of the deoxyribose
ring [2’,3’-dideoxyinosine (ddI)]
ii. Conversion of the Deoxyribose to Another Sugar

- ex: arabinose sugar
- ex: adenine arabinoside (vidarabine, araA)
- antiviral agent
cytosine arabinoside (cytarabine, araC)
- anticancer chemotherapy
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iii. Chemically Modifying Sugar Moiety

- ex: zidovudine (AZT)
acyclovir
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3. Proofreading of Newly Synthesized DNA
- template sequence misreading  deleterious or lethal mutation
a. DNA Polymerase III Activities
i. 5’  3’-Polymerase
- replace the correct base
ii. Proofreading Activity (3’  5’ Exonuclease)
- hydrolytically removes incorrectly added base
- excision done in the reverse direction from that of synthesis
- ensure correct base pairing
- edits mistakes
- does not degrade correctly paired nucleotides
F. RNA Primer Excision and Its Replacement by DNA

- DNA polymerase III continues to synthesize DNA until it is blocked by proximity to RNA primer 
RNA excision  gap filled by DNA polymerase I
1. 5’  3’ Exonuclease Activity of DNA Polymerase I
- other activities of DNA polymerase I
- 5’ 3’ polymerase activity (synthesize DNA)
- 3’5’ exonuclease activity (proofreading of newly synthesized DNA chain)
- hydrolytically removes RNA primer upon completion of leading and lagging strand synthesis
- locates the space (“nick”) between the 3’-end of the DNA and the 5’-end of the adjacent RNA
primer  remove RNA nucleotides  replaces with deoxyribonucleotides synthesizing
DNA in the 5’  3’ direction (5’  3’ polymerase activity) at the same time
proofreading using 3’  5’ exonuclease activity  gap filled
- synthesis of each new Okazaki fragments takes place until it reaches the RNA primer of
the preceding Okazaki fragments and the RNA primer  nick-translation
properties of Pol I  RNA hydrolysis  replacement with DNA
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2. 5’  3’ Exonuclease Activity of DNA Polymerase I vs. 3’  5’ Exonuclease Activity by DNA

Polymerase I and III
a. 5’  3’ Exonucleases
- of DNA polymerase I
- can remove a nucleotide at a time from a DNA region that is properly base-paired
- nucleotides removed
- ribonucleotides
- deoxyribonucleotides
- can remove groups of altered nucleotides in the 5’  3’ direction
- remove up to 10 nucleotides at a time
- important in the repair of damaged DNA (ex: removal of pyrimidine dimers)
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G. DNA Ligase
- does the phosphodiester linkage between the 5’-phosphate group of the DNA chain synthesized by
DNA polymerase III and the 3’-hydroxyl group of the DNA synthesized by DNA polymerase I
- exergonic reaction consuming ATP  AMP + PPi
H. Replisome
- large molecular complex
- consists of
- all replication enzymes including two Pol III enzymes
- all replication factors
- may be attached to the membrane  DNA is passed through the stationary replisome
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I. Termination of Replication
1. Ter Sites
- termination sequences
- signal the termination of DNA synthesis
2. Ter-Binding Protein
- binds to termination sequences  prevents helicase dnaB protein from further DNA
unwinding  termination of replication
3. Tus Protein
- binds to a Ter site  prevents strand displacement by DnaB  arrest of DNA replication
EUKARYOTIC DNA REPLICATION

- not well described as in prokaryotes
- fundamentally proceed by a mechanism similar to that of prokaryotic replication
- semiconservative
- proceeds bidirectionally from multiple replication origins
- functions analogous to prokaryotic DNA replication
- eukaryotic single-stranded DNA-binding proteins
- ATP-dependent DNA helicases
- RNase H
- remove RNA primers
- steps involved in eukaryotic DNA replication
A. Replicons
- basic units of replication
1. Function
- encompasses all of the DNA replicated from the growing replication forks from a single origin
2. Size - 50-120 m
- large number of replicons to replicate the mammalian genomes in a reasonable period of time
- 8 hours to replicate the human genome
3. Replication Rate
a. Prokaryotes
- 1000 base pairs/second
b. Eukaryotes
- 10x slower than prokaryotic replication rate
- 100 base pairs/second
- each replicon completes synthesis in about 1 hour
- during the total eukaryotic replication period, all replicons are not active
- slow eukaryotic replication rate due to interferences of
- nucleosomes
- chromosomal proteins
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B. Multiple Eukaryotic DNA Polymerases
1. Nuclear DNA Polymerases
a. Classification
- on the basis of
- molecular weight
- cellular location
- sensitivity to inhibitors
- templates or substrates on which they act
2. Mitochondrial DNA Polymerase
3. DNA Polymerases
- categorized according to
- molecular weight
- cellular location
- sensitivity to inhibitors
- templates or substrates they act
a. Pol  and Pol 
- major polymerases of DNA replication in eukaryotes (work together to synthesize both
the leading and lagging strands)
- members of the -family of eukaryotic DNA polymerases
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i. Pol  - essential for replication
- multisubunit enzyme
- 1 have primase activity
- initiates strand synthesis
- on the leading strand
- at the beginning of each Okazaki fragment on the
lagging strand
- synthesize a short RNA primer
- 5’3’ polymerase activity
- extends the RNA primer
- adds a short piece of DNA
- analogous to E. coli pol I
- component of lagging strand synthesis
- lacks exonuclease activity
ii. Pol  - main polymerase for the leading and lagging strands
- analogous to E. coli pol III
- completes DNA synthesis on the leading strand
- elongate each Okazaki fragment
- 3’5’ exonuclease activity to proofread the newly synthesized DNA
- unlimited processivity when it is in complex with proliferating cell nuclear
antigen (PCNA) which functions as a sliding clamp
b. Pol 
- no role in replication
- excise primers
- carry out DNA repair
c. Pol  - appears to have a regulatory function
- carry out DNA repair
- may substitute for DNA polymerase δ in certain cases
d. Pol γ - resides in and replicates mitochondrial DNA
4. Comparison of Prokaryotic and Eukaryotic DNA Polymerases
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C. Other Factors Involved in Eukaryotic Replication

1. 5’  3’ Exonucleases
- one is associated with DNA polymerase
2. 3’  5’ Exonuclease Activity
- DNA polymerase
- other enzymes have been isolated exhibiting the same activity
3. Ligase
- 2 ligases isolated
- 1 involved in replication
- 1 involved in DNA repair synthesis
4. Helicases
5. Single-Stand Binding Proteins
6. Topoisomerases
- several well-characterized topoisomerases
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REVERSE TRANSCRIPTASE
A. Human Immunodeficiency Virus (HIV)
- retrovirus
- carries in its genome in the form of single-stranded RNA molecules
B. Reverse Transcriptase
- viral RNA-directed DNA polymerase
- uses a tRNA primer to replicate the viral genome
- moves along the template in the 3’5’ direction synthesizing DNA in the 5’3’ direction
- no primase or 3′ → 5′ exonuclease activity
- lack of proofreading  high mutation rate
- contain ribonuclease (RNase H) activity
- essential tool for cloning mRNA sequences  synthesize complementary DNA molecule (cDNA)
- eukaryotic reverse transcriptase activity
- associated with telomerase (hTRT)
- encoded by retrotransposons (residual viral genomes permanently maintained in human DNA)
that play a role in amplifying certain repetitive sequences in DNA
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C. Therapeutic Drugs
- prevent progression of HIV infection to acquired immunodeficiency syndrome (AIDS)
a. Reverse Transcriptase Inhibitors
- nucleoside
- non-nucleoside
b. Protease Inhibitors
- target another HIV maturation enzyme
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DRUGS THAT AFFECT REPLICATION

- inhibitors of replication
- some antibacterial drugs
- some antiviral drugs
- many chemotherapeutic drugs
- classified according to the mechanism by which they inhibit replication
A. Antimetabolites
- reduce or inhibit the production of a substrate for replication (dNTPs)
- without substrates  DNA polymerases cannot make DNA
(Refer to INHIBITORS OF PURINE and PYRIMIDINE METABOLISM - chapter of nucleotide

metabolism)
B. Substrate (Nucleoside) Analogues

- can be incorporated into DNA by DNA polymerases  inhibit further replication  slowed division
of rapidly growing cells and viruses
- modified in the sugar portion of the nucleoside
1. Azidothymidine (AZT) / Zidovudine
a. Structure
- azido (N3) group replaces the hydroxyl group on the 3’ position of thymidine
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b. Therapeutic Use
- potent antiviral drug
- for human immunodeficiency virus (HIV) infections
c. Mechanism of Action
- AZT is phosphorylated  incorporated into DNA
- azido group in the 3’ position is an unacceptable primer for further DNA synthesis
i. Phosphorylated AZT
- effective antiviral drug
- accepted as a substrate by a viral DNA polymerase better than the human DNA
polymerases
ii. Reverse Transcriptase
- viral DNA polymerase
- uses RNA as a template to synthesize DNA  RNA-dependent DNA
polymerase
2. Cytosine Arabinoside (araC) / Cytarabine
a. Structure
- 2’-hydroxyl group of the sugar is in the trans configuration
b. Therapeutic Use
- for acute myelocytic leukemia
i. Adenine Arabinoside (araA)
- analogue of araC
- antiviral
c. Mechanism of Action
- exact mechanism of action is unknown
- phosphorylated  incorporated into DNA  slows replication rate
alters DNA structure  prone to
breakage
3. 2’,3’-Dideoxyinosine (DDI)
- hydroxyl group removed from the 3’-carbon of the deoxyribose ring
4. Adenine Arabinoside (araA)

- antiviral
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C. Inhibitors That Interact Directly with DNA
1. Intercalators
- drugs
- usually with aromatic rings
- insert between adjacent stacked base pairs
a. General Mechanism of Action
- causes a physical block and disruption or change in the DNA conformation 
inhibits the action of replication enzymes
- many are mutagenic and induce mistakes in replication  disease
b. Anthracycline Glycosides
i. Structure
- commonly used
- daunorubicin
- doxorubicin
- antibiotics produced by a strain of streptomyces
- planar tetracycline ring intercalates between DNA bases
ii. Therapeutic Use

iia. Daunorubicin
- for leukemia
iib. Doxorubicin
- for wide range of cancers
- derivative show potent anticancer activity
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c. Actinomycin D
i. Structure
- antibiotic produced by a strain of streptomyces
- planar phenoxazone ring intercalates between DNA bases
ii. Therapeutic Use

- cancer chemotherapy for Wilm’s tumor in children in combination with
- surgery
- radiotherapy
- other chemotherapeutic drugs
2. Drugs that Damage DNA
a. Alkylating Agents
- act as strong nucleophiles
- evolved from sulfur and nitrogen mustard gases of World Wars I and II
i. General Mechanism of Action
- become linked to many cellular nucleophiles (7th nitrogen on the purine ring
of guanine in DNA)
- alkyl linkage  mispairing with guanines  mutations
DNA breakage
cross-linking of the double helix
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ii. Therapeutic Use

- for many different cancers
b. Platinum-Coordination Complexes
- ex: Cisplatin
i. Mechanism of Action
- platinum-containing compounds react with nucleophiles (7th nitrogen on the
purine ring of guanine)  cross-linking between adjacent guanines in
DNA
ii. Therapeutic Use
- effective chemotherapeutic drug especially in conjunction with other
chemotherapeutic drugs
- for - testicular cancers
- ovarian cancers
- enhance toxic effect of radiotherapy in cancer cells
c. Bleomycins
i. Structure
- complex compounds
- isolated from a strain of streptomyces
- made with a wide number of side groups  production of related agents with
different toxic effects for different tumors
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ii. Mechanism of Action

- bind to DNA  interact with oxygen and Fe++  DNA breakage
iii. Therapeutic Use
- used in conjunction with other chemotherapeutic drugs
- for - testicular cancers
- other cancers
D. Inhibitors of Replicative Enzymes
1. DNA Polymerase Inhibitors
- very limited clinical use
2. Topoisomerase Inhibitors
- ex: nalidixic acid
fluoroquinolones
- inhibit bacterial topoisomerase DNA gyrase  no relief of positive supercoils
- effective in the treatment of urinary tract infections
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TELOMERES and IMMORTALITY
A. Telomeres
- repetitive noncoding DNA sequences complexed with proteins  protects the ends of the
chromosomes from attack by nucleases
- found at the extreme end of the 5’-end of the lagging strand following removal of the RNA primer
(ends of chromosomes)
- DNA consists of repetitive sequences of 1-4 Ts and 1-4 Gs base-paired to a complementary chain of As
and Cs
- TG strand
- longer than its complement
- single-stranded DNA region at the 3’-end of the double helix

- few hundred nucleotides long
- folds back on itself stabilized by a protein  complex  protect the end of the
chromosome
- susceptible to single-stranded DNA degradation back to double-stranded DNA
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B. Replication of Linear Chromosomes
- starts at coding sites (origins of replication) within the main body of chromosomes (not at the two
extreme ends)
C. Cell Senescence (Aging)

- telomeres (ends of chromosomes) get slightly shorter with each cell division (consequential telomere
shortening)  chromosome instability  cell aging or death
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D. Stem Cells
- have longer telomeres than their terminally differentiated daughters
- have great therapeutic potential
1. Give Rise to
a. Daughter cell that will differentiate, given the appropriate signals, into any cell type (except
fetal placental cells)
- limited ability to replicate when differentiated
b. Daughter cell which will not differentiate and has the infinite ability to replicate (self-
renewal)
2. Source Categories of Stem Cells in Mammals
a. Embryonic Stem Cells
- derived from blastocysts
b. Adult Stem Cells
- found in adult tissues
c. Cord Blood Stem Cells (Embryonic-Like Stem Cells)
- found in the umbilical cord
- not as primitive as ES cells
- can differentiate into many more cells types than adult progenitor cells
d. Induced Pluripotent Stem Cells (IPSC)
- by reprogramming adult cells to regain stem-like properties
3. Subcategory by “Potency”
- specifies the differentiation potential (potential to differentiate into different cell types) of the
stem cell
a. Totipotent Stem Cells
- produced from the fusion of an egg and sperm cell
- cells produced by the first few divisions of the fertilized egg are also totipotent
- differentiate into embryonic and extraembryonic cell types
- differentiate into any cell type in the adult body
b. Pluripotent Stem Cells
- descendants of totipotent cells
- differentiate into cells derived from any of the three germ layers
c. Multipotent Stem Cells
- produce only cells of a closely related family of cells (haematopoietic stem cells
differentiate into red blood cells, white blood cells, platelets)
d. Unipotent Cells
- produce only one cell type
- have the property of self-renewal (distinguishes them from non-stem cells)
E. Telomerase
- enzyme contained by cells that do not age
- germ-line cells
- cancer cells
- contains
- short RNA template complementary to the DNA telomere sequence
- telomerase reverse transcriptase activity (hTRT)
- replaces the lost ends  maintain the telomeres
- act as template primer at the extreme ends of the chromosomes  protects against telomere
shortening
- carries its own RNA molecule (about 150 nucleotides long)
- contain copies of A and C sequences complimentary to the T and G repeat sequences
- base-pairs with the terminal nucleosides at the single-stranded 3’-end of the DNA 
serve as template for extending the DNA strand
- after the repeat sequence is completed  telomerase RNA is translocated to the newly
synthesized end of the DNA  hydrogen bonding
- transient expression of telomerase in various stem and daughter cells is part of their normal biology
- inability to activate telomerase (in addition to overexpression) in cells (ex: immune system)  disease
pathologies
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F. Germ Cells
- express telomerase  replicate without shortening of their telomeres
G. Somatic Cells
- most switch off the activity of telomerase after birth and die as a result of apoptosis
H. Cancer Cells
- many reactivate telomerase  contribute to immortality
I. Progeria
- premature ageing syndrome
- cells have extremely short telomeres
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SUMMARY
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MOLECULAR BIOLOGY:
RNA STRUCTURE
RNA STRUCTURE
- unbranched polymeric structure
- composed of mononucleotides joined together by phosphodiester bonds
- most RNAs exist as single strands capable of folding into complex structures
1. Three Major Types of RNA that Participate in Protein Synthesis
- rRNA
- tRNA
- mRNA
a. Differ in Terms of
- size
- function
- structural modifications
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2. Differences From DNA
a. Size - considerably smaller than DNA
b. Sugar
- contain ribose sugar (instead of deoxyribose)
c. Nucleotide
- contain uracil (instead of thymine)
d. Form
- usually exists in single-strand form
A. Primary Structure
1. RNA - initially synthesized as single-stranded polymer by the process of transcription
2. Ribonucleotides
- linked into a polar molecule by phosphodiester bonds
a. Phosphodiester Bonds
- between
- 3’-hydroxyl on the sugar of 1 ribonucleotide through a phosphate to
- 5’-hydroxyl on the sugar of another ribonucleotide
b. Sugar-Phosphate Linkages
- form symmetrical backbone
- 5’-end of 1 sugar linked through a phosphate to
- 3’-end of adjacent sugar
c. Bases - variable
- stick out from the backbone
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B. Secondary Structure
1. Double-Stranded RNA
- RNA is single-stranded but can form regions of double helix by folding back on itself
a. Base Pairing
- complementary RNA sequences can base pair
i. Adenine with Uridine
ii. Guanine with Cytosine
b. A-Form Helix
- 2’-hydroxyl groups on the ribose sugar sterically hinders formation of B-form 
double helical regions assume conformations resembling A-DNA
- nature of RNA double helix is similar to DNA
- strands must be antiparallel
c. DNA-RNA Hybrids
- show A-form conformations
2. Other Structures
- varied shapes due to various sections of RNA forming double-stranded regions via specific
base pairing
a. 5S Ribosomal RNA (rRNA)
- contains helices, hairpin loops, internal loops, and bulges
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b. Transfer RNA (tRNA)

- base pairing and extensive stacking interactions  compact shape
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C. Tertiary Structure
a. Roles of Some RNAs
- structural
- interact extensively with specific proteins
- catalytic functions  form some very complex structures
1. RNA Modifications
- terminal additions
- base modifications
- methylations
- at numerous positions of the different bases
- most common of the modifications
- allow for unusual base pairings that enhance RNA structural
diversity
- trimming
- internal segment removal
- splicing
- after RNA synthesis
- convert inactive primary transcript  functional molecule
2. Transfer RNA
- most heavily modified
MAJOR CLASSES of RNA

- 3 functionally distinct classes of RNA are produced in prokaryotes
- 4 are produced in eukaryotes
A. Messenger RNA (mRNA)
- most heterogeneous type in terms of
- size (500-6000 nucleotides)
- base sequence
- carries genetic information from DNA  cytosol  ribosomes  template for protein synthesis
1. Prokaryotic mRNA
a. Basic Features
- not all portions code for polypeptides
i. Polycistronic
- carry information for the production of multiple polypeptides
ia. Cistrons
- sequences that code for proteins
ii. Leader Sequence/5’-Untranslated Region (5’-ULR)
- contain sequences that are never translated into protein
iii. Trailer Sequence/3’-Untranslated Region (3’-UTR)
- contain sequences that are never translated into protein
iv. Intercistronic Regions / Spacers
- sequences between cistrons
b. Abundance
- 5% of total cellular RNA
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c. Stability
- stable for just a few minutes
- lifetime is short
2. Eukaryotic mRNA
a. Basic Features
i. Monocistronic
- carry information for the production of a single polypeptides
ii. Precursor Form

- heterogeneous nuclear RNA (hnRNA)
- most but not all eukaryotic mRNA arise by extensive post-transcriptional
processing of large precursors
iii. Leader Sequence (5’-ULR)
iv. Trailer Sequence (3’-UTR)
v. Polyadenylate (Poly-A) Tail
- most but not all eukaryotic mRNA
- long adenylate residues (200-300)
- on the 3’-end of the RNA chain
vi. Cap - on the 5’-end of eukaryotic mRNAs
- consists of a 7-methylguanylate molecule attached backward through a 5’ to
5’ triphosphate linkage
b. Abundance
- no more than 5% of the cell RNA
- precursor hnRNA
c. Stability
- relatively stable
- half-lives of hours to days
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B. Ribosomal RNA (rRNA)
- found in association with different proteins as components of the ribosomes (site of protein synthesis)
a. Functions
i. Structural
ii. Ribozyme
- catalytic for some of the translation reactions
- 80% of the ribosomal mass
1. Prokaryotic rRNA
a. Basic Features
i. 3 Types
ia. 23S rRNA
- 2904 nucleotides
- component of the large 50S ribosomal subunit
ib. 16S rRNA
- 1541 nucleotides
- component of the small 30S ribosomal subunit
ic. 5S rRNA
- 120 nucleotides
“S” - Svedberg unit

- related to
- molecular weight
- shape of the compound
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b. Abundance
- most abundant
- 80% of total prokaryotic cellular RNA
2. Eukaryotic rRNA
- bigger than those of prokaryotes
a. Basic Features
i. 4 Types
ia. 28S rRNA
- 4718 nucleotides
ib. 18S rRNA
- 1874 nucleotides
- component of the small 40S ribosomal subunit
ic. 5.8S rRNA
- 160 nucleotides
id. 5S rRNA
- 120 nucleotides
- transcription product of a separate gene
b. Abundance
- 4% of total eukaryotic cellular RNA is 40S precursor rRNA
- 71% is fully processed rRNAs
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C. Transfer RNA (tRNA)
- smallest (4S)
- 74-95 nucleotide residues
- 1 specific tRNA type for each of the amino acids
- contain unusual bases (ex: pseudouracil)
- extensive intrachain base pairing
- serve as adaptor molecule  carry specific amino acid (covalently attached to its 3’-end) to the site of
protein synthesis  facilitates incorporation of amino acids into newly synthesized proteins in a
template-dependent manner
- 15% of the total RNA in the cell
1. Prokaryotic tRNA
a. Basic Features
i. Size - small
- average of 80 nucleotides
ii. Structure
- all tRNAs have common structural features  function in the ribosome
- unique structural features necessary for recognition by the enzyme that
catalyze amino acid attachment to tRNAs
- sequences that pair with appropriate codons in ribosomes are unique for each
tRNA
iii. Processing
- arise from processing of large precursor tRNAs
iv. Modification
- heavily modified post-transcriptionally
b. Abundance
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2. Eukaryotic tRNA
a. Basic Features
- similar to prokaryotes in
- size
- structural features
- heavily modified post-transcriptionally
b. Abundance
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EUKARYOTIC SMALL RNAs
- variety of functions
- classified into 2 broad types according to where they are located
A. Small Cytoplasmic RNAs (scRNAs)
1. 7S RNA
- major scRNA
- 294 nucleotides
- RNA component of signal recognition particles
B. Small Nuclear RNAs (snRNAs)
- associated with proteins in small nuclear ribonucleoprotein particles (snRNPs - pronounced “snurps”)
1. snRNPs
- function in the splicing reactions needed to process hnRNA to mRNA
SUMMARY
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MOLECULAR BIOLOGY:
TRANSCRIPTION
OVERVIEW
A. DNA - contains the genetic master plan of the organism
B. RNA - “working copies” of the DNA that the master plan is expressed
C. Genes
1. Description
- portion of DNA that contains the codes for a polypeptide sequence
- consist of lengths of DNA that contain sufficient nucleotide triplets to code for the appropriate
number of amino acids in the polypeptide chains of a particular protein
2. Size - vary greatly
- most extend over 20-40 kbp
- few (ex: gene for muscle protein dystrophin) can extend over millions of base-pairs
3. Coding Sequences
a. Bacteria
- continuous
b. Higher Organisms
- coding sequences (exons) interrupted by intervening sequences that are non-
coding (introns) at various positions
a. Code for RNA Molecules
- will not be further translated into proteins
i. rRNA and tRNA
- vital roles in polypeptide synthesis
ii. MicroRNAs
- single-stranded RNA molecules
- about 22 nucleotides
- inactivate specific messenger RNAs  disrupt expression of their proteins 
regulate cell proliferation and apoptosis
- can be inactivated by DNA methylation
b. Code for Proteins
D. Transcription
- the conversion of genetic information to polypeptides and proteins relies on the transcription of
sequences of bases in DNA to messenger RNA molecules
1. RNA Polymerases
a. Reaction Catalyzed
(RNA)n residues + NTP  (RNA)n+1 residues + PPi
b. Prokaryotes
- one species of RNA polymerase synthesizes all of the RNA except for the short RNA
primers needed for DNA replication
i. Primase
- synthesize RNA primers
2. Messenger RNAs (mRNAs)
- transcripts of certain DNA regions
- translated into sequences of amino acids (polypeptide chains)
a. Initial or Primary mRNA
- complete copy of one strand of DNA
- contains both introns and exons
- complementary to the DNA template (antisense) strand
- identical to the coding (sense/antitemplate) strand (with U replacing T)
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3. Other RNAs
- ribosomal RNAs (rRNAs )
- transfer RNAs (tRNAs)
- small RNA molecules
- perform specialized structural and regulatory functions without translation
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4. Important Features of Transcription

a. High Selectivity
- many transcripts are made of some regions of the DNA
- few or no transcripts made from other regions
- due to
i. Signals
- embedded in the nucleotide sequence of the DNA
- instruct the RNA polymerase
- where to start
- how often to start
- where to stop transcription
ii. Regulatory Proteins
- results to the biochemical differentiation of an organism’s tissues
b. Posttranscriptional Modification
- while still in the nucleus
- converts inactive primary transcript to functional molecule
i. Terminal Additions
ia. Capping
- at the 5′ end
ib. Poly-adenylation
- at the 3′ end
ii. Base Modifications
iii. Trimming
iv. Internal Segment Removal
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v. Splicing
- introns are removed
- achieved by small nuclear RNA in association with specific proteins
- alternative splicing is possible whereby an entire exon can be omitted  more
than one protein can be coded from the same gene
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5. Untranslated Regions
- first 20 or more nucleotides
a. Recognition and Regulatory Sequences
- necessary for translation and possibly earlier transcription
b. Poly A Tail
- not translated (3′ untranslated region)
- preceded by stop codon (UAA, UAG or UGA)
6. AUG (Methionine) Codon

- start of translation
STEPS in RNA SYNTHESIS

A. Template Recognition
- involves binding of RNA polymerase II to a specific sequence of DNA molecules (DNA promoter
region)
1. Polymerase-Binding Site
- defines the starting point of transcription
- bacterial RNA polymerase binds to a specific region of about 60 base pairs of the DNA
2. Promoter Sequences
- transcription does not require a primer
- responsible for directing RNA polymerase to initiate transcription at a particular point
- differ between prokaryotes and eukaryotes
- DNA sequence that specifies the site of RNA polymerase binding from which transcription is
initiated
- organized into several regions with sequence homology
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a. Nomenclature and Numbering Conventions
- used to avoid confusions in the description of sequences
- sequence of only 1 DNA strand is presented
- RNA synthesis occurs in a 5’ to 3’ direction  sequences are written in a 5’ to 3’
direction
 sequence of the DNA strand that is identical to the RNA transcript is
presented
- position 1 of a gene is the base that is equivalent to the 1st base of the 5’-end of the
RNA transcript of that gene
- sequences preceding the 1st base
- numbered negatively
- said to be upstream of the initiation point
- sequences following the 1st base
- numbered positively
- said to be downstream of the initiation point
- 1st base at the transcription start site is assigned a position of +1
b. Consensus Sequences in the Promoter Region

- do not tolerate mutational changes  remain constant even in evolutionarily remote
organisms (the sequences are conserved in evolution)
- mutations at different sites upstream of a gene have different effects depending on
where they are located
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c. Prokaryotic Promoters
- contain “consensus” nucleotide sequence of six base pairs TATAAT
- highly conserved
- many different promoters contain some very similar or identical
sequences
i. 3 Sequence Elements
ia. Initiation Site (Startpoint / Transcription Start Site)
- most gene transcriptions always start at the same base (position 1)
- usually a purine
ib. TATAAT or Pribnow or -10 Box
- stretch of 6 nucleotides (TATAAT)
- exists about 9-18 base pairs upstream of the start point (10 base pairs
above the starting point)
- has been called -10 sequence
- usually found 10 base pairs upstream of the start point
- mutation at this region can affect gene transcription controlled by
the promoter
ic. TTGACA or -35 Box
- another region of conserved sequences
- typically found 35 base pairs upstream of the start point
- also recognized by RNA polymerase
- mutation at this region can affect gene transcription controlled by
the promoter
ii. Two Important Regulatory Sequences

iia. -35 Box
- 5’-TATTGACA-3’
iib. -10 Box (TATA Box)
- 5’-TATAAT-3’
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d. Eukaryotic Promoters
- each type of the eukaryotic RNA polymerase uses a different promoter
- promoters used by RNA polymerase I and II
- upstream of the start point
- promoters used by RNA polymerase III
- usually downstream of the start point
i. Initiation Site / Start Point
- promoters of all eukaryotic RNA polymerases direct the initiation of
transcription to a particular start point
- usually a purine (pyrimidine is not uncommon)
ii. RNA Polymerase I
- has a bipartite promoter
- one part 170 to 180 bp upstream (5’ direction)
- other from about 45 bp upstream to 20 bp downstream (3’ direction)
(core promoter)
- requires two ancillary factors
i. UPE1 (upstream promoter element 1)
ii. SL1
- located in the nucleolus
- synthesizes ribosomal RNA (accounts for about 50-70% of the relative
activity)
iii. RNA Polymerase II

- requires a transcription factor complex (TFIID) that binds to a single upstream
promoter
- located in the nucleoplasm
- represents 20-40 % of cellular activity
- responsible for the synthesis of heterogeneous nuclear RNA (hnRNA) which
is the precursor of mRNA
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iv. RNA Polymerase II Promoters
iva. TATA or Hogness Box
- a consensus sequence (nearly the same in all organisms)
- highly conserved in evolution
- about 4-8 base pairs
- usually followed by three or more adenine bases
- sequence of DNA nucleotides that is almost identical to the pribnow
box
- binding site of RNA polymerase II
ivai. Location
- 20-30 (25-35) bases upstream of the transcriptional start site
ivaii. 5’-TATAAA-3’
- core DNA sequence
ivaiii. TATA Binding Protein
- normally bind TATA box in the process of transcription
- unwinds the DNA and bends it through 80o
ivaiv. AT-Rich Sequence
- facilitates easy unwinding (due to 2 hydrogen bonds
between bases as opposed to 3 between GC pairs)
ivb. CAAT Box and CG Box
- 2nd consensus sequence
- distinct pattern of nucleotides with a GGNCAATCT sequence
- occurs 50-130 (40-200) bases upstream of the initial transcription
site
- signals the binding site for the RNA transcription factor
- typically accompanied by a conserved sequence
- genes that have this element seem to require it for transcription in
sufficient quantities
ivc. Cis-Acting Genetic Elements

- term for the promoter DNA sequences that are on the same DNA
molecule as the genes being transcribed
- binding site for general transcription factors
ivd. Trans-Acting Genetic Elements
- transcription factors encoded by a gene on different chromosome
- synthesized in the cytosol
- can diffuse through the cell to points of action including to different
chromosomes
- stimulate or inhibit transcription of particular genes
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v. RNA Polymerase III
- uses either upstream promoters or two internal promoters downstream of
the transcription start site
- responsible for the synthesis of
- tRNAs
- other small RNAs
- contributes only minor activity of about 10%
va. 3 Transcription Factors Required with Internal Promoters
vai. TFIIIA
- a zinc finger protein
vaii. TFIIIB
- a TBP and two other proteins
vaiii. TFIIIC
- a large protein
- > 500 kD
vi. RNA Polymerase III Promoters

- downstream of the start point
via. 5S rRNA Promoter
- made of 2 sequence elements in the transcribed portion of the gene
- sequence 50-70 base pairs downstream of the start point
- sequence 80-90 base pairs downstream of the start point
vib. tRNA Promoters

- made of 2 sequence elements
- sequence between +8 and +30
- sequence between +50 and +70
- downstream of the start point
- within the transcribed portion of the gene
B. Initiation
- opening of DNA double helix  initiation complex makes the template strand available for base pairing
- begins with synthesis of the first RNA molecules at the initiation complex
- the RNA polymerase remains at the promoter while it synthesizes the first nine nucleotide bonds
- requires several other proteins (activators and transcription factors)
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1. Initiation Factors
- needed to initiate transcription
i. Prokaryotes
- only a single factor is needed which is sigma () factor
ii. Eukaryotes
- multiple factors are required
a. Prokaryotic  Factor
- required for accurate initiation of transcription
i. Function
- enables the RNA polymerase holoenzyme to recognize and bind tightly to
the promoter sequences
ii. Process
iia. Upon binding  opening or melting of DNA double helix
iib. Enzyme  phosphodiester bond formation between the 1st 2 bases

- 1st base is usually a purine nucleoside triphosphate - pppA or
- pppG
iic. Elongation  10 nucleotides added   factor dissociates  core
enzyme continues to elongate the transcript
iid. Released  factor  combine with free core enzyme  holoenzyme
formation  able to initiate transcription
b. Assembly of Eukaryotic General Transcription (Initiation) Factors
- the general transcription factors (interact with each other and with RNA polymerase
II) and RNA Polymerase II are needed to initiate transcription from TATA
box promoters
i. Transcription Factor IID (TFIID)
- transcription factor D for polymerase II
- recognizes and binds to the TATA box sequences independently of RNA
polymerase II
ia. TATA-Binding Protein (TBP)
- small
- 30-kD
- recognizes TATA Box
- part of one of the many subunits of TFIID
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ii. Transcription Factor IIA (TFIIA)
- associates with TFIID
- binds to TATA box upstream of TFIID
iii. Transcription Factor IIB (TFIIB)
- binds to RNA polymerase II and TATA box downstream of TFIID
- facilitates binding of RNA polymerase II to TFIID and TFIIA complex to
the TATA box
- is an ATPase
- cleaves ATP upon formation of the preinitiation complex of RNA
polymerase II, TFIID, TFIIA and TFIIB
iv. Transcription Factor IIE (TFIIE)
- binds to the preinitiation complex
- transcription begins at the start point in the presence of ribonucleoside
triphosphates
iv. TFIIH
- escorted by TFIIF
- ensure that Pol II is attached to the promoter
- other activities
- helicase
- ATPase
v. Polymerase II
- activated by phosphorylation  transcription begins
va. Polypeptide Tail
- site of phosphorylation
- composed in mammals of 52 repeats of the amino acid sequence
YSPTSPS
- serine (S) and threonine (T) side chains are phosphorylated
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c. Role of Enhancers in Eukaryotic Gene Regulation

i. Enhancers
- special cis-acting DNA sequences
ia. Function
- increase the rate of initiation of transcription by RNA polymerase
II when bound by transcription factors
ib. Location
- on the same chromosome as the gene whose transcription they
stimulate
ibi. “Upstream” (to the 5′-side) or “downstream” (to the 3′-side) of

the transcription start site
ibii. Close to or thousands of base pairs away from the promoter
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ibiii. On either strand of the DNA

ic. Response Elements
- DNA sequences contained by enhancers
- bind specific transcription factors (activators)
ii. Silencers
- act over long distances
- reduce the level of gene expression when bound
d. Inhibitors of RNA Polymerase II
i. α-Amanitin
- enzyme
- potent toxin
- produced by the poisonous mushroom Amanita phalloides (“death cap” or
“destroying angel”)
- forms a tight complex with the polymerase  inhibition of mRNA synthesis
and protein synthesis
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e. RNA Polymerase I and III
- also need specific transcription factors to initiate transcription from their respective
promoters
C. Elongation
- promoter region recognition by holoenzyme  synthesis of DNA sequence transcript by RNA
polymerase with release of  subunit
- begins when the enzyme moves along the DNA  extend the RNA chain
- as the enzyme moves, it unwinds the DNA double helix
- DNA helicases aid in the process
- the DNA that has been transcribed rewinds into the double helix behind the polymerase
1. RNA Polymerase
- does not require a primer
- no known endo- or exonuclease activities  no ability to proofread or repair mistakes in the
RNA
- utilizes ribonucleoside triphosphates
- release of pyrophosphate each time a nucleotide is added to the growing chain
- binding with the DNA template  DNA helix unwinding
- as it pushes its way between the strands of the double helix  creates:
- positive supercoils ahead of the transcription site
- will be relaxed by gyrase
- negative supercoils behind it
- will be relaxed by topoisomerases I and II
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2. Requirements
- same in eukaryotes and prokaryotes
3. Template
- single strand of DNA acts as template to direct the formation of complementary RNA during
transcription
4. Substrate
- 4 ribonucleotide triphosphates
- adenosine triphosphate (ATP)
- guanosine triphosphate (GTP)
- cytidine triphosphate (CTP)
- uridine triphosphate (UTP)
- cleavage of the high-energy phosphate bond between the  and  phosphates provides the
energy for the addition of nucleotides to the growing RNA chain
5. Direction of Synthesis
- except for the 1st nucleoside triphosphate
- subsequent nucleotides are added to the 3’-hydroxyl of the preceding nucleotide (5’  3’
direction)
6. Enzyme
a. Properties of Prokaryotic RNA Polymerase
- 1 species of RNA polymerase responsible for all cellular RNA synthesis
- except for the short RNA primers needed for DNA replication
- multisubunit enzyme
- recognize and binds to nucleotide sequences (promoter region) at the beginning of the
length of DNA to be transcribed
- makes complementary RNA copy of the DNA template
- recognize the end of the DNA sequence to be transcribed (termination region)
- RNA is synthesized from its 5’-end to its 3’-end (antiparallel to its DNA template)
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ai. Transcription Unit

- extends from the promoter to the termination region
aii. Primary Transcript

- product of the process of transcription by RNA polymerase
i. RNA Polymerase Holoenzyme in E. coli
- required for proper initiation of transcription
- binds DNA relatively weakly except at specific promoter sequences
recognized by the σ subunit
ia. Components
iai. Core Enzyme
- required for the elongation steps of RNA synthesis
- peptide subunits
-2 - 40,000 MW
-1 - 155,000 MW
- 1 ’ - 160,000 MW
- responsible for the 5’  3’ RNA polymerase activity
- lacks specificity (cannot recognize the promoter region of
the DNA template)
- binds DNA tightly
- half-life of ~60 min
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ibi.  Subunit (Sigma Factor)
- 85,000 MW
- enables the polymerase to recognize the promoter region of
the DNA template
- different  factors recognize different groups of genes
- dissociates from the complex on the initiation of
transcription, leaving the core enzyme to continue
transcription
ii. Termination Factor

- some DNA regions that signal termination of transcription are recognized by
the RNA polymerase itself
- others are recognized by specific termination factors
- ex: rho () factor of E. coli
b. Eukaryotic RNA Polymerases
i. Mitochondrial RNA Polymerase
- single specie only
- resembles bacterial polymerase more closely than it does the eukaryotic
enzyme
ii. Nuclear RNA Polymerases
- distinct enzymes that function to synthesize different RNAs
iia. Structure
- large enzymes >500,000 MW
- each has very complex subunit composition
- with >10 subunit polypeptides
iib. Location
- subnuclear localization
iii. 3 Classes of Eukaryotic Nuclear RNA Polymerases
- large enzymes with multiple subunits
- each unit recognize particular types of genes
iiia. RNA Polymerase I
- located in the nucleolus
- synthesizes the precursor of large ribosomal RNAs in the
nucleolus
- 28S
- 18S
- 5.8S
- mRNA and tRNA
- synthesized in the nucleoplasm
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iiib. RNA Polymerase II
- synthesizes
- precursors of mRNAs
- small nuclear RNA (snRNA)
- used by some viruses to produce viral RNA
- general transcription factors
- required for simple basal transcription of most class II genes
a. Promoters for Class II Genes
i. TATA or Hogness Box
ii. CAAT Box
- may serve as recognition sites for eukaryotic promoters
b. RNA Polymerase II Inhibitors
i. -Amanitin
- bicyclic octapeptide
- potent toxin by mushroom Amanita phalloides
(called death cap or destroying angel)
- forms a tight complex with the polymerase 
mRNA synthesis inhibition  protein
synthesis inhibition
iiic. RNA Polymerase III
- produces the small RNAs
- tRNAs
- 5S rRNA
- some snRNAs
.
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D. Termination
- RNA polymerase is removed from the DNA
- the formation of the unstable primary transcript is complete
- immediately translated in prokaryotes
- modified (processed) in eukaryotes
1. Prokaryotes
a. -Independent Termination
- particular sequences can cause the core enzyme to terminate transcription
- required structural features of the newly synthesized RNA molecule
- requires that a sequence in the DNA template generate a sequence in the nascent RNA
that is self-complementary
- RNA polymerase halts when it encounters a hairpin-forming GC-rich region that is

followed by a series of U residues on the DNA sense strand
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i. Stable Hairpin Turn
- slows down the progress of RNA polymerase and pauses
temporarily
- complementary to a region of the DNA template near the termination
region that exhibits twofold symmetry (due to presence of a
palindrome)
ia. Palindrome
- region of double-stranded DNA
- each of the 2 strands contain stretches that have the same
nucleotide sequence when read in the same direction
ib. Guanosine-Cytosine (G-C)-Rich Stem-Loop Base

- important in the termination of transcription
- very stable G-C base pairs
- makes the formation of a stem-loop structure
favourable or more stable
ii. String of Us
- following the hairpin turn
- bonding of U to the corresponding DNA template A is weak 
facilitates separation of the newly synthesized RNA from the
DNA template
- termination becomes favourable when the RNA polymerase pauses

- no specific base where transcription stops
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b. -Dependent Termination
i.  Factor
- hexameric ATPase
- binds to a C-rich “rho recognition site” near the 3’-end of the nascent RNA
 migrates until it reaches the RNA polymerase paused at the
termination site in the 5’3’ direction  ATP-dependent RNA-DNA
helicase activity of rho separates the RNA-DNA hybrid helix 
RNA release
2. Eukaryotes
- few identified termination sequences
- transcription continues for up to several thousand base pairs beyond the point of polyadenylation
E. Transcription Units
- segment of DNA between the sites of initiation and termination of transcription
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F. Determining Transcription Start Sites

1. Nuclease S1 Protection Assay
- used to analyze a segment of DNA suspected to contain a transcription start site
2. Endonuclease S1
- enzyme present in the mold Aspergillus oryzae
- cleaves single-stranded DNA and RNA but not double-stranded
- all single-stranded DNA will be removed  allow relevant DNA to be identified
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G. Footprinting
- technique used to identify the promoter region
- RNA polymerase and DNA (containing the putative promoter)  incubated with alkylating reagent at
DNA bases that are not protected by contact with the RNA polymerase  cleavage of the DNA
backbone at the alkylated residues  products analyzed by electrophoresis to determine the
region of DNA that binds to RNA polymerase
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H. Action of Antibiotics
- some antibiotics prevent cell growth by RNA synthesis inhibition
1. Rifampin
- rifampicin derivative
- bind to the -subunit of the prokaryotic RNA polymerase (when the polymerase is in the
holoenzyme form)  interference with the formation of the 1st phosphodiester bond
 inhibition of the initiation of transcription
- no effect on eukaryotic nuclear RNA polymerases
- treatment of tuberculosis
2. Dactinomycin (Actinomycin D)
- binds to DNA template  interferes with RNA polymerase movement along the DNA
- tumor chemotherapy
POST-TRANSCRIPTIONAL RNA MODIFICATION

1. Primary Transcript or Heterogeneous Nuclear RNA (hnRNA)
- linear copy of a transcriptional unit
- RNA immediately produced by RNA polymerase (before it undergoes modification)
2. Transcriptional Unit
- DNA segment between specific initiation and termination sequences
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3. Primary Transcripts of tRNAs and rRNAs

- post-transcriptionally modified by cleavage of the original transcripts by ribonucleases
4. Prokaryotic mRNA
- generally identical to its primary transcript (no modification)
5. Eukaryotic mRNA
- extensively modified post-transcriptionally
A. Prokaryotes
1. Messenger RNA (mRNA)
- not post-transcriptionally processed
- functional immediately after synthesis
- translation often begins before transcription is complete
2. Ribosomal RNA (rRNA)
a. Preribosomal RNAs
- long precursor molecules of ribosomal RNAs
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b. 7 Genes Produce rRNA
- each gene produces 30S precursor rRNA which is processed into functional rRNA
- contain sequences that become
- 23S rRNA
- 16S rRNA
- 5S rRNA
- within the transcribed portion are some of the tRNA genes
- different rRNA genes contain different tRNA genes
c. Cleavage
- by - ribonuclease P
- ribonuclease III
- non-functional spacer sequences are removed
d. Base Modification
- by methylation  functional rRNAs
3. Transfer RNA (tRNA)
a. Large Precursor Transcripts
- give rise to tRNAs not formed from processing
b. tRNA Genes
- clustered
- contain sequences for 2-7 tRNAs
c. Cleavage
- by - ribonuclease P
- ribonuclease D
- removal of the portions of the transcript that form functional tRNAs
d. Addition of Sequence -CCA to the 3’-End
- by tRNA nucleotidyl transferase
- CCA sequence is common to all tRNAs
e. Base Modification
-by - methylation
- more extensive modifications
- production of unusual bases
- necessary for the tRNAs to adopt their unique, functional conformations
B. Eukaryotes
- RNAs are processed during their transport from the nucleus to cytosol
- needed to be functional in the cytoplasm
- allows for another level of gene regulation
1. Ribosomal RNA (rRNA)
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a. Preribosomal (45S) RNAs

- precursor molecule
- no tRNA sequences
- made by the transcription of hundreds of separate rRNA genes
- in the nucleolus
- by RNA polymerase I
- highly methylated before it is cleaved to the functional rRNAs
- spacer sequences are removed by endonucleolytic cleavage by specific endonucleases
- 5.8S rRNA base pairs with the 28S rRNA during formation of the ribosomal subunits
i. Yields - 28S rRNA

- 18S
- 5.8S
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b. 5S rRNA
- synthesized by the transcription of 5S gene by RNA polymerase III
- modified separately
2. Messenger RNA (mRNA)
- formed from extensive processing of hnRNA
a. Heterogeneous Nuclear RNA (hnRNA)
- primary RNA transcript molecule synthesized by RNA polymerase II
- contains the sequences that are found in cytosolic mRNA
- extensively modified after transcription which may include
i. 5’ “Capping”
- 1st of the processing reactions for hnRNA
- begins during transcription or immediately thereafter
- all mRNAs are capped
ia. Cap - 7-methylguanosine
- attached backward through a triphosphate linkage to the 5’-
terminal of mRNA  unusual 5’5’ triphosphate
linkage
- addition of guanosine triphosphate cap
- catalyzed by nuclear enzyme guanylyltransferase
- methylation of terminal guanine
- occurs in the cytosol
- catalyzed by guanine-7-methyl-transferase
- S-adenosylmethionine as the methyl donor
- additional methylation steps may occur
ib. Addition of 7-Methylguanosine Cap
- facilitate more efficient initiation of translation
- helps to stabilize mRNA
- protection from digestion by ribonucleases that
degrade RNAs from their 5’-end
(5’-exonucleases)
- lack of cap  inefficient translation
ic. 3 Possible Cap Structures
- depending on the presence or absence of additional methyl
groups on the 2 nucleosides adjacent to the
7-methylguanylate
ici. Cap 0
- when there is no additional methylation beyond the
methylated guanosine
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icii. Cap 1
- when the 1st ribose sugar adjacent to the
7-methylguanylate is methylated
iciii. Cap 2
- when the 1st and 2nd ribose sugars adjacent to the
7-methylguanylate are methylated
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b. Poly-A Tail Addition (Polyadenylation)
- necessary for all hnRNAs to be successfully converted to mRNAs
- chain of 200-300 adenine nucleotides attached to the 3’-end
- not transcribed from the DNA (template independent)
- added after transcription by nuclear polyadenylate polymerase
- help stabilize mRNAs
- protects the mature mRNA from ribonuclease activity in cells
- facilitate their exit from the nucleus
- gradually shortened after entering the cytosol
- endonuclease cuts the molecule on the 3′ side of the sequence
AAUAAA then poly-A polymerase adds the poly-A tail
i. Polyadenylation Signal Sequence (AAUAAA)

- consensus sequence
- near the 3’-end of the RNA molecule
- signals that a poly-A tail is to be added to the mRNA
ii. Signal That Identifies the Site of Polyadenylation
- lies within the RNA
iii. Poly-A Polymerase
- nuclear enzyme
- catalyzes the polymerization of adenylate residues onto the
free 3’-end of the mRNA
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iv. Occurs
- after capping
- before splicing
c. Splicing
- process of removing non-coding sequences or introns
- all sequences necessary to form mRNA that codes for a protein
product are contained in the hnRNA
- coding sequences are often split or separated by non-coding
sequences
i. Introns
- intervening RNA sequences between exons
- do not code for proteins from the primary transcript
- transcribed portions of the genes that are removed in the
processing of hnRNA to mRNA
- different genes have different number of introns of different
sizes
ia. -Globin Genes
- 2 introns
ib. LDL Receptor Gene
- 17 introns
ii. Exons
- protein-coding sequence
- expressed portions of the genes
- spliced together forming the mature mRNA
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iii. Intron-Exon Junctions
- all hnRNA introns have
iiia. Guanine-Uracil (G-U) Sequence
- on the 5’ border
iiib. Adenine-Guanine (A-G) Sequence
- on the 3’ border
- G-U and A-G sequences are flanked by sequences that are
identical to or similar in all introns
- branch site
- within introns
- conserved sequence
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iv. Mechanism of Splicing
- catalyzed by spliceosome
iva. Spliceosome
- large (50S to 60S) ribonucleoprotein complex
- made of 5 snRNPs that contain 5 snRNAs
- U1
- U2
- U4
- U5
- U6
- binding of snRNPs  bring together RNA sequences into
perfect alignment for splicing
- 2’-hydroxyl group of an adenosine residue (branch site) in
the intron
- attacks and forms a phosphodiester bond with the
phosphate at the 5’-end of the intron
- newly-freed 3’-OH of the upstream exon forms
phosphodiester bond with the 5’-end of the
downstream exon
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v. Role of Small Nuclear RNAs (snRNAs)

- in association with proteins forming small nuclear
ribonucleoprotein particles (snRNPs)
- for recognition of the conserved sequences in the introns
- facilitate the splicing of some exon segments by forming
base pairs with the consensus sequences at each end
of the intron
vi. Systemic Lupus Erythematosus
- often fatal inflammatory disease
- results from autoimmune response in which the patient
produces antibodies against snRNPs
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vii. Effect of Splice Site Mutations
- lead to improper splicing  production of aberrant
proteins
- 15% of all genetic diseases are results of mutations that
affect RNA splicing
- some cases of  thalassemia
- incorrect splicing of -globin mRNA
viii. Regulation of Gene Expression at the Level of Transcription
viiia. Promoters
- critical for initiation of transcription
- mutations may decrease the quantity of gene
transcribed
viiib. Enhancer
viiic. Silencer
ix. Transport
- after intron removal  mature mRNA molecule  pores in
the nuclear membrane  cytosol
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x. Alternative Splicing of mRNA Molecule

- important mechanism for generating multiple protein
isoforms from a single gene (multiple variations of
mRNA and its protein products in different tissues)
- resulting proteins differ slightly in their amino acid
sequence  small functional differences
(often these differences are restricted to
certain tissues)
- allows for a high degree of functional flexibility 
evolutionary advantage
- ex: different types of muscle cells
- produce the same primary transcript from
tropomyosin gene
- different patterns of splicing in the different cell
types  production of tissue-specific
tropomyosin protein molecules
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xa. Calcitonin Gene

- the primary transcript for the calcitonin gene
contains six exons  splicing  two types
of mature mRNA
xai. Calcitonin
- consisting of exons 1-4 (excluding exons 5
and 6)
- produced in the thyroid
xaii. Calcitonin-Like Protein (Calcitonin Gene-
Related Product, CGRP)
- in the hypothalamus
- consisting of exons 1, 2, 3, 5, and 6 and
excluding exon 4
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3. Transfer RNA (tRNA)

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a. Large Precursor RNAs
- may contain 1 or more tRNA sequences
i. tRNA Sequences
- excised from the precursor
- by specific endonucleolytic cleavage
ii. CCA Sequence

- added to the 3’-end after the tRNA is cleaved from the precursor
iii. Extensive Modification

 adopt final functional structure
b. Introns
- may be contained by some tRNAs
- small (14-50 nucleotides)
- no sequence homology in different tRNAs
- splicing
- different from mRNA
- enzymes recognize characteristic features of the tRNA to identify the intron-
exon junctions
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SUMMARY
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MOLECULAR BIOLOGY:
PROTEIN SYNTHESIS
INTRODUCTION
A. Genetic Information
- stored in the chromosomes
- transmitted to daughter cells through DNA replication
- expressed through transcription to RNA
- translation into polypeptide chains
- any alteration in the nucleic acid sequence  improper amino acid inserted into the polypeptide chain
 disease or death of the organism
B. Translation
- process by which ribosomes convert the information carried by mRNA to the synthesis of new
proteins
- requires the cleavage of “high-energy” phosphoanhydride bonds  endergonic
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RIBOSOMES and PROTEIN ASSEMBLY
1. Ribosomes
- large ribonucleoprotein particles
- coordinate the interaction of mRNA and tRNAs during protein synthesis
- products of individual genes (ribosomal genes)
2. Proteome
- all proteins produced by a cell at any given time
A. Structure and Components of Ribosomes
1. Ribosomes
- made of a small and a large subunit
2. Prokaryotic Ribosomes
- a bacterial cell contains about 20000 ribosomes (about 25% of its mass)
a. Contain
- three different types of rRNA molecules
- up to 83 proteins
b. Escherichia coli Ribosome
- sedimentation coefficient of 70 S
- approximately
- 65% RNA
- 35% protein
Sedimentation Coefficient
- measure of the rate of sedimentation in an ultracentrifuge of a
molecule suspended in a less dense solvent
- measured in Svedberg units (S)
- S values are not additive
Dalton - unit of atomic or molecular mass
c. Prokaryote 70 S Ribosome
- molecular weight of 2.5 million daltons (MDa)
- can be dissociated into
i. 30 S - smaller subunit
- contains
- large 16 S rRNA
- 21 proteins
- site where genetic information is decoded
- has a proofreading mechanism
ii. 50 S - larger subunit
- consists of
- smaller rRNAs (5 S) with 120 ribonucleotides
- larger rRNAs (23 S) with ~2900 ribonucleotides
- 33-35 proteins
- provides peptidyltransferase activity
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3. Eukaryotic Ribosome
- much larger (80 S, 4.2 MDa)
- consists of
a. 40 S Subunit
- contains
- 18 S rRNAs (1900 bases)
- 33 proteins
b. 60 S Subunit
- contains
- 5.8 S rRNAs (160 bases)
- 50 proteins
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4. Mitochondria and Chloroplasts
- have their own ribosomes
- resemble those of prokaryotes
- structure
- sensitivity to antibiotic inhibitors of translation
B. From Gene to Protein
1. Cell Nucleus
- directs the production of endogenous proteins (protein synthesis)
2. Nuclear RNA
- bound to nuclear RNA-binding proteins for stabilization
3. Mature RNA
- released from the nucleus into the cytoplasm
- associates with ribosomes
C. Nucleolus and Ribosomes

1. Nucleolus
- morphologically and functionally specific region in the cell nucleus
- where ribosomes are synthesized
a. In Humans
- rRNA genes (200 copies per haploid genome)  transcribed by RNA polymerase I 
formation of 45 S rRNA precursors  packaged with ribosomal proteins (from
the cytoplasm)  cleavage  three of the four rRNA subunits  transfer
from the nucleus and released into cytoplasm with the separately synthesized 5 S
subunits  formation of functional ribosomes
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2. Small RNAs
a. Small Nuclear RNAs (snRNA)
- family of RNA molecules
- bind specifically with small number of nuclear ribonucleoprotein particles (snRNP,
pronounced “snurps”)
- important roles in the modification of RNA molecules after transcription
(posttranscriptional modification)
- base-pair with pre-mRNA and with each other during RNA splicing
b. Small Nucleolar RNAs (snoRNA)
- assist in
- processing of pre-rRNAs
- assembly of ribosomes
D. Medical Relevance
- a variety of chemical compounds (naturally as poisons or synthetic products) are used for cancer therapy
by inhibition of transcription or translation
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The GENETIC CODE
- system of RNA sequences that designate particular amino acids in the process of translation
A. Codons
- genetic words
- composed of 3 nucleotide bases
- usually presented in the mRNA language of
- adenine (A)
- guanine (G)
- cytosine (C)
- uracil (U)
- nucleotide sequences are written from the 5’-end to the 3’-end
- 4 nucleotide bases
- used to produce the 3-base codons  64 different combinations
1. How to Translate a Codon
2. Termination (Stop or Nonsense) Codons

- UAG
- UGA
- UAA
- do not code for amino acids
- signals that synthesis of the peptide chain coded for by that mRNA is completed (termination of
translation)
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3. Consequences of Altering the Nucleotide Sequence
- changing a single nucleotide base on the mRNA chain (point mutation)  1 of the following
results
a. Silent Mutation
- the new codon may code for the same amino acid
b. Missense Mutation
- the new codon may code for a different amino acid
c. Nonsense Mutation
- the new codon may become a termination codon
d. Other Mutations
- can alter the amount or structure of the protein produced by translation
i. Trinucleotide Repeat Expansion
ii. Splice Site Mutations
iii. Frame-Shift Mutations
4. Nonrandom Arrangement of Codons
a. Most Synonyms
- share the first two nucleotides
b. Codons with Different Nucleotides in the 1st Position
- tend to specify chemically similar amino acids
c. Codons with 2nd Position Pyrimidines
- encode mostly hydrophobic amino acids
d. Codons with 2nd Position Purines
- encode mostly polar amino acids
 nonrandom evolution of the genetic code  minimizes the deleterious effects of point
mutations
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B. Characteristics of the Genetic Code
1. Specificity
- specific (unambiguous)
- a specific codon always codes for the same amino acid
2. Near Universality
- the specificity of the genetic code has been conserved from very early stages of evolution
- only slight differences in the manner the code is translated
- exceptions in the universality of the genetic code are found in human mitochondria
- UGA - designates tryptophan
- AUA - codes for methionine
- additional stop codons
- AGA
- AGG
3. Redundant/Degenerate Code
- more than 1 codon can specify a single amino acid
- all amino acids, except methionine and tryptophan, have more than one codon
a. Synonyms
- codons that designate the same amino acid
- synonymous codons usually differ only in the 3rd base of the codon
b. Amino Acids Having More Than One Codon
- first 2 bases in the codon are usually the same
- base in the third position often varies
4. Unambiguous Codons
- each codon specifies no more than one amino acid
5. Nonoverlapping and Commaless
- the code is read from a fixed starting point as a continuous sequence of bases, taken 3 at a time,
and without spacer bases
- if 1 or 2 nucleotides are deleted or inserted to the interior of a message sequence  frameshift
mutation  reading frame is altered
- if 3 nucleotides are added  new amino acid is added to the polypeptide (reading frame is not
affected)
- if 3 nucleotides are deleted  an amino acid is lost (reading frame is not affected)
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C. Colinearity of Gene and Product

- the product of the gene is the peptide specified by the sequence of the expressed regions of the genes
D. Overlapping Genes
- some viruses code for more proteins than would be predicted from their nucleotide content
- some of the expressed portions of the viral genome overlap and code for multiple products in
different reading frames
COMPONENTS REQUIRED for TRANSLATION

A. Amino Acids
- all amino acids must be present
- amino acid deficiency or absence  translation stops at the codon specifying that amino acid
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B. tRNA
- 1 specific type of tRNA is required per amino acid
- humans
- at least 50 species of tRNA
- bacteria
- 30-40 species
- only 20 amino acids  some amino acids have more than 1 specific tRNA molecule
- called adaptor molecules
- carry a specific amino acid
- recognize the codon for that amino acid
1. Amino Acid Attachment Site
- at the 3’-end of the tRNA
- carboxyl group of the amino acid is in an ester linkage with the 3’-hydroxyl of the ribose moiety
of the adenosine nucleotide in the -CCA sequence at the 3’-end of the tRNA
- when a tRNA has a covalently attached amino acid  said to be “charged”
- when a tRNA is not bound to an amino acid  said to be “uncharged”
- the amino acid that is attached to a tRNA  said to be “activated”
2. Anticodon
- 3-base nucleotide sequence of the tRNA molecule
- recognizes a specific codon on the mRNA which specifies the insertion into the growing
peptide chain of the amino acid carried by the tRNA
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C. mRNA
- template for the synthesis of the desired polypeptide chain
D. Aminoacyl-tRNA Synthetases
E. Functionally Competent Ribosomes
a. Ribosomes
- large complexes of
- protein
- rRNA
- consist of 2 subunits
- large
- small
b. Prokaryotic and Eukaryotic Ribosomes
- similar in structure
- serve the same function (factories for protein synthesis)
c. Ribosomal Subunits
i. Large Ribosomal Subunit
- catalyzes formation of the peptide bonds that link amino acid residues in a
protein
ii. Small Ribosomal Subunit
- binds mRNA
- responsible for the accuracy of translation by ensuring correct base-pairing
between the codon in the mRNA and the anticodon of the tRNA
1. rRNA
- have extensive regions of secondary structure arising from the base pairing of
complementary sequences of nucleotides of different portions of the molecule
2. Ribosomal Proteins
- greater amount in eukaryotic than prokaryotic ribosomes
- role in the structure and function of the ribosomes and its interactions with other components
of the translation system
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3. A, P, and E Sites on the Ribosomes
- binding sites for tRNA molecules
- together, they cover 2 neighboring codons
a. Acceptor (A) Site
- binds an incoming aminoacyl-tRNA
b. Peptidyl (P) Site
- occupied by peptidyl-tRNA
- tRNA carries the chain of amino acids that has already been synthesized
c. Exit (E) Site
- occupied by the empty tRNA as it is about to exit the ribosome
4. Cellular Location of Ribosomes

a. Eukaryotic Cells
- free in the cytosol or in close association with the endoplasmic reticulum (rough
endoplasmic reticulum)
i. Rough Endoplasmic Reticulum-Associated Ribosomes
- responsible for synthesizing proteins that are to be:
- exported from the cell
- integrated into
- plasma membrane
- endoplasmic reticulum membrane
- Golgi membrane
- incorporated into lysosomes
ii. Mitochondria
- have their own set of ribosomes
F. Protein Factors
- initiation factors
- elongation factors
- termination or release factors
- required for peptide synthesis
- functions
- catalytic
- stabilize synthetic machinery
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G. ATP and GTP as Energy Sources
- cleavage of 4 high energy phosphate bonds for the addition of 1 amino acid to the growing polypeptide
chain
- 2 from ATP in the aminoacyl-tRNA synthetase reaction
- 1 in PPi removal
- 1 in the subsequent PPi hydrolysis to inorganic phosphate by pyrophosphatase
- 2 from GTPs
- 1 for binding the aminoacyl-tRNA to the A site
- 1 for the translocation step
- additional ATP and GTP molecules required for initiation in eukaryotes
- additional GTP molecule required for termination in both eukaryotes and prokaryotes
AMINO ACID ACTIVATION: FORMATION of AMINOACYL-tRNAs

A. Adaptor Molecules
- aminoacylated tRNAs (charged tRNAs)
- link between the message to protein
- tRNAs to which amino acid has been covalently attached
B. Aminoacyl-tRNA Synthetases
- required for attachment of amino acids to their corresponding tRNAs
- implement the genetic code
- responsible for the activation and attachment of the amino acids to the 3’-terminal adenosine of their
corresponding tRNAs
1. Morphology
- vary in - molecular weight
- number of subunits
- amino acid composition
2. Specificity
- very specific in their attachment of the correct amino acid to the correct tRNA
- codon recognition is entirely due to the tRNA  fidelity of protein synthesis depends largely
on the high specificity of tRNA synthetases
a. Selectivity
- the very high specificity of tRNA synthetases
- due to the high selectivity of the enzyme for the
- amino acid to be activated
- tRNA to which the amino acid is to be attached
b. Proofreading and Editing Capability
- if - incorrectly activated the wrong amino acid
- adds the wrong amino acid to the tRNA
 deactivate the amino acid by hydrolysis
hydrolyze the amino acid from the tRNA
3. Classes
- two structurally unrelated classes of aminoacyl-tRNA synthetases
i. Class I
ii. Class II
a. Differences
- mechanism by which they recognize their tRNA substrates
- initial site of aminoacylation on the tRNA
- amino acid specificity
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4. Aminoacylation Reaction
- attachment of an amino acid to its proper tRNA
- catalyzed by tRNA synthetases
a. Aminoacyl-Adenylate Complex Formation
b. Aminoacyl Group Transfer to the 2’ or 3’-Hydroxyl Group of the 3’-Adenosine
Aminoacyl-AMP + tRNA  Aminoacyl-tRNA + AMP
c. Sum of the Above Reactions

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d. Pyrophosphate (PPi) Hydrolysis
- forms 2 free phosphates
- makes the overall reaction irreversible
- 2 high-energy phosphate bonds are expended in the formation of a single
aminoacyl-tRNA
CODON RECOGNITION by tRNA (CODON-ANTICODON RECOGNITION)

- recognition of a particular codon in mRNA is accomplished by the anticodon sequence of tRNA
- some tRNAs recognize >1 codon for a given amino acid
A. Antiparallel Binding Between Codon and Anticodon
1. Rules - complementarity
- antiparallelism
2. Codon-Anticodon Pairing
- mRNA codon is read 5’  3’ by an anticodon (of tRNA) pairing in the flipped (3’  5’)
orientation
3. Base Pairings
- 1st 2 bases of the codon and the last 2 bases of the anticodon
- by normal base pairing
- guanine-cytosine (G-C)
- adenine-uridine (A-U)
- last base of the codon with the 1st base of the anticodon
- follows less rigid requirements
- allows some tRNAs to base pair with >1 codon
4. Writing the Sequences of Both Codons and Anticodons
- the nucleotide sequence must always be listed in 5’  3’ order
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B. Wobble Hypothesis
- mechanism by which tRNAs can recognize >1 codon for a specific amino acid
- base at the 5’-end of the anticodon (1st base of the anticodon)
- not as spatially defined as the other 2 bases
- movement (“wobble”) of the 1st base
- allows non-traditional base pairing with the 3’-base of the codon (last base of the
codon)
- allows a single tRNA to recognize >1 codon
 wobbling  there need not be 61 tRNA species in order to read the 61 codons coding
for amino acids
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1. Rules Governing Base Pairing of the Codons 3rd Base Position
- 1st base in the anticodon is cytosine or adenine  pairing in the 3rd base of the codon is
normal
- 3rd base would be guanine or uracil respectively
- 1 base in the anticodon is uracil  3rd base of the codon can be either of the purines (adenine
st
or guanine)
- 1st base in the anticodon is guanine  3rd base of the codon can be either of the pyrimidines
(uracil or cytosine)
- 1st base in the anticodon is inosine (which is possible because tRNAs have many unusual bases)
 3rd base of the codon can be
- adenine
- cytosine
- uracil
PROTEIN SYNTHESIS in PROKARYOTES

- nucleotide sequence on the mRNA is translated into the language of amino acid sequence
- mRNA is translated from 5’-end to 3’-end  synthesis of proteins from the amino-terminal end to
the carboxyl-terminal end
- eukaryotic protein synthesis resembles that of prokaryotes in most details
A. Initiation
1. 30S Initiation Complex Formation
- 1st event in protein synthesis
a. Requirements
i. mRNA Strand
- to be translated
ii. Initiation Factors
- IF-1
- IF-2
- IF-3
- facilitate initiation complex assembly
iii. GTP - provides energy for the process
iv. 30S Ribosomal Subunit
iva. 70S Particle Dissociation
IF-3
70S  50S + 30S  30S IF-3 + 50S
IF-1
IF-3 - act as antiassociation factor

IF-1 - increases the forward rate of reaction
v. Formylmethionine-tRNAf (fmet-tRNAf)
va. tRNAf
- initiator tRNA
- brings modified methionine (fmet) to the 30S initiation complex
- different sequence than the tRNA (tRNAm) that inserts methionine in
internal positions of the peptide chain
vb. Aminoacyl-tRNA Synthetase
- links methionine to both
- tRNAf
- tRNAm
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vc. Transformylase
- adds formyl group from N10-formyltetrahydrofolate to the amino
group of methionine that is attached to the tRNAf  N-fmet-
tRNAf
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2. Steps in the 30S Initiation Complex Formation

a. 30S Subunit Binds to a Specific mRNA Site
- in association with
- IF-1
- IF-2
- IF-3
i. IF-3 - required for binding mRNA to the 30S subunit
ii. 2 mechanisms by which the ribosomes recognizes the nucleotide sequence that
initiates translation
iia. Shine-Dalgarno Sequence (in E. coli)
- purine-rich sequence
- identical with or similar to the sequence 5’-UAAGGAGGU-3’
- base pairs with a pyrimidine sequence in the 16S rRNA
- 16S ribosomal RNA component of the 30S ribosomal
subunit
- has a nucleotide sequence near its 3’-end that is
complementary to all or part of the Shine-
Dalgarno sequence
- located 6-10 bases upstream of the initiator (AUG or GUG) codon on
the mRNA molecule (near its 5’-end)
- places the initiator codon in the proper position in the 30S subunit to
bind to the initiator tRNA (fmet-tRNAf)
 the mRNA 5’-end and the 3’-end of the 16S ribosomal RNA can
form complementary base pairs  facilitated binding and
positioning of the mRNA on the 30S ribosomal subunit
- when fmet-tRNAf is the initiator codon
- pairs with GUG (even though GUG is normally a valine
codon)
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iib. Initiation Codon

iibi. AUG Codon
- at the beginning of the message
- recognized by a special initiator tRNA
- facilitated by IF-2 in E. coli
- goes to the P site
- all other charged tRNAs enter at the A site
iibii. In Bacteria and Mitochondria
- initiator tRNA carries an N-formylated methionine
- formyl group is added to methionine
- after the amino acid is attached to the
initiator tRNA
- by transformylase
- uses N10-tetrahydrofolate as the carbon
donor
- N-terminal methionine is usually removed before the protein
is completed
b. To Complete 30S Initiation Complex Formation

- fmet-tRNAf binds to the 30S particle
- IF-2 - in association with GTP
- proper binding of the initiator tRNA to the 30S particle
- IF-3 - dissociates from the 30S initiation complex upon binding of the fmet-tRNAf
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3. IF-3 Release
- allows the 50S subunit to bind to the 30S initiation complex to form the 70S initiation
complex
a. GTP Hydrolysis
 GDP + Pi upon formation of the 70S initiation complex
b. Released from the 70S Initiation Complex
- IF-1
- IF-2
- GDP
- inorganic phosphate (Pi)
c. 2 Sites of the 70S Ribosome that can be Occupied by Aminoacylated tRNAs
- peptide (P) site
- amino (A) site
d. P Site - occupied by fmet-tRNAf
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B. Elongation
1. Aminoacylated tRNA
- complementary to the codon adjacent to the initiator codon (ex: AUG)
- inserted in the A site  starting the process of elongation
a. Elongation Factors Required
- EF-Tu
- EF-Ts
- EF-G
- abundant proteins
- present in the cell at levels of 5-10% of all proteins
b. EF-Tu
- effects delivery of an aminoacyl-tRNA to the empty A site
c. GTP - bound to EF-Tu
- hydrolyzed
d. GDP - from GTP hydrolysis
- remains associated with EF-Tu until displaced by EF-Ts
e. EF-Tu and EF-Ts
- forms a complex that is split by the binding of another GTP  EF-Tu-GTP complex
formation for the delivery of the next aminoacyl-tRNA
f. EF-Tu-GTP
- delivers all aminoacyl-tRNA except fmet-tRNAf to the A site
2. Peptide Bond Formation
a. Amino Acid Transfer
- the activated amino acid attached to the tRNA in the P site, initially fmet-tRNAf, is
transferred to the amino group of the aminoacyl-tRNA in the A site
- addition of amino acids to the carboxyl end of the growing chain
b. Peptidyl Transferase
- catalyzes peptide bond formation
- activity intrinsic to the 23S rRNA (ribozymes) found in the 50S ribosomal subunit
c. Result of the Reaction
- 2 amino acids being attached to the tRNA (dipeptidyl-tRNA) in the A site
- leaves an uncharged tRNA in the P site
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3. Translocation
a. Ribosomal Movement
- moved 3 nucleotides in a 5’  3’ direction along the mRNA
- results in the following
i. Movement of the uncharged tRNA from the P site to the E site
ii. Movement of the dipeptidyl-tRNA to the P site
iii. Unoccupied A site
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b. EF-G - catalyzes translocation
- forms a complex with GTP during translocation
- GTP  GDP + Pi
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C. Termination
- occurs when 1 of the termination codons moves into the A site
1. Release Factors
- no tRNAs pair with stop codons
a. Stop Codons
- are recognized by release factors
- RF-1 or
- RF-2
i. RF-1 - recognizes the termination codons
- UAA
- UAG
ii. RF-2 - recognizes
- UGA
- UAA
iii. RF-3 - binds GTP
- stimulates the activities of RF-1 and RF-2  promoting termination
2. Binding of Release Factors
- induces peptidyl transferase to release the polypeptide from the tRNA in the P site by
hydrolysis
- tRNA is also released from the P site
3. Ribosomal Subunit Separation
- GTP-hydrolysis-dependent
a. 30S Subunit
- may move along the mRNA until another Shine-Dalgarno sequence is encountered 
resumption of translation
- may completely dissociate from the mRNA
D. Energy Requirements for Protein Synthesis
- each peptide bond formed requires 5 (7) high energy phosphate bonds
1. tRNA Aminoacylation
- ATP  AMP + 2 phosphates for every amino acid attached to its cognate tRNA
2. fmet-tRNAf Binding to the P Site
- GTP  GDP + phosphate upon initiation of every polypeptide synthesized
3. Aminoacyl-tRNA Binding to the A Site
- GTP  GMP + 2 phosphate for every aminoacyl-tRNA bound to the A site
4. Translocation
- GTP  GDP + phosphate for every translocation step
5. Termination
- GTP  GDP + phosphate for every polypeptide synthesis terminated
E. Role of High Energy Requirements During Translation
1. High Translation Fidelity
- protein synthesis in E. coli
- error frequency of <1 misincorporation per 2000 amino acids coupled
- high energy expenditure  low error rate
a. tRNA Aminoacylation Accuracy
- requires cleavage of 2 high energy phosphate bonds in ATP
b. Codon-Anticodon Recognition
- GTP hydrolysis is coupled to a proofreading function that “tests” a codon-anticodon
interaction for a mismatch
2. High Translation Rate
- protein synthesis is rapid  requires high energy input
a. E. coli
- a single ribosome can couple 15 amino acids/second
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F. Polysomes
- complex of
- 1 mRNA
- number of ribosomes
- because of the length of the mRNA, >1 ribosomes at a time translate the mRNA
PROTEIN SYNTHESIS in EUKARYOTES

- eukaryotic protein synthesis resembles that of prokaryotes in most details
- the differences are a consequence of the more complex cellular organization of the eukaryotic cell
A. Initiation
- same basic events occur
a. Requirements
i. mRNA Strand
- monocistronic
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- use only 1 initiator codon (AUG)

- do not use Shine-Dalgarno sequence to direct initiation to the initiator codon
- less rigidly defined sequences are known to be involved to direct
initiation to the initiator codon
- have 7-methylguanylate cap on their 5’-ends  role in the initiation of
translation
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ii. Initiation Factors
- at least 10 initiation factors (eIF)
iii. 40S Ribosomal Subunit

iiia. 60S Ribosomal Subunit
- bound by eIF-6  60S subunit prevented from binding to the 40S
subunit
iiib. 40S Ribosomal Subunit
- bound by eIF-4C  role in the association of the 2 ribosomal
subunits
iv. met-tRNAi
iva. Initiator tRNA
- carries unmodified methionine (not formylated) to the 40S initiation
complex
ivb. 2 Structurally Different tRNAs
- recognize AUG codons
ivbi. tRNAi
- recognizes the initiator codon
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ivbii. tRNAm
- recognizes the internal noninitiator methionine codons
ivc. Methionyl-tRNA Synthetase
- adds methionine residue to either
- tRNAi or
- tRNAm
v. Energy Requirements
- GTP
- ATP
2. Steps in the 40S Initiation Complex Formation
a. Preinitiation Complex Formation
Ternary Complex + 40S Subunit  Preinitiation Complex
Ternary Complex
- formed between
- eIF-2
- GTP
- met-tRNAi
b. mRNA Binding to the Preinitiation Complex of the Ribosome
- not entirely understood
- exact events
- functions of the initiation factors
i. Initiation factors
- all function during mRNA binding
- includes - eIF-3
- eIF-4A
- eIF-4B
- eIF-4C
- eIF-4F
ii. Eukaryotic Initiation Factor (eIF) 4A
- includes a subunit that acts as a cap-binding protein (CBP)
iia. CBP- may initiate mRNA binding by interacting with 5’-cap structure
- promotes binding of eIF-4A and eIF-4B to mRNA
iib. Importance of Cap
- cap analogue 7-methylguanosine monophosphate is a potent
initiation inhibitor
iii. ATP - hydrolyzed and bound by eIF-4A  unwinding of the secondary structure in
the 5’-untranslated region of the mRNA
iv. 40S Initiation Complex Formed
c. Initiation Codon Recognition
- 5’-untranslated region of the mRNA
- variable length
- between 40-80 nucleotides (regions longer than 700 nucleotides are known to
occur)
- no Shine-Dalgarno sequence is present
- less rigidly defined sequences are known to be involved in the process
of initiator codon selection
i. Initiation
- occurs at the 1st AUG codon from the 5’-end
ii. Kozak Consensus Sequence
- recognized by the ribosome as the translational start site (sequence is
A or G - CCAUGG)
- aids in defining the initial AUG codon for translation
- loss of the sequence  reduced efficiency of translational initiation
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iii. 40S Ribosomal Subunit
- binds near the 5’-end of the mRNA
- scans the mRNA in the 3’-direction until it finds the 1st AUG in the proper
sequence context
- ATP hydrolysis
- essential for the scanning process
- requires the actions of
- eIF-5
- eIF-4C
60S Subunit + 40S Subunit  80S Initiation Complex
a. Before 60S Subunit Joins the 40S Subunit

- removed are
- eIF-2GDP
- eIF-3
- requires eIF-5
b. After Release
- eIF-2GDP  eIF-2GTP by the action of eIF-2B
c. Upon Completion of 80S Initiation Complex Formation
- all other initiation factors are removed
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B. Elongation
- similar mechanism as in prokaryotes
1. Necessary Components
- 80S Initiation Complex
- aminoacyl-tRNA
- GTP
- eukaryotic elongation factors
- eEF-1
- eEF-1
- eEF-2
2. Aminoacyl-tRNAs
- bound to the P site as ternary complexes (eEF-1 GTP aminoacyl-tRNA)
- GTP hydrolysis  GDP
3. Analogies
- eEF-1 to prokaryotic EF-Tu
- eEF-1 to prokaryotic EF-Ts
a. eEF-1
- catalyzes the GDP to GTP exchange in the eEF-1
4. Peptide Bond Formation
- same mechanism as in prokaryotes
5. Translocation
- requires
- eEF-2 GTP complex
- GTP hydrolysis
- analogous to prokaryotic translocation mediated by EF-G-GTP complex
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C. Termination
- similar mechanism as in prokaryotes
1. Single Release Factor (eRF)
- with an associated GTP
- recognizes all 3 termination codons
2. Peptidyl Transferase
- of the ribosome
- activity effects termination when the release factor binds to the ribosome
3. GTP Hydrolysis
- required for
- termination and
- dissociation of the ribosomal subunits
D. Gene Expression Regulation

1. Transcriptional Level
- gene expression is most commonly regulated at this level
2. Translational Level
- rate of protein synthesis is also sometimes regulated
- accomplished in eukaryotes is by covalent modification of eIF-2 (phosphorylated eIF-2 is
inactive)
3. Binding of Regulatory Proteins to mRNA
- both eukaryotes and prokaryotes
a. Results
- block translation
- extend mRNA use by protecting it from degradation
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PROTEIN LOCALIZATION in EUKARYOTES

A. Eukaryotic Protein Synthesis
- all translation of eukaryotic nuclear genes begins on ribosomes free in the cytoplasm
- proteins translated on free cytoplasmic ribosomes
1. Cytoplasmic Proteins
2. Mitochondrial Proteins
- encoded by nuclear genes
- proteins being translated may belong in other locations
- certain proteins are translated on ribosomes associated with the rough endoplasmic reticulum
1. Secretory Proteins
- extracellular matrix protein collagen
2. Serum Proteins
- immunoglobulins
- peptide hormones
- serum albumin
3. Organelle Proteins
- as those of the mitochondria
- >90% have to be imported from the cytoplasm
4. Integral Membrane Proteins
- have to be inserted into the correct intracellular membrane with the correct
orientation to properly function
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B. Protein Synthesis on Membrane-Bound Ribosomes
- proteins synthesized in the endoplasmic reticulum are made in a precursor form that is processed before
they reach their final destination
1. Signal Hypothesis
a. Leader or Signal Sequence
- sequence of the proteins to be made on the endoplasmic reticulum on their amino-
terminal end
- directs the protein synthesis in the endoplasmic reticulum
- usually 15-30 amino acids long
- contains a stretch of hydrophobic amino acids
b. Steps in the Leader Sequence Recognition and Direction of Protein Synthesis to the
Endoplasmic Reticulum
i. Signal Sequence Recognition by the Signal Recognition Particle (SRP)
ia. SRP - elongated complex of
- 6 non-identical proteins
- small cytoplasmic RNA (scRNA)
ii. SRP-Ribosome Complex Binding to the Endoplasmic Reticulum and
Continuation of Protein Synthesis into the Lumen of the Endoplasmic
Reticulum
iia. The translation-arrested SRP-ribosome complex (docking proteins)
interacts with a SRP receptor on the endoplasmic reticulum
iib. SRP Receptor
- made of 2 dissimilar, integral membrane proteins
iic. SRP-Ribosome Complex and SRP Receptor Interaction
 signal sequence insertion into the membrane
protein synthesis resumption
iid. GTP Cleavage
- just prior to the resumption of protein synthesis
- by the SRP receptor
 SRP dissociation from the ribosomes
iie. During signal sequence insertion into the membrane
- signal sequence associates with another integral membrane protein
(signal sequence receptor)
c. Completion of Synthesis into the Endoplasmic Reticulum
i. Signal Peptidase
- on the luminal side of the endoplasmic reticulum
(- while - the signal sequence is attached to the signal sequence
receptor
- remainder of the protein is being synthesized)
- cleaves the signal sequence from the protein
ii. Protein passes through the membrane into the lumen during synthesis
iii. For Some Proteins
- integral membrane proteins may form a passage or channel
through which they may pass during synthesis and enter the
lumen
d. Signal Membrane Protein Synthesis
- similar except for stop-transfer signals
i. Stop-Transfer Signals
- sequence of hydrophobic amino acids within the protein
- halts the transfer of the protein across the membrane
- function as membrane-binding sequence
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C. Protein Targeting
- many proteins are destined to perform their functions within specific cellular organelles
1. Amino Acid Sequences
- some proteins
- direct proteins to their final locations
a. Nuclear Proteins
- contain nuclear localization signal
b. Mitochondrial Proteins
- have mitochondrial entry sequence
2. Other Signals
a. N-terminal Hydrophobic Signal Sequence
- ensure translation on the RER
- found on proteins destined to be secreted (insulin), placed in the cell membrane
(Na+-K+ ATPase), or ultimately directed to the lysosome (sphingomyelinase)
b. Phosphorylation of Mannose Residues
- direct an enzyme to a lysosome
i. Lysosomal Enzymes and Phosphorylation of Mannose
- lysosomal enzymes are glycosylated and modified
- Golgi apparatus  specific mannose residues located in N-linked
oligosaccharide chains phosphorylated by N-acetylglucosamine-1
phosphotransferase  mannose-6-phosphate in the oligosaccharide
chain  protein removed from the secretion pathway  directed to
lysosomes
ia. I-Cell Disease
- genetic defect affecting phosphorylation  lysosomal enzymes
released into the extracellular space  inclusion bodies
accumulate in the cell  compromised function
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iai. Manifestations
- coarse facial features, gingival hyperplasia, macroglossia
- craniofacial abnormalities, joint immobility, clubfoot, claw-
hand, scoliosis
- psychomotor retardation, growth retardation
- cardiorespiratory failure, death in first decade
- bone fracture and deformities
- mitral valve defect
- secretion of active lysosomal enzymes into blood and
extracellular fluid
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POSTTRANSLATIONAL MODIFICATION of POLYPEPTIDE CHAINS
A. Post-Translational Modification of Polypeptide Chains in the Endoplasmic Reticulum
- covalent modifications either
- while still attached to the ribosome
- after completion of synthesis
1. Folding into Tertiary and Quarternary Conformations
a. Proper Disulfide Bond Formation
- important component in the formation and maintenance of the proper tertiary and
quarternary conformations of many proteins
- occurs in the lumen of the endoplasmic reticulum
- catalyzed by protein disulfide isomerase
b. Multimeric Protein Assembly
- occurs in the lumen of the endoplasmic reticulum
c. Mutated Proteins
- do not allow proper folding
- often not transported from the endoplasmic reticulum
- ex: 1-antitrypsin
- major cause of emphysema in caucasians
- protease inhibitor
- produced in the endoplasmic reticulum of the
- liver
- macrophages
- as the neutrophils work in the lung neutrophils  elastase released  destroy
lung cells
- released elastase blocked by circulating α1-antitrypsin
- single mutation  improper folding  lack of antiprotease activity 
development of emphysema caused by proteolytic destruction of lung
cells  reduction in the expansion and contraction capability of the
lungs
2. Covalent Alterations
a. Phosphorylation
- occurs on hydroxyl groups of
- serine
- threonine
- tyrosine
- catalyzed by protein kinases
- reversed by protein phosphatases
- may increase or decrease the functional activity of the protein
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b. Glycosylation
- most proteins synthesized in the endoplasmic reticulum are glycosylated
- proteins destined to become
- part of plasma membrane
- secreted from the cell
- have carbohydrate chains (oligosaccharides) attached to:
- hydroxyl groups (O-linked) of
- serine
- threonine
- amino group (N-linked) of asparagine within the sequences:
- Asn-X-Ser
- Asn-X-Thr
- core oligosaccharide
- transferred as a complete unit from an activated form
- activated core oligosaccharide
- linked by a high-energy pyrophosphate linkage to a lipid donor
molecule (dolichol) within the endoplasmic reticulum
membrane
- glycosyl transferase
- catalyzes the transfer
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- much glycosylation (N-linked and O-linked)

- occurs after proteins leave the endoplasmic reticulum and enter the Golgi
apparatus
- N-linked glycosylation
- begins in the endoplasmic reticulum as proteins are being synthesized
i. Solely N-glycosylated
- transferrin
ii. Solely O-glycosylated
- heparin
iii. Both N- and O-glycosylated
- LDL receptor
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c. Hydroxylation
- in the endoplasmic reticulum
- proline and lysine residues of -chains of collagen
d. Other Covalent Modifications

- for functional activity of a protein
- biotin - must be covalently bound to protein component of carboxylase enzymes to be
catalytically active
i. Carboxylation
- carboxyl groups added to glutamate residues by vitamin K dependent
carboxylation  -carboxyglutamate residues (essential for the
activity of several of the blood-clotting proteins)
ii. Lipidation
- farnesyl groups  help anchor proteins in membranes
iii. Acetylation
- many proteins acetylated posttranslationally
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iv. Biotinization
B. Post-Translational Modification of Polypeptide Chains Beyond the Endoplasmic Reticulum

1. Transport to the Golgi Complex
- by vesicles
a. Golgi Complex
- principal director of the intracellular movement of macromolecules
- signal for the movement are not well defined
- responsible for the completion of the glycosylation of proteins that began in the
endoplasmic reticulum
2. Trimming or Proteolytic Cleavage
- for the proteins to become active
a. Proteins Destined for Secretion from the Cell
- initially made as large, precursor molecules which are not functionally active
- portions removed by endoproteases  release of active molecule
- cellular site of the cleavage reaction depends on the protein to be modified
- endoplasmic reticulum
- Golgi apparatus
- developing secretory vesicles (insulin)
- some cleaved after secretion
b. Zymogens
- inactive precursors of secreted enzymes
- activated through cleavage once they have reached their proper sites of action
- ex: Trypsinogen  trypsin in the small intestines
- synthesized as zymogens to protect the cell from digestion by its own products
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c. Insulin
- synthesized in the -cells of the islets of Langerhans in the pancreas
i. mRNA for Human Insulin
- specifies 110 amino acids (preproinsulin)
ii. Signal Sequence
- formed by the 24 amino acids on the amino-terminal end
- removed during synthesis in the endoplasmic reticulum
iii. Proinsulin
- formed by the remaining 86 amino acids
- packed into secretory granules
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iv. Endoproteases and Exoprotease

- within the secretory granules
- hydrolyze bonds  active insulin
inactive C peptide
4 amino acids
v. Active Insulin
- made of
- 21 amino acid A chain
- 30 amino acid B chain
- held together by disulfide bonds that were formed in the lumen of the
endoplasmic reticulum
vi. Active Insulin and C Peptide

- secreted in the plasma upon demand
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d. Glucagon
- synthesized as a large preproglucagon in the -cells of the islets of Langerhans in the
pancreas
i. Preproglucagon Signal Peptide
- removed during synthesis in the endoplasmic reticulum  proglucagon
ii. Endoproteolytic Cleavages
- within the secretory granules
- remove 29 amino acids (active glucagon) from within proglucagon precursor
C. Protein Degradation
1. Ubiquitin
- small
- highly conserved protein
2. Ubiquitination
- attachment of a ubiquitin
- marking of proteins that are defective or destined for rapid turnover  rapidly degraded by
proteasome
3. Proteasome
- complex, ATP-dependent, proteolytic system
- located in the cytosol
- role in producing antigenic peptides for presentation by class I MHC molecules
DRUGS and INHIBITORS of PROTEIN SYNTHESIS in EUKARYOTES

A. Therapeutic Drugs
1. Streptomycin
- aminoglycoside
- used to treat
- heart infections
- tuberculosis
- prevents binding of fmet-tRNAf to the P site of the initiation complex  inhibits initiation of
protein synthesis
- effect localized to the 12S protein of the 30S ribosomal subunit
- causes misreading of the mRNA sequence
2. Tetracycline
- wide-spectrum antibiotic
- binds to 30S ribosomal subunit
- inhibits binding of aminoacyl-tRNAs
3. Chloramphenicol
- wide-spectrum antibiotic
- blocks peptidyl transferase reaction
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a. Gray Baby Syndrome

- dangerous condition in newborns (especially premature babies) due to chloramphenicol
(drug for bacterial infections including meningitis)
- UDP-glucuronyl transferase deficiency  drug not excreted  toxicity
i. Manifestations
- blue lips, nail beds, and skin (cyanosis)
- death
- hypotension
4. Erythromycin
- binds to 50S ribosomal subunit  inhibits translocation reaction
B. Inhibitors
- antibiotics
- inhibit protein synthesis
- for research purposes
- not proven to be good therapeutic drugs
1. Cycloheximide
- inhibits peptidyl transferase reaction
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2. Puromycin
- analogue of the aminoacyl-adenosine moiety of aminoacyl-tRNA
- structure is very similar to the 3’ end of an aminoacyl-tRNA  bind to the ribosomal A
site  peptide bond formation  peptidyl-puromycin
- resembles only the 3’ end of the tRNA

- does not engage in translocation
- dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the
peptide
 prematurely terminates polypeptide synthesis
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C. Toxins
1. Diphtheria Toxin
a. Source
- produced by lysogenic bacteriophage that infects Corynebacterium diphtheriae
- C. diphtheriae
- can infect the nasopharynx
b. Mechanism of Action
i. Elongation Factor-2 (eEF-2) Inactivation by ADP Ribosylation
- ADP-ribose transfer from oxidized nicotinamide-adenine-dinucleotide (NAD+)
to an already post-translationally modified histidine in eEF-2
 inhibits translocation step of elongation
c. Clinical Aspects
- infected patients  sore throat  severe edema and airway blockage
- may progress to heart infections  high death rate
2. Pseudomonas Toxin
- elongation factor-2 (eEF-2) inactivation by ADP ribosylation  inhibits translocation step of
elongation
3. Ricin
a. Source
- protein produced by castor beans
b. Mechanism of Action
i. Ricin Toxin
- N-glycosidase
- removes a single adenine from 28S rRNA (depurination)  inhibits
ribosomes  inhibits protein synthesis in eukaryotes
c. Clinical Aspects
i. Ricin - protein component of castor oil (distasteful laxative)
- extremely toxic  should not be administered in a prolonged period of time
ii. Long-Term Castor Oil Use
 persistent diarrhea and loss of intestinal function
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4. Ribosomal RNA Specific Nucleases
- specific cleavage of rRNAs  inhibit protein synthesis
a. -Sarcin
- fungal toxin
- cleaves 28S rRNA  inhibition of aminoacyl-tRNA binding
b. Colicin E3
- secreted by some Escherichia coli strains
- cleaves 16S rRNA  inhibition of initiation  inhibits protein synthesis in other
bacteria
c. Shiga Toxin and Verotoxin
i. Shiga Toxin
- produced by Shigella dysenteriae
ii. Verotoxin
- shiga-like toxin
- produced by enterohemorrhagic E. coli
- subunits A (RNA glycosylases)  remove a single adenine residue from the 28S
rRNA (cuts 28S rRNA)  inactivate the 28S rRNA in the 60S subunit of the
eukaryotic ribosome  inhibition of aminoacyl-tRNA binding to A site 
inhibition of eukaryotic protein synthesis
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SUMMARY
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MOLECULAR BIOLOGY:
MUTATION and DNA REPAIR
MUTATION
- heritable
- any change in the sequence of DNA base pairs that is permanent
- arises by chance
- to be considered a mutation, the change in the base-pair sequence must not be a result of recombination
A. Types of Damage to DNA
B. Origins of Mutations
1. Spontaneous Cellular Events
a. Errors in Replication
- if added base during replication is noncomplementary to the template  mispairing
 mutation during the next round of replication if not repaired
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i. Proofreading
- component activity during replication
- reduces the number of bases that are misincorporated or rare tautomers of
bases may become incorporated during replication
ii. Postreplicative Repair Systems
- correct base mispairing
iii. Prokaryotes
- errors of this type occurs in less than once for every 10,000,000,000 bases
replicated
b. Replication Slippage
- does not involve an alteration of individual nucleotides
- results from incorrect alignment between allelic and nonallelic DNA sequences during
replication
- when the template strand contains short tandem repeats (CA repeats as in
microsatellites)  newly replicated strand and the template strand may shift
their positions relative to each other (microsatellite instability)
- with replication or polymerase slippage (leading to incorrect pairing of repeats)  some
repeats are copied twice or not at all depending on the direction of the shift
i. Backward Slippage
- the new strand results in the addition (insertion) of nucleotides to the new
strand
ii. Forward Slippage
- of the new DNA strand
- results in the loss (deletion) of nucleotides from the new DNA
2. Random Cellular Events

a. Errors Due to Recombination Events
i. DNA of Living Cells
- mobile
- often rearranged or recombined
- ex: ia. Immunoglobulin Gene Diversity
- arises in part from translocation and rearrangement of
immunoglobulin genes
ib. Chromosomes
- sometimes cross over and recombine during meiosis
ic. Viruses
- of many species
- capable of moving their DNA in and out of the host cells’
genomic DNA
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id. Some Pieces of DNA
- mobile
- can move in different positions in the genome
ie. Reverse Transcription
- by some viral species
- some RNAs  reverse transcription  DNA formation 
inserted into the genome
ii. Typical DNA Rearrangements

- leave no changes or only a few small changes that can be repaired
- some rearrangements  extensive change in the cell’s DNA that cannot be
repaired  severe or lethal mutations
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b. Chromosomal Translocations
- exchange of genetic material between two nonhomologous chromosomes
i. Reciprocal (Non-Robertsonian) Translocation
- results in a true exchange of DNA fragments between two chromosomes
- can lead to the formation of new fusion genes, or a changed level of
expression of existing genes
ii. Robertsonian Translocation
- large fragment of a chromosome attaches to another chromosome
- no DNA is attached in return
- common translocations are confined to the acrocentric chromosomes
iia. Minority of Cases of Down Syndrome

- caused by the Robertsonian translocation of approximately 1/3 of
chromosome 21 on to chromosome 14
c. Interstitial Deletions
- deletions of large DNA fragments on a single chromosome  results in the pairing
of two genes that are not normally in sequence
- can lead to the formation of fusion oncogenes
d. Chromosomal Inversions
- large segment of a single chromosome becoming reversed within the same
chromosome
- usually resulting from a rearrangement following chromosomal breakage
- can create fusion genes
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3. Chemical Mutagens
a. Result
- alteration of
- DNA bases
- DNA structure
b. Produce 2 Classes of DNA Damage
i. Point Mutations
- one base pair replaces another
ii. Insertion/Deletion Mutations
- one or more nucleotide pairs are inserted or deleted
c. Chemical Alteration of Bases in DNA
- deamination
- oxidation
- alkylation
- the base itself may be lost by hydrolysis of the glycosidic bond
d. Example
i. Nitrous Acid
ii. Intercalating Agents
- can generate insertion/deletion mutations
4. Irradiation
- alter DNA structure  mutations if not repaired
a. UV Light
- can fuse 2 pyrimidines adjacent to each other in the DNA
b. Ionizing Radiation
- high-energy
- cause double-strand breaks
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5. Spontaneous Biochemical Changes
 mutations if not repaired
a. Deamination and Methylation
i. Cytosine Deamination  Uracil
- if not repaired  adenine pairs with the template strand (containing the uracil)
instead of guanine
ii. Methylation
- methylation of the carbon atom at position 5 of cytosine  5-methylcytosine
 deamination  change to thymine containing an oxygen at position
4 instead of an amino group
- mutation will not be corrected because thymine is a natural base
iii. Adenine Deamination
- deaminated at position 6  hypoxanthine which contains an oxygen in this
position instead of an amino group and which pairs with cytosine
instead of thymine
- resulting change after DNA replication is a cytosine instead of a thymine in
the mutant strand
b. Spontaneous Depurination
- purines are less stable than pyrimidine under normal cellular conditions
- glycosidic bond linking the purine to the sugar phosphate backbone is often broken
- if not replaced before replication  any base may be added to complement the missing
base during replication
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c. Alkylation of Guanine
- alkylation
- introduction of a methyl or an ethyl group into a molecule
- involves reaction with the ketone group at position 6  6-methylguanine
- cannot form a hydrogen bond  unable to pair with cytosine (instead it will
pair with thymine)  after the next replication the opposite cytosine
(C) is replaced by a thymine (T) in the mutant daughter molecule 
molecule contains an abnormal GT pair instead of GC
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- important alkylating agents

- ethylnitrosourea (ENU)
- ethylmethane sulfonate (EMS)
- dimethylnitrosamine
- N-methyl-N-nitro-N-nitrosoguanidine
6. Addition or Deletion of One or More Nucleotides
C. Types of Mutations
a. 2 Categories
i. Small-Scale Mutations
- single or a few base changes
ia. Base Substitution
iai. Divisions
iaia. Transition
iaib. Transversion
iaii. Classification
- depending on the consequences of a mutation
iaiia. Silent
iaiib. Missense
iaiic. Nonsense
ib. Base Deletion
ic. Base Insertion
- base deletion and insertion  codon frame shifts  change in reading
frame of base sequence of a gene (frameshift mutations) 
synthesis of completely different protein
- 3 base insertions or deletions do not change codon frames for protein
synthesis
ii. Large-Scale/Chromosomal Mutations
iia. Translocations
- interchanges of large segments of chromosomal DNA
iib. Inversions
- inversion of chromosomal segments to opposite orientation through
chromosomal rearrangements
iic. Deletions of Chromosomal Segments
- also be caused by chromosomal rearrangements during meiosis
iid. Nondisjunction of Chromosomes
- pairs of chromosomes fail to separate properly during cell divisions
 genetic diseases (Down’s, Turner’s, triple-X syndromes)
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1. Point Mutations
- most common type of mutation
- when a single DNA nucleotide base is replaced by a different nucleotide (are also known as
base substitutions)
- occasionally, single nucleotide deletions or insertions  also considered point mutations, but
they cause reading frame shifts
a. 3 Types of Point Mutations
- possible if base substitution occurs in the coding region of a gene
i. Missense Mutation
- replacement of a single nucleotide base with a different one  change in the
codon so that it codes for a different amino acid
- common types of mutations
ia. Effect
- no effect or
- very serious consequences
ib. Clinical Example
- sickle cell anemia
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ii. Nonsense Mutation

- a change in one DNA base pair  conversion of an amino acid codon to a stop
codon  shortened or truncated protein
ia. Effect
- protein products are usually non-functional
ib. Clinical Example
- thalassemias
iii. Silent Mutation
- leads to formation of a codon synonym (the genetic code is redundant)
- no change in the amino acid sequence of the gene product
- most often, this results from a base change in the 3rd position of the codon
(wobble position)
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b. 5 Types of Point Mutations by Biochemical Origin
i. Transitions
- 1 purine is replaced by another purine or
- 1 pyrimidine is replaced by another pyrimidine
- ex: replacement of a G-C pair by an A-T pair would result in a transition
ii. Transversions
- purine is replaced by pyrimidine or
- pyrimidine is replaced by purine
- A-T pair replaced by either a T-A or C-G pair
iii. Tautomerism
- modification of a base caused by migration of a proton or a hydrogen bond
 switching of an adjacent single and a double bond
iv. Depurination
- caused by a spontaneous hydrolysis of a purine base (A or G)
- deoxyribose-phosphate backbone remains intact
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v. Deamination
- spontaneous reaction that can result in the following conversions
- cytosine into uracil (C to U)
- 5-methylcytosine into thymine
- adenine into hypoxanthine (A to HX)
- since uracil can be recognized (thymine and hypoxanthine are not detected
as errant) deamination of C to U is the only of these that can be
corrected
2. Frame-Shift Mutations
- if one or two nucleotides are either deleted from or added to the coding region of a message
sequence  altered reading frame  product with a radically different amino acid
sequence, or a truncated product due to the creation of a termination codon
a. Insertion of One or More Base Pairs
- three nucleotides are added  new amino acid added to the peptide (reading frame is
not affected)
b. Deletion of One or More Base Pairs

- small deletions may remove one or a few base pairs
- larger deletions can remove an entire gene or several neighbouring genes
- three nucleotides deleted  amino acid is lost
- no change in the reading frame
- results to deletion of 1 or more amino acids
i. Cystic Fibrosis (CF)
- hereditary disease
- primarily affects the pulmonary and digestive systems
- most commonly caused by deletion of three nucleotides from the coding
region of a gene  loss of phenylalanine at the 508th position
(ΔF508 mutation) in the protein coded  prevents normal folding of
the CF transmembrane conductance regulator (CFTR) protein 
destruction by the proteasome
ia. CFTR
- normally functions as a chloride channel in epithelial cells
- loss  thick, sticky secretions in the lungs and pancreas  lung
damage and digestive deficiencies
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3. Trinucleotide Repeat Expansion
- a sequence of three bases that is repeated in tandem will become amplified in number  too
many copies of the triplet occur
- if occurs within the coding region of a gene  the protein will contain many extra copies of
one amino acid
a. Nucleotide Repeats
- short DNA sequences that are repeated a number of times in a row
b. Trinucleotide Repeat Expansion
- made up of 3 bp sequences
i. Human Genome
- contains tandem repeats of trinucleotides (triplets)
ii. Trinucleotides
- triplets normally occur in groups of 5-35 repeats
- usually transmitted stably
- can become unstable and expand to pathological lengths
- can expand abnormally within or near certain genes, interfere with gene
expression, and cause disease depending on the gene involved (triplet
diseases)
- most of these diseases affect the central nervous system
- in some diseases the repeat consists of more than three nucleotides
- once the normal, variable length has expanded, the number of repeats tends to
increase even more when passed through the germline  earlier onset
of the disease than in the preceding generations (anticipation)
iii. Different Types of Trinucleotide Repeats and Their Expansions
- trinucleotide repeats can be distinguished according to their location with
respect to a gene
iiia. Very Long Expansions
- outside the coding region of a gene
- noncoding
- the increase in the number of these repeats can be drastic (up to 1000
or more repeats)
iiiai. First Stages of Expansion
- do not usually lead to clinical signs of a disease
- predispose to increased expansion of the repeat in the
offspring of a carrier (premutation)
iiib. Modest Expansions

- within a gene
- effect is dramatic (as in several severe neurological diseases) because
they result in expanded glutamine tracts
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iv. Unstable Trinucleotide Repeats in Different Diseases

- disorders due to pathological expansion of trinucleotide repeats are classified
according to the type of trinucleotide repeat
- sequence of the three nucleotides
- location with respect to the gene involved
- clinical features
- all involve the central or the peripheral nervous system
iva. Type I Trinucleotide Diseases
- characterized by CAG (codes for glutamine) trinucleotide expansions
within the coding regions of different genes
ivai. Normal
- about 20 CAG repeats occur normally in these genes 
about 20 glutamines occur in the gene product
ivaii. Disease State
- number of glutamines is greatly increased in the protein 
polyglutamines disorders
ivaiii. Huntington Disease
- amplification of CAG codon  insertion of many
glutamine residues in the huntington protein 
neurodegenerative disorder (Huntington disease)
- additional glutamine residues  unstable protein 
accumulation of protein aggregates
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ivb. Type II Trinucleotide Diseases
 decreased amount of protein produced
- characterized by expansions of CTG, GAA, GCC, or CGG
trinucleotides within a noncoding region of the gene
involved, either at the 5’ end (GCC in fragile X syndrome type
A, FRAXA), at the 3’ end (CGG in FRAXE; CTG in
myotonic dystrophy), or in an intron (GAA in Friedreich
ataxia)
ivbi. Fragile X Syndrome and Myotonic Dystrophy
- trinucleotide repeat expansion occurs in the untranslated
portion of a gene  decrease in the amount of
protein produced
c. Tetranucleotide Repeat
- is made up of 4 bp sequences
4. Splice Site Mutations
- can alter the way in which introns are removed from the pre-mRNA molecule  aberrant
proteins
a. Lost Splice Site Through Mutation
 spliceosomes may
i. Delete nucleotides from the adjacent exon
ii. Leave nucleotides of the intron in the processed mRNA
iii. Use next normal upstream or downstream splice site  exon deleted from the
processed mRNA
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b. Example Diseases
- -thalassemia
- Gaucher disease
- Tay-Sachs
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5. Amplifications
- cellular events
- result in multiple copies of whole DNA segments (including all the genes located on them)
- usually caused by a disproportionately high level of DNA replication in a limited portion of
the genome  multiplied genes are effectively amplified  higher number of copies of
the encoded protein
- can alter the phenotype of the affected cell
- ex: drug resistance in certain cancers is linked to amplifications of genes that confer
resistance to chemotherapeutic agents by preventing their uptake into the
cell
6. Large Segment Deletions from a Chromosome
- during an unequal crossover in meiosis
a. Normal Crossover/Recombination Event
- between homologous chromosomes
- normal part of meiosis I
- generates genetic diversity in reproductive cells (egg and sperm)
- homologous maternal and paternal chromosomes exchange equivalent segments
- no genetic information lost from either one
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b. Unequal Crossover/Recombination Event

- rare
- one of the two homologs loses (deletion) some of its genetic information
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i. α-Thalassemia
- deletion of one or more α-globin genes from chromosome 16
ii. Cri-du-chat Syndrome
- terminal deletion of the short arm of chromosome 5
iia. Manifestations
- mental retardation, microcephaly, wide-set eyes, characteristic kitten-
like cry
7. Type of Mutation According to Cell of Origin
a. Germline Mutation
- mutation arises in a germ cell
- can be passed to the offspring
b. Somatic Mutation
- mutation in a somatic cell
- cannot be passed on to offspring
- passed on to the somatic daughter cells of the organism
- ex: cancers resulting from somatic mutations
D. Mutagens
- agents that directly cause or increase the likelihood of changes in the DNA sequence
1. Chemical Agents
a. Base Analogues
- purines or pyrimidines
- similar to one of the four nucleotide bases found in DNA  can be incorporated into
DNA during replication  inhibition of replication
mispairing  mutagenic
- differ enough chemically  cause mismatch during base pairing  mutations in
daughter DNA strands
Antimetabolites
- share similarity with regularly occurring nucleotides
- incorporation into DNA  inhibit further replication 
competitive inhibitors of DNA replication
- used as anticancer chemotherapeutics
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i. 5-Bromouracil/Bromodeoxyuridine (BrdU)
- used by researchers to identify dividing cells because it is incorporated into
the DNA during replication
- thymine analogue
- contains a bromine atom instead of the methyl group in position 5 
ambiguous and often incorrect base pairing
- can exist in the enol form as of thymine

- 5-bromo substitution  more often exists in the rare enol tautomer more
often than thymine
- pairs with guanine instead of adenine
- DNA incorporation  transition of adenine-thymine to guanine-cytosine

ii. 2-Aminopurine
- analogue of adenine
- forms a one-hydrogen bond base pair with cytosine instead of forming a
base pair with thymine
- DNA incorporation  transition of guanine-cytosine to adenine-thymine
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b. Chemical Mutagens
i. Nonalkylating Agents
ia. Formaldehyde (HCHO)
- reacts with amine groups
- cross links
- DNA
- RNA
- proteins
ib. Hydroxylamine (NH2OH)
- reacts with cytosines  form derivatives that pair with adenines
instead of guanines  transversion
ic. Nitrous Acid (HNO2) and Hydroxylamine (NH2OH)
- oxidatively deaminates
- cytosines  uracils
- adenines  inosines/hypoxanthines
- guanines  xanthines
- result to transition of guanine-cytosine to adenine-thymine 
point mutations
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ii. Alkylating Agents

- transfer alkyl groups to other molecules, including DNA
- cross-link guanine nucleotides in DNA  DNA damage  mutations in
both replicating and nonreplicating cells
- introduce sufficient DNA damage  cell unable to divide  used as
anticancer drugs (cisplatin, carboplatin)
iia. N-Methyl-N’-Nitro-N-Nitrosoguanidine (MNNG)
- highly mutagenic
- leads to
- transitions
- transversions
- act primarily at replication forks  not likely to be repaired before it
fixed by replication
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iib. Methylnitrosamines
- release the reactive methyl cation (CH3+)  methylates OH- and
NH2 groups in DNA
iic. Benzo[a]pyrene
- carcinogen
- aromatic hydrocarbon
- converted into the active form in the organism
- multiple hydroxylation of one of the rings  reactive epoxide 
react with NH2 groups in guanine residues
- free radicals of benzo[a]pyrene also contribute to its toxicity
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iii. Intercalating Agents

- insert themselves between two nucleotide base pairs  physically interferes
with DNA transcription and replication  DNA damage 
mutation events
iiia. Acridines
- tend to cause frameshift mutation
iiib. Ethidium Bromide
- fluorescent DNA dye
- commonly used in research laboratories
iiic. Aflatoxin
- carcinogen produced by a fungus from the genus Aspergillus
iiid. Doxorubicin and Daunorubicin
- used as cancer chemotherapeutics
iiie. Thalidomide
- teratogen
- associated with numerous cases of phocomelia (very short or absent
long bones and flipper-like appearance of hands and/or feet) in
the 1960s
iiiei. Current Uses
- as a last resort anti-inflammatory agent in the treatment of
erythema nodosum leprosum and sarcoidosis
- salvage chemotherapeutic agent in patients with multiple
myeloma
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iv. Methylating Agents

- transfer methyl groups to DNA nucleotide bases  mutations
- typically not used as anticancer agents because do not cause cell death
iva. Ethylmethane Sulfonate (EMS)
- adds methyl group to the nitrogen in the 7th position of the purine ring
of - guanine
- adenine
- methylation  labile N-glycosidic bond  hydrolysis 

depurinated site
- if not repaired  any base may pair at this site
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v. DNA Cross-Linking Agents

- act as mutagens
- form covalent bonds between nucleotide bases in DNA  interfere with
replication and transcription
va. Platinum
vb. Cisplatin
- platinum derivative
- cancer chemotherapeutic agent
vi. Reactive Oxygen Species
- free radicals
- rendered highly reactive by the presence of unpaired electrons
- "steal" electrons from DNA to become more stable ROS  DNA damage
- ex: Superoxide
H2O2
Hydroxyl radicals
 important in age-related cellular damage
2. Short-Wavelength UV Light
- type of electromagnetic radiation with a shorter wavelength (200-400 nm)
- higher energy than that of visible light
- induce formation of covalent bonds between adjacent thymine nucleotides  bulky thymine
dimers  DNA damage
- mutagenic effects, mainly in skin cells (sunburn)  increased risk of skin cancer
- ex: DNA damage to melanocytes  loss of control mechanisms for cellular growth 
uninhibited growth patterns seen in various types of melanomas
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a. Thymine Dimers Formation (Dimerization)
- most common chemical change due to UV exposure
- two neighboring thymine bases become covalently linked to one another (direct
mutation)  DNA structural distortion
transcription inhibition
replication disruption
3. Ionizing Radiation
- roentgen rays (X-rays)
- α, , and radiation
- produced by radioactive materials
- electromagnetic radiation
- with energy high enough to ionize a molecule or atom by removing an electron from its orbit 
production of extremely reactive free radicals (molecules with unpaired electrons) 
extensive DNA damage including purine ring opening, depurination and phosphodiester
bond breakage  mutations and eventual cell death
- potentially very dangerous
- used in targeted cancer treatment and radiography (X-rays)
E. Mutagenesis and Carcinogenesis
- most mutagens  cancer formation  carcinogenic
- many chemicals are not mutagenic in the form they enter the body but are carcinogenic
1. Types of Carcinogens
a. Direct Carcinogens
- exist in the mutagenic form when they enter the body
b. Indirect Carcinogens
- not mutagenic in the form they enter the body  converted to mutagens upon
metabolism in the body
i. Liver - has a very active detoxification system  converts many inactive mutagens to
active mutagens
ia. Cytochrome P450 Oxidase System
- endoplasmic reticulum-bound enzyme
- converts lipophilic compounds to hydrophilic compounds by
hydroxylation or other chemical modifications  excretion
- this process may convert inactive mutagens to active species
DNA REPAIR
- responsible for minimizing the negative effects that DNA damage has on the cell
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- may not occur properly when certain tumor suppressor genes have been inactivated through mutation or
deletion
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1. Environmental Insults to DNA
 alteration or removal of nucleotide bases
i. Damaging Agents
- radiation
- chemicals
2. Bases - also altered or lost spontaneously from the mammalian DNA (many thousand/cell/day) 
mutation  deleterious effects
cancer
3. Repair Enzymes are Involved in
- recognizing the lesion
- excising the damaged section
- filling the gap using the sister strand as template
4. Major DNA Repair Systems
a. Direct Repair
b. Repair of Single-Strand Damage
c. Recombination Repair
5. When DNA Damage Surpasses a Certain Threshold

a. Causes
- too much accumulated damage
- DNA repair mechanisms no longer effective
b. Fates
i. Senescence
- cell enters a irreversible dormant state
- main cellular processes and functions are suspended
ii. Apoptosis
- cell undergoes programmed cell death, or suicide, by activating
specialized signal cascades
iii. Cancer
- cell starts undergoing unregulated cell division  neoplasia and tumor
growth
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A. Direct Repair
- some base modifications can be repaired without excision repair
- when specific DNA nucleotides are damaged by chemical modification, the resulting molecular species
are specific to the nucleotide that was damaged
- the cell can use this information to determine the original nucleotide  directly reverse the damage
using mechanisms specific to the type of damage
1. Photoreactivation
a. DNA Photolyase
- enzyme found in all organisms
- binds at the defect
- when illuminated, cleaves covalent bonds that form thymine-thymine dimers  two
single bases again
- activated by visible light at 400-600 nm
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2. Dealkylation of Guanines
a. Guanine Alkyltransferases
- remove the methyl and ethyl groups from alkylated guanines
- alkylate themselves in the process  inactivated (takes 1 protein to dealkylate 1
guanine)
b. Methylguanine Methyltransferase (MGMT)

- remove the methyl groups from methylated guanines
B. Repair of Single-Strand Damage
- only one strand of the DNA double helix is damaged
- the complementary base on the opposite strand can be used as a template for repair
1. Base Excision Repair
a. Causes of Base Alterations
- single nucleotides damaged by
- spontaneous alteration (cytosine  loss of amino group  uracil)
- action of deamination or alkylating compounds
- ex: nitrous acid
- formed by the cell from
- nitrosamines
- nitrites
- nitrates
- removes amino group from
- cytosine
- adenine
- guanine
- spontaneously lost bases
- improper incorporation of dUTP instead of dTTP during DNA synthesis
b. Repair Enzymes
i. DNA Glycosylase
- remove abnormal bases
- hydrolytic cleavage from the deoxyribose-phosphate backbone  apurinic or
apyrimidinic (AP) site
ii. Apurinic or Apyrimidinic (AP) Endonuclease
- recognize a missing base
- makes endonucleolytic cut just to the 5’-side of the AP-site
iii. Deoxyribose-Phosphate Lyase
- removes the single, empty, sugar-phosphate residue
iv. 5’  3’ DNA Polymerase (DNA Polymerase I in E. coli)
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v. DNA Ligase
c. Process
- damaged base removed by a DNA glycosylase  endonuclease cleaves the
backbone at the resulting abasic site  exonuclease removes several
additional residues  gap filled in by a DNA polymerase  nick sealed by
DNA ligase
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2. Nucleotide Excision Repair (NER)

- excise and replace longer stretches of nucleotides (2-30 bases)
- more sophisticated method to correct pyrimidine (thymine-thymine) dimers or other DNA
lesions involving several base pairs
- generally involves more proteins than either mismatch or base excision-repair
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a. Thymine-Thymine Dimer
- UV light  covalent joining of 2 adjacent pyrimidines (thymine)  dimer  prevent
DNA polymerase from replicating the DNA strand beyond the dimer
b. Repair Enzymes
i. Endonucleases
ia. Human Cells
- XPA
- XPB
- XPC
ib. uvrABC Excinuclease
- uvrA
- uvrB
- uvrC
- UV-specific endonuclease
- recognizes the damaged base  makes a break several bases
upstream (toward the 5' side)  excises an oligonucleotide
(12 or 13 nucleotides in prokaryotes, 27 to 29 nucleotides in
eukaryotes) containing the damaged bases
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ii. 5’  3’ Exonuclease
- activity of DNA polymerase I
iii. 5’  3’ DNA Polymerase
iiia. DNA Polymerase I
- in prokaryotes
iiib. DNA Polymerase /
- in humans
- fill the gap using the sister strand as template
iv. DNA Ligase
- catalyses the formation of a phosphodiester linkage (seals the nick) to restore
the intact DNA molecule
c. Process
- defect recognition  unwinding of the DNA encompassing the defect  excision
nuclease (exinuclease) cuts the DNA upstream and downstream of the defective
region  gap formed  gap filled in by a polymerase  religation
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d. UV Radiation
- can form dimers in the skin cells
i. Xeroderma Pigmentosum
- rare genetic disease
- autosomal recessive disease
- may involve up to 9 genes
- results to hypersensitivity of the skin to UV light damage
ia. Etiology
- deficiency in the excision-repair system of thymine-thymine
dimers
- cells cannot repair the damaged DNA  extensive accumulation of
mutations  skin cancers
- absence of UV-specific endonuclease
ib. Clinical Presentation
- many skin and eye problems
- extreme sensitivity to sunlight, skin freckling and ulcerations, high
incidence of skin cancers due to UV light exposure
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3. Mismatch Repair (MMR)/(Methyl-Directed Mismatch Repair System)

- used when there is an error in the pairing (base pair mismatch) of nucleotides secondary to
DNA replication or recombination
- detects errors that escaped DNA polymerase proofreading during DNA replication
- corrects insertions and deletions up to 4 base pairs in length
- defects in enzymes in this system have been implicated in high incidence of cancer (including
hereditary nonpolyposis colorectal cancer syndrome)
a. Mismatch Repair Systems in E. coli
i. Long Patch System
- can replace 1 kb of DNA and more
ii. Short Patch System
iii. Very Short Patch System
b. Identification of the Mismatched Strand
- mismatch  Mut proteins that identify the mispaired nucleotide(s) must be able to
discriminate (based on the degree of methylation) between the correct strand
and the strand with the mismatch
i. GATC Sequences
- found approximately once every thousand nucleotides
- methylated on the adenine residue
- methylation is not done immediately after synthesis  newly synthesized
DNA is hemimethylated
- parental strand is methylated (assumed to be correct)
- newly synthesized DNA is not methylated (daughter strand with
mismatch  repaired)
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c. Repair Enzymes
i. Endonuclease
- nicks the mismatched strand/s  mismatched base/s removed  gap formed
ia. Bacteria
iai. MutS Homodimer
- recognizes and binds to mismatched base pairs
iaii. MutL Homodimer
- forms a tetramer with MutS to loop out the region of DNA
containing the mismatched base pairs in a ATP-
driven reaction
 MutS/MutL Tetramer
- recruits MutH and activates MutH endonuclease activity
iaiii. MutH
- makes a single-strand cut (nick) adjacent to an
unmethylated GATC sequence
ib. Human
ibi. hMSH1
- cleave DNA and remove the strand with erroneous bases
ibii. hMLH1
- cleave DNA and remove the strand with erroneous bases
ibiii. hMSH2
- bind to mismatched base pairs
ii. UvrD Helicase

- unwind the DNA
iii. 5’  3’ Exonuclease
- activity of DNA polymerase I
- remove the defect
iv. 5’  3’ DNA Polymerase III (DNA Polymerase I in E. coli)
- fill the gap using the sister strand as template
v. DNA Ligase
- splice the γ’-hydroxyl of the newly synthesized DNA to the 5’-phosphate of
the remaining stretch of the original DNA
For Your Eyes Only
d. Process
- mismatched strand is nicked with endonuclease  mismatched base(s) is/are
removed  DNA polymerase fills in the gap sister strand is used as a
template  religation
e. Hereditary Nonpolyposis Colon Cancer (HNPCC)/Lynch Syndrome

- one of the most common inherited cancers
- gene mutation (usually hMLH1 or hMSH2 - encode enzymes for DNA mismatch
repair)
- inheritance of one nonfunctional, deleted copy of hMLH1 or hMSH2 gene  birth 
somatic mutation in the other copy  loss of mismatch repair function 
chromosomes retain errors (mutations) in many other loci (some may contribute
to cancer progression)
- manifested in intestinal cells (constantly undergoing cell division)
i. Microsatellite Instability
- prominent type of error that accompanies DNA replication
- lack of mismatch repair  replicated DNA vary in number of repeats at a
locus (ex: TGTGTGTGTGTG or TGTGTG) (this variation is
microsatellite instability)
- may be used as diagnostic tool
ia. Microsatellites (Short Tandem Repeats)
- di-, tri-, and tetranucleotide repeats dispersed throughout the DNA
usually in noncoding regions
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C. Recombination Repair
For Your Eyes Only
1. Genetic Recombination
- an exchange between two homologous DNA molecules
- provides the means to restructure genetic information
- confers an evolutionary advantage
- help eliminate unfavorable mutations
- maintain and spread favorable mutations
- endow each individual with a unique set of genetic information
- must occur between precisely corresponding sequences (homologous recombination) to
ensure that not one base pair is lost or added
- the newly combined (recombined) stretches of DNA have to retain their original structure 
function properly
2. Two Types of Recombination
a. Generalized or Homologous Recombination (Recombinatorial Repair or Crossing Over)
- occurs between two sections of DNA with extensive homology
- uses the enzymes that normally perform genetic recombination between homologous
chromosomes during meiosis
- used predominantly by the lower eukaryotes to repair double-strand breaks
i. Recombination Initiated From Single-Strand DNA Breaks
- aligned strands of DNA cleaved  strands cross over  form a
four-branched structure (Holliday junction)  junction can
dissociate into two new duplexes
ii. Recombination Initiated From Double-Strand Breaks
iia. Double-Strand Breaks
- occur in meiosis and DNA repair
- common consequence of γ-radiation
- other causes
- high-energy radiation
- oxidative free radicals
- naturally during gene rearrangements
- some chemotherapeutic agents
iib. Repair of Double-Strand Breaks
- no direct template exists to guide the cell's repair process
iic. Example of Important Human Pathway for Repair
iici. Three Central Proteins
- encoded by the genes
ATM - from the disease ataxia-telangiectasia that
result from mutation in this gene
- member of a protein kinase family
- activated in response to DNA damage
- the active form phosphorylates BRCA1 at
specific sites
BRCA1 - from the hereditary predisposition to breast
cancer that result from mutation in
this gene
- phosphorylated form
- induces homologous
recombination in
cooperation with BRCA2
- involved in transcription and
transcription-coupled
DNA repair
BRCA2 - from the hereditary predisposition to breast
cancer that result from mutation in
this gene
RAD51 - mammalian homologue of E. coli RecA
repair protein
For Your Eyes Only
For Your Eyes Only
For Your Eyes Only
b. Site-Specific Recombination (Nonhomologous End-Joining Repair - NHEJ)

- ends of the 2 DNA fragments brought together by a group of proteins
- does not require that the 2 DNA sequences have any sequence homology
- main repair mechanism in humans
- error prone
- always mutagenic
- defects in this system are associated with predisposition to
- cancer
- immunodeficiency syndromes
i. Two Proteins Involved
ia. Ku - heterodimer of 70 kDa and 86 kDa subunits
- binds to free DNA ends
- has latent ATP-dependent helicase activity
- bind DNA  recruits DNA-PK
ib. DNA-Dependent Protein Kinase (DNA-PK)
- has a binding site for DNA free ends and another for dsDNA just
inside these ends
- allows for the approximation of the two separated ends
- kinase activity activated by the free end DNA-Ku-DNA-PK complex
- reciprocally phosphorylates Ku and the other DNA-PK molecule on
the opposing strand, in trans  dissociates from the DNA and
Ku  activation of the Ku helicase  unwinding of the two
ends  approximated DNA forms base pairs
For Your Eyes Only
ii. Exonuclease
- remove the extra nucleotide tails
iii. DNA ligase
- fill the gaps
3. Transposition
- utilizes recombination to insert one DNA sequence into another without regard to sequence
homology
- widespread spontaneous process in living organisms by which a DNA sequence (transposon or
transposable element) inserts itself at a new location in the genome
a. Transposons
- does not have any sequence relationship with the target site
- major source of genetic variation
- important role in the evolution of genomes
- DNA segments in prokaryotes and eukaryotes that moves directly from one site of the
genome to another without an intermediary such as plasmid or phage DNA
For Your Eyes Only
i. Promote
- inversions, deletions, and rearrangements of host DNA  affect gene
expression and permit chromosome evolution
ii. Most Eukaryotic Transposons
- resemble the DNA from retroviruses rather than bacterial transposons (hence
called retrotransposons)
iia. Transposition of a Retrotransposon
- begins with its transcription to RNA  synthesis of DNA from the
RNA template by reverse transcriptase  DNA then inserts
randomly into the host genome
iii. Result
- change the functions of target sequences
- cause disease when inserted into a functioning gene
iv. Examples of Transposons

iva. Insertion Sequences (IS)
- simplest
- contain a transposase gene that mediates the recombination
reactions involved in transposition
- recombination occurs between short target sequences located at the
ends of the transposon
ivb. Replicative and Nonreplicative Transposons (Tn)
ivc. Transpositions of Retroelements via RNA Intermediates
For Your Eyes Only
ivd. More Complex Transposons
- contain genes required for transposition as well other genes (ex:
antibiotic resistance genes)
4. Thymine-Thymine Dimer Recombination Repair
For Your Eyes Only
4. Thymine-Thymine Dimer Recombination Repair
- provides a method to replicate around a thymine-thymine dimer that is not repaired prior to
replication
- thymine-thymine dimers cannot serve as templates during replication
- replication can bypass the region containing the thymine-thymine dimers and the dimer can be
repaired after replication
5. Recombination in E. coli
a. RecA - polymerizes on single-stranded gaps in DNA duplexes
- partially unwinds the duplexes and exchanges two strands in an ATP-dependent
reaction
b. Additional Proteins Needed to

- further unwind DNA
- produce nicks
- maintain proper supercoiling
- drive branch migration
- seal nicks
D. Translesion Synthesis
- last resort as means of continuing DNA replication
- DNA damage is extensive enough to prevent the replication machinery from advancing
- special DNA polymerases (repair the damaged DNA)  insert nucleotide bases (not completely
arbitrary, not based on a template)  introduce mutations by necessity, but enables the cell to
continue DNA replication
Short quiz on Basic Concepts of Metabolism and
Metabolic Effects of Insulin & Glucagon
Total points 15/15
The respondent's email (meds_nivetha.jothivanan@unp.edu.ph) was recorded on

submission of this form.
Which statement about Catabolic pathways is false? * 1/1
Catabolic reactions are reactions that break down complex molecules and serve to
capture chemical energy in the form of adenosine triphosphate (ATP) from the
degradation of energy- rich fuel molecules.
Catabolism allows molecules in the diet (or nutrient molecules stored in cells) to be
converted into building blocks needed for the synthesis of complex molecules.
Energy generation by degradation of complex molecules occurs in four stages
Catabolic pathways are typically oxidative, and require coenzymes such as NAD+
Which is not considered a stage of Catabolism? * 1/1
Hydrolysis of complex molecules
Conversion of building blocks to simple intermediates
Oxidation of Acetyl CoA
Oxidation of FADH2
Regulation of Metabolism is characterized by the following except: * 1/1
Signals from within the cell (intracellular)
Communication between cells (intercellular)
Second messenger systems
Phosphodiesteraise inositol pathway
These organs play a dominant role in fueling metabolism, except: * 1/1
Liver
Adipose
Brain
Adrenal glands
Phosphodiesterase is inhibited by the following, except: * 1/1
Allopurinol
Theophylline
Caffeine
Theobromine
Which is true about Insulin? 1/1
Insulin is a polypeptide hormone produced by the β cells of the islets of Langerhans

– which make make up only about 2-4% of the total cells of the pancreas.
Insulin’s metabolic effects are anabolic, favoring, synthesis of glycogen,

triacylglycerols, and protein.
Insulin is composed of 52 amino acids arranged in two polypeptide chains,

designated A and B, which are linked together by two disulfide bridges.
Pig (porcine) and beef (bovine) insulin differ from human insulin at one and three
amino acid positions, respectively.
Which is true about Inhibition of Insulin secretion? * 1/1
The synthesis and release of insulin are decreased when there is abundance of
dietary fuels, and also during periods of stress.
The synthesis of Insulin is mediated primarily by epinephrine, which is secreted

by the adrenal medulla in response to stress, trauma, or extreme exercise.
Epinephrine has a direct effect on energy metabolism, causing a slow mobilization

of energy- yielding fuels, including glucose from the liver and fatty acids from
adipose tissue
Epinephrine can override the normal glucose-stimulated release of insulin. Thus, in

emergency situations, the sympathetic nervous system largely replaces the plasma
glucose concentration as the controlling influence over β-cell secretion.
Glucagon is a polypeptide hormone secreted by the α cells of the 1/1
pancreatic islets of Langerhans. Which is true about Glucagon? *
Glucagon, along with epinephrine, cortisol, and growth hormone (the “counter-
regulatory hormones”), opposes many of the actions of insulin.
Glucagon acts to maintain blood glucose levels by activation of adipose tissues

glycogenolysis and gluconeogenesis.
Glucagon is composed of 32 amino acids arranged in a single polypeptide chain.
Glucagon is synthesized as a large precursor molecule (proglucagon) that is

converted to glucagon through a series of selective proteolytic cleavages
Glucagon is secreted by the following, except: * 1/1
Low blood glucose:
Amino acids
Epinephrine
Carbohydrate-rich meal
Which is not an Adrenergic symptom of Hypoglycemia? * 1/1
Confusion
Anxiety
Palpitation
Tremor
Neuro glycopenic symptoms often result from a gradual decline in blood 1/1
glucose, often to levels below ____ : *
30 mg/dl
40 mg/dl
50 mg/dl
60 mg/dl
These statements describe alcohol intoxication except: * 1/1
Alcohol intoxication can precipitate hypoglycemia, particularly in individuals who

have depleted their stores of liver glycogen.
Increased availability of OAA allows acetyl CoA to be diverted to ketone body

synthesis in liver and can result in alcoholic ketoacidosis
Alcohol consumption can also increase the risk for hypoglycemia in patients using
insulin
Chronic alcohol consumption can also result in alcoholic fatty liver due to increased
synthesis of triacylglycerols as a result of decreased fatty acid oxidation due to a fall
in the NAD+/NADH ratio, and increased lipogenesis due to the increased availability
of fatty acids and of glyceraldehyde 3-phosphate
In the well-fed state, Carbohydrate metabolism is described by the 1/1
following, except: *
Increased phosphorylation of glucose
Increased glycogen synthesis
Increased activity of the hexose monophosphate pathway (HMP)
Decreased glycolysis
The energy metabolism of skeletal muscle is unique in being able to 1/1

respond to changes in the demand for ATP that accompanies muscle
contraction. At rest, muscle accounts for approximately 30% of the
oxygen consumption of the body, whereas during vigorous exercise, it is
responsible for up to _____ of the total oxygen consumption. *
80%
85%
90%
95%
Which is a false statement re Brain metabolism in the well fed state? * 1/1
Although contributing only 2% of the adult weight, the brain accounts for a
consistent 30% of the basal oxygen consumption of the body at rest.
Normally, glucose serves as the primary fuel for the brain.
If blood glucose levels fall below approximately 40 mg/100 ml, cerebral function is
impaired.
If the hypoglycemia occurs for even a short time, severe and potentially irreversible
brain damage may occur
Other:
This form was created inside of University of Northern Philippines.
Forms
Biochem
1. Which statement is true regarding 0/1 the Fuel stores of a 70 kg man? *
● Fat is 15 kg while glycogen is only 0.2 kg.
● Protein is 8 kg.
● Although glycogen is listed as an energy source, only about one third of
the body's glycogen can be used for energy production without fatally
compromising vital functions
● Enormous caloric stores are available in the form of Carbohydrates
2. These are expected in the skeletal 1/1 muscle during fasting, except: *
● After about 3 weeks of fasting, muscle decreases its use of ketone bodies and
oxidizes fatty acids almost exclusively.
● During the first 1 week of fasting, muscle uses fatty acids from adipose
tissue and ketone bodies from the liver as fuels
● During the first few days of fasting, there is a rapid breakdown of muscle protein,
inorder to provide amino acids that will be then used by the liver for
gluconeogenesis.
● Glucose transport into skeletal muscle cells via insulin-sensitive GLUT-4 proteins
in the plasma membrane and subsequent glucose metabolism are depressed
because of low levels of circulating insulin.
3. Which statement is true regarding 0/1 the Effects of Insulin on lipid metabolism? *
● Insulin increases the level of circulating free fatty acids by inhibiting the
activity of hormone sensitive lipase that degrades triacylglycerol in adipose
tissue.
● Adipose tissue responds within seconds to administration of insulin, which
causes a significant reduction in the release of fatty acids.
● Insulin increases the transport and metabolism of glucose into adipocytes,
providing the substrate glycerol 3-phosphate for triacylglycerol synthesis.
● Insulin also increases the lipoprotein lipase activity of adipose tissue by
decreasing the enzyme's synthesis, thus providing fatty acids for esterification
4. 64-year-old John is presented to 0/1 his family doctor with complaints of frequent
episodes of dizziness and of numbness in his legs. During a routine history and physical
examination, the doctor finds that the patient leads a sedentary lifestyle, is obese (body
mass index of 32), and has hypertension (blood pressure of 200/120 mm Hg). The
patient is asked to return to the clinic a week later in the fasting state, during which time
a blood specimen is obtained, and a glucose tolerance test is glucose tolerance test is
performed. Blood analysis reveals fasting hyperglycemia, hyperinsulinemia,
dyslipidemia, and glucose intolerance.The diagnosis is type Il diabetes mellitus.
Alterations in substrate metabolism within which of the following organs can be a cause
for the observed blood analysis? *
● Brain
● Liver
● Kidney
● Heart
5. These correctly explains Fat Metabolism during the well fed state, except: *
● De novo synthesis of fatty acids from acetyl CoA in adipose tissue is low in
humans, except when refeeding a previously fasted individual
● Increased TAG synthesis: After consumption of a lipid-containing meal,
hydrolysis of the TAG of chylomicrons (from the intestine) and VLDL (from the
liver) provides adipose tissue with fatty acids (see Figure 24.5,).
● Because adipocytes lack glycerol kinase, glycerol 3-phosphate used in TAG
synthesis comes from the metabolism of glucose. Thus, elevated levels of
glucose and insulin favor storage of TAG
● Elevated insulin favors the phosphorylated (active) form of
hormone-sensitive lipase
6. Which is true about Inhibition of 0/1 Insulin secretion? *

● The synthesis and release of insulin are decreased when there is abundance of
dietary fuels, and also during periods of stress.
● Epinephrine can override the normal glucose-stimulated release of insulin. Thus,
in emergency situations, the sympathetic nervous system largely replaces the
plasma glucose concentration as the controlling influence over B-cell secretion.
● Epinephrine has a direct effect on energy metabolism, causing a slow
mobilization of energy- yielding fuels, including glucose from the liver and fatty
acids from adipose tissue
● The synthesis of Insulin is mediated primarily by epinephrine, which is
secreted by the adrenal medulla in response to stress, trauma, or extreme
exercise.
7. Maria has joined a beauty pageant 1/1 and decided to go on an "after six diet", thus
scheduling her early supper at 5:30 pm. Despite her new program, she claims to be still
feeling fine when she gets up at around 5:00 am to start her training. Maria feeling
"okay" is explained by the following except:
● liver glycogen is nearly exhausted only after 18-20 hours of fasting
● The liver first uses glycogen degradation and then gluconeogenesis to maintain
blood glucose levels in the fasted (postabsorptive) state.
● increased glucagon to insulin ratio causes a rapid mobilization of liver glycogen
stores due to phosphorylation (activation) of glycogen phosphorylase
● Gluconeogenesis, begins 4-6 hours after the last meal and becomes fully active
as stores of liver glycogen are depleted.
1 .How does cAMP regulate the action of Protein kinase A (PKA)?
1. Four molecules of CAMP bind to PKA and dissociate it into 2 catalytic subunits and 2
regulatory subunits
2. CAMP phosphorylates PKA which sets it into action.
3. CAMP does not affect the action of PKA
4. CAMP is initially bound to PKA to O prevent its action, and when it dissociates PKA is
able to function.
2.These statements describe alcohol intoxication except:
1. Increased availability of OAA allows acetyl CoA to be diverted to ketone body synthesis in liver
and can result in alcoholic ketoacidosis.
2. Alcohol intoxication can precipitate hypoglycemia, particularly in individuals who have depleted
their stores of liver glycogen.
3. Chronic alcohol consumption can also result in alcoholic fatty liver due to increased synthesis of
triacylglycerols as a result of decreased fatty acid oxidation due to a fall in the NAD+/NADH
ratio, and increased lipogenesis due to the increased availability of fatty acids and of
glyceraldehyde 3 phosphate
4. Alcohol consumption can also increase the risk for hypoglycemia in patients using insulin
3.Which of the following complications is less likely to occur in type Il diabetics, as opposed to type I
diabetics? *
1. Neuropathy
2. Retinopathy
3. Hypoglycemic coma
4. Cardiovascular disease
4. Which is not considered a stage of Catabolism? *
1. Conversion of building blocks to simple intermediates

2. Oxidation of FADH2
3. Oxidation of acetyl CoA
4. Hydrolysis of complex molecules
5. Which is NOT true regarding DM Type 1 *
1. There is partial deficiency of insulin caused by an auto-immune attack on the ß cells of the
pancreas, that over a period of years leads to gradual depletion of the B-cell population.
2. Symptoms appear abruptly when 80-90% of the B cells have been destroyed
3. Persons with type 1 diabetes constitute approximately 10% of diabetics.
4. Beta Cell destruction requires both a stimulus from the environment and a genetic determinant
can neither respond to variations in circulating fuels nor maintain a basal secretion of insulin.
6. Which is a false statement re Brain 1/1 metabolism in the well fed state?
1. If the hypoglycemia occurs for even a short time, severe and potentially irreversible brain
damage may occur
2. Although contributing only 2% of the adult weight, the brain accounts for a consistent 30% of
the basal oxygen consumption of the body at rest.
3. If blood glucose levels fall below approximately 40 mg/100 ml, cerebral function is impaired.
4. Normally, glucose serves as the primary fuel the brain.
1. After the concert, Dexter and his friends decided to proceed to a bar for some drinking spree. Later
then, Dexter starts to have mild tremors, palpitation and sweating. Dexter now is experiencing
hypoglycemia. Which statement is FALSE regarding alcohol metabolism resulting to hypoglycemia?
a. The abundance of NADH favors the reduction of lactate to pyruvate, and of malate to oxaloacetate.
b. Ethanol-mediated increase in NADH causes the intermediates of gluconeogenesis to be diverted

into alternate reaction pathways, resulting in the decreased synthesis of glucose.
c. Ethanol is first converted to O acetaldehyde by alcohol dehydrogenase.
d. Acetaldehyde is subsequently O oxidized to acetate by aldehyde dehydrogenase.
2. When you eat a large meal, the ______activity called digestion takes the biomolecules you consumed
and breaks them down into smaller molecules.
a. Respiratory
b. Catabolic
c. Anabolic
d. Metabolic
3. Which of the following is true regarding second messengers?
a. They are activated by voltage gated ion channels
b. They are activated by ligand gated and voltage-gated ion channels
c. None of these
d. They are activated by ligand gated ion channels
4. Glucagon is a polypeptide hormone secreted by the a cells of the pancreatic islets of Langerhans.
Which is true about Glucagon?
a. Glucagon is synthesized as a large precursor molecule (proglucagon) that is converted to glucagon

through a series of selective proteolytic cleavages
b. Glucagon acts to maintain blood glucose levels by activation of adipose tissues glycogenolysis and
gluconeogenesis.
c. Glucagon is composed of 32 amino acids arranged in a single polypeptide chain.
d. Glucagon, along with epinephrine, cortisol, and growth hormone (the "counter-regulatory
hormones"), opposes many of the actions of insulin.
5. A 17-year-old male presents complaining of an inability to perform strenuous exercise without
bringing on painful muscle cramps and weakness. He indicated that mild to moderate exercise resulted
in noproblems. When he was administered an ischemic exercise test, his serum lactate concentrations
did not increase significantly. A deficiency in which of the following enzymes is most likely the cause
ofthe patient's muscle cramps?
a. Carnitine palmitoyl transferase II
b. Glucose-6-phosphatase
c. Glycogen synthase
d. Glycogen phosphorylase
6. Which statement about Anabolic pathways is false?
a. Anabolic reactions combine small molecules, such as amino acids, to form complex molecules, such
as proteins.
b. Anabolism is a divergent process in which a few biosynthetic precursors form a wide variety of
polymeric or complex products.
c. Anabolic reactions require energy, which is generally provided by the breakdown of ATP to
adenosine diphosphate (ADP) and inorganic phosphate (Pi).
d. Anabolic reactions often involve chemical reductions in which the reducing power is most
frequently provided by the electron donor FADH.
7. Which is not an Adrenergic symptom of Hypoglycemia?
a. Tremor
b. Palpitation
c. Confusion
d. Anxiety
8. Philip has decided to train for an upcoming marathon. Nearing the age of 50, Phil figures that after he
trains he should be able to maintain a 9 minute-per-mile pace, which would mean that he would finish
the race in approximately 4 hours. He would be adequately hydrating himself at the various water
stations along the way, as he is about to finish the 26 mile 385 yard course. What is the primary fuel that
his leg muscles would be using?
a. Fatty acids from the blood
b. Glycerol from the blood

c. Ketone bodies from the blood
d. Glycogen stored in muscle
9. Neuroglycopenic symptoms often result from a gradual decline in blood glucose, often to levels below
a. 40 mg/dl
b. 60 mg/dl
c. 30 mg/dl
d. 50 mg/dl
1.You were scheduled to take your 1/1 long exam on Biochemistry on the last day of the exam week.
You spent only an approximately 4-5 hours of sleep for the past 3 days. You then decided to drink 1 cup
of kapeng barako to help you feel awake for the night. Which statement is false?
• Cyclic adenosine monophosphate (CAMP) and cyclic guanosine monophosphate (CGMP) are
intracellular second messenger molecules degraded and inactivated by the enzyme
phosphodiesterases(PDE).
• Phosphodiesterase inhibitors exert their effects on their targeted phosphodiesterase enzymes

preventing cGMP or CAMP degradation.
• Caffeine exerts its effects on phosphodiesterase enzymes by nonselective inhibition of PDE1,

and PDE5, promoting CAMP accumulation.
• Caffeine also inhibits adenosine and gamma-aminobutyric acid (GABA) receptors and
suppresses intracellular calcium release, improving and enhancing cognition, memory, energy,
sleep, fatigue, and drowsiness.
2.Flow of intermediates through the 0/1 pathways of energy metabolism is controlled by these
mechanisms, except:
• Polar modification of enzymes

• The availability of substrates
• Induction-repression of enzyme synthesis
• Allosteric regulation of enzymes
3.Dexter, who is an avid fan of Lady 0/1 Gaga, bought a ticket for her idol's concert. He was so excited
and made sure he memorized all Lady Gaga's songs to be able to sing out loud during the concert.
Because of his excitement, Dexter finished all his office work early, to be just in time for Lady Gaga's
concert at 8:00 pm. Dexter however failed to eat his lunch and dinner. But despite this, Dexter was still
able to dance and sing his heart out initially during the concert. Which is FALSE regarding the
glucoseregulating systems?
• Cortisol and growth hormone are less important in the short-term maintenance of blood glucose
concentrations. They do, however, play a role in the long-term management of glucose
metabolism
• Epinephrine promotes glycogenolysis and lipolysis, inhibits insulin secretion, and stimulates
the insulin-mediated uptake of glucose by peripheral tissues.
• Glucagon stimulates hepatic glycogenolysis and gluconeogenesis.
• Glucagon and Epinephrine are most important in the acute, short term regulation of blood
glucose levels.
4.Manang Thelma works as an orderly at a local hospital for 8 hours each day. She is given an hour for
her lunch break and a little “siesta”. According to Manang Thelma, lunch is her favorite meal. She then
makes sure to have a complete and well balanced meal every lunch time, to provide her the energy that
she needs for her remaining work. These statements are true regarding the Well fed state, Except:
• Elevated levels of glucose within the hepatocyte (as a result of elevated extracellular levels)
allow glucokinase to phosphorylate glucose to glucose 6-phosphate
• During this interval, transient increases in plasma glucose, amino acids, and triacylglycerols
(TAG) occur, the latter primarily as components of VLDL synthesized by the intestinal mucosal
Cells
• The absorptive (fed) state is the two-to four-hour period after ingestion of a normal meal.
• Islet tissue of the pancreas responds to the elevated levels of glucose and amino acids with an
increased secretion of insulin and a decreased release of glucagon.
5.A 62 y/o male patient who has 40 0/1 pack smoking history complained of difficulty of breathing. He
then consulted with a medical specialist and was diagnosed with Chronic Obstructive Pulmonary
disease. He was given medications including Theophylline. Why is Theophylline considered an effective
medication for COPD?
• Methylxanthines, the most common methylxanthine is theophylline, is commonly used only for
short term treatment of COPD by inducing bronchodilation
• PDE-4 is an enzyme found in cells of the lungs, and PDE-4 inhibitors, including Theophylline,
inhibit the degradation of intracellular cyclic adenosine monophosphate(CAMP) and increase
cAMP levels in target cells, further causing bronchial muscle relaxation.
• Phosphdiesterases hydrolyze the phosphodiester bond of CAMP and CGMP to form the active
5’-AMP and 5’-GMP
• Phosphodiesterase are a class of medications that promote blood vessel dilation (vasodilation)
and smooth muscle relaxation in certain parts of the body, such as the heart, lungs, and genitals.
Aling Marites is a 58 y/o patient diagnosed with End stage renal disease and is on regular Hemodialysis
for the past 2 years already. She had previous history as well of metabolic acidosis where she presented
with increased and acidotic breathing.She was previously advised by her Nephrologist to take small
frequent feedings and to avoid fasting. During her Dialysis sessions, they would need to travel
approximately 2 hours from home to the hospital, and each session entails 4 hours. Aling Marites then
makes sure to bring snacks during each session. The kidneys plays a very vital role during fasting, thus
Chronic kidney disease patients are not advised prolonged fasting. Which statement is false.
A) The glutamine released from the muscle’s metabolism of branched chain amino acids is taken
up by the kidney and acted upon by renal Oglutaminase and glutamate dehydrogenase,
producing a ketoglutarate that can be used as a substrate for gluconeogenesis, plus ammonia
(NH3).
B) Kidney also provides compensation for the acidosis that accompanies the increased production
of ketone bodies.
C) In long-term fasting, there is a switch from nitrogen disposal in the form of urea to disposal in
the form of ammonia.
D) The kidney expresses the enzymes of gluconeogenesis, including glucose 6-phosphatase, and in
late fasting about 30% of gluconeogenesis even occurs here.
Bianca, a 6 year old patient, was brought to the emergency room presenting with vomiting and deep
gasping breath. Physical exam reveals a fruity smelling breath. Her blood glucose was checked which
revealed a Random blood sugar level of 635 mg/dl. Arterial Blood gas was likewise facilitated showing
metabolic acidosis. Urine Ketones were likewise positive. Which FALSELY explains hyperglycemia and
ketoacidosis.
A) DKA is treated by replacing fluid and electrolytes, and administering long acting insulin to
gradually correct hyperglycemia without precipitating hypoglycemia
B) Ketosis results from increased mobilization of fatty acids from adipose tissue, combined with
accelerated hepatic fatty acid ß oxidation and synthesis of 3 hydroxybutyrate and acetoacetate.
C) Elevated levels of blood glucose and ketones are the hallmarks of untreated type 1 diabetes
mellitus
D) Diabetic ketoacidosis occurs in 25 40% of those newly diagnosed with type 1 diabetes, and may
recur if the patient becomes ill or does not comply with therapy.
Which statement is FALSE regarding Insulin resistance and obesity.
A. In the absence of a defect in B-cell function, diabetic, obese individuals can compensate for
insulin resistance with elevated levels of insulin.
B. Obesity is the most common cause of insulin resistance; although most people with obesity and
insulin resistance do not become diabetic.
C. insulin secretion is two to three times higher in obese subjects than it is in lean individuals as a
compensatory mechanism
D. insulin resistance is characterized by uncontrolled hepatic glucose production, and decreased
glucose uptake by muscle and adipose tissue.
Which statement about Catabolic pathways is false? *
A. Catabolic reactions are reactions that break down complex molecules and serve to capture
chemical O energy in the form of adenosine triphosphate (ATP) from the degradation of energy-
rich fuel molecules.
B. Catabolic pathways are typically oxidative, and require coenzymes such as NAD+
C. Catabolism allows molecules in the diet (or nutrient molecules stored in cells) to be converted
into building blocks needed for the synthesis of complex molecules.
D. Energy generation by degradation of complex molecules occurs in four stages
Second messenger cascades are frequently initiated by activation of a G protein-coupled receptor

(GPCR). Ligand binding to the extracellular domain of the GPCR triggers a conformation change in the
GPCR that permits activation and dissociation of the G protein to which it is associated. What is the
biochemical change catalyzed by the activated GPCR that permits activation of its associated G protein?
A. The GPCR phosphorylates protein kinases, which phosphorylate and activate the G protein
B. The GPCR exchanges the G protein's bound GDP for a GTP
C. The GPCR breaks covalent bonds between the intracellular domain and the G protein
D. The GPCR opens ion channels, and O influx of calcium activates the G
Intensive treatment with insulin delays the onset and slows the progression of long-term complications.
These statements are true, except: *
A. Glycated proteins mediate some of the early microvascular changes of diabetes.

B. Clinical evidence supports initiating intensive therapy with the goal of lowering HbA1C levels to
below 7.5% as early as possible in the course of diabetes.
C. The benefits of tight control of blood glucose outweigh the increased risk of severe
hypoglycemia in most patients
D. Incidence of retinopathy decreases as control of blood glucose improves and HbA1C levels also
decrease
1. You then advise John the following, except: *
A. Weight reduction, exercise, and medical nutrition therapy (dietary modifications)
B. Ophthalmologist consult 5 years after the initial diagnosis.
C. Fasting Blood glucose of 80-130 mg/di
D. Postprandial Blood Glucose of < 160 mg/dL
2. Which is NOT true about the regulation of metabolism?
A. mgells function in isolation so they could interact with the other tissues.
B. Regulatory signals that inform an individual cell of the metabolic state of the body as a whole include
hormones, neurotransmitters, and the availability of nutrients
C. Regulatory signals influence signals generated within the cell.
D. The pathways of metabolism must be coordinated so that the O production of energy or the synthesis
of end products meets the needs of the cell.
3. Protein kinase A is _____.
A Activated by covalent binding of cyclic AMP
B Allosterically activated by cyclic AMP
C. Completely inhibited by cyclic AMP
D. Affected by cyclic AMP only under unusual circumstances
4. Regulation of Metabolism is characterized by the following except:
A Signals from within the cell (intracellular)
B. Communication between cells (intercellular)
C. Phosphodiesteraise inositol pathway
D. Second messenger systems
5. Which is true about Insulin?
A Insulin is composed of 52 amino acids arranged in two polypeptide chains, designated A and B, which
are linked together by two disulfide bridges.
B Insulin's metabolic effects are anabolic, favoring, synthesis of glycogen, triacylglycerols, and protein.
C Pig (porcine) and beef (bovine) insulin differ from human insulin at one and three amino acid
positions, respectively.
D Insulin is a polypeptide hormone produced by the B cells of the islets O of Langerhans - which make
make up only about 2-4% of the total cells of the pancreas.
6. These statements describe the Mechanism of insulin action, except:
A Autophosphorylation initiates a cascade of cellsignaling responses, including phosphorylation of a

family of proteins called insulin receptor substrates (IRS)
B. The insulin receptor is synthesized as a single polypeptide that is glycosylated and cleaved into a and
3 subunits, which are then assembled into a tetramer linked by disulfide Bonds
C Insulin binds to specific, high affinity receptors in the cell membrane of most tissues, including liver,
muscle, and adipose.
D The binding of insulin to the a subunits of the insulin receptor induces conformational changes that
are transduced to the a subunits promoting a rapid auto phosphorylation of specific tyrosine residues
ofreach subunit protein
7. Glucagon is secreted by the following, except:
A Low blood glucose
B Epinephrine
C Carbohydrate-rich meal
D Amino acids
8. Which is FALSE regarding the Diagnosis of type 1 diabetes
A Symptoms of abrupt polyuria, polydipsia and uropathy often triggered by stress or an illness are
significant.
B When the diagnosis of type 1 diabetes is uncertain by clinical presentation, testing for circulating islet-
cell antibodies is recommended.
C Diagnosis is confirmed by a fasting blood glucose (FBG) ≥ 126 mg/dl, commonly accompanied by
ketoacidosis.
D Other accompanying symptoms are fatigue, weight loss, and weakness.

Biochemistry 2nd Sem 21-22 Midterm Quiz
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Biochemistry 2nd Sem 21-22 Midterm Quiz
Dr. Brendo V. Jandoc
GENERAL INSTRUCTIONS
Select A if statements A, B and C are correct
Select B if statements A and C are correct
Select C if statements B and D are correct
Select D if only statement D is correct
Select E if all of the statements are correct
Select F if all of the statements are incorrect
Functions of vitamin D: A. Increase intestinal calcium uptake B. Stimulate 1 point

bone resorption C. Minimize renal calcium loss D. Maintain adequate
calcium plasma levels *
E
F
Na+, K+, and Cl-: A. Maintain ECF and ICF osmolarity B. Maintain water 1 point
balance C. Neutralize positive and negative charges on proteins and other
molecules D. Regulate pH *
Minerals as cofactors may A. form complexes with substrates B. play direct 1 point
role in catalysis C. serve as redox reagent D. serve as precursors of
coenzymes for the enzymes of intermediary metabolism *
F
Vitamin B12 deficiency: A. Alter the folate enterohepatic cycle B. More 1 point
commonly due to vitamin malabsorption C. Interfere with the
enterohepatic cycle D. Rarely due to absence of the vitamin *
Retinoic acid: A. Support spermatogenesis in the male B. Essential for 1 point

normal reproduction C. Prevent fetal resorption in the female D. Promotes
growth and differentiation of epithelial cells *
D
Retinol: A. Contains b-ionone ring with an unsaturated side chain B. High 1 point
affinity binding to specific receptor proteins in the nucleus of target
tissues C. Found in animal tissues as a retinyl ester with long-chain fatty
acids D. Mediates most of the actions of the retinoids except vision *
Ascorbic acid: A. Coenzyme in hydroxylation of prolyl and lysyl residues 1 point

during collagen biosynthesis B. Reducing agent in several different
reactions C. Protective agent against free radical damage D. Aids dietary
iron absorption from the intestines *
B
Biochemical indicators of pyridoxine deficiency: A. Decreased blood PLP 1 point
levels B. Decreased RBC transaminase activity C. Decreased urinary
pyridoxic acid excretion D. Increased urinary cystathionine excretion *
Vitamin deficiency due to inadequate absorption: A. Biliary obstruction B. 1 point

Pernicious anemia C. Regional Ileitis D. Renal malfunction *
F
Biotin functions as: A. carrier of activated carbon dioxide B. receives 1- 1 point
carbon fragment from donors C. coenzyme in carboxylation reactions D.
transfers 1-carbon fragment to intermediates *
Wernicke's encephalopathy: A. Confusion B. Ophthalmoplegia C. Truncal 1 point

ataxia D. Neuropathy *
A
Thiamine as coenzyme: A. Conversion of a-ketoglutarate to succinyl-CoA 1 point
B. Conversion of pyruvate to acetyl-CoA C. Creation of branched-chain
amino acids leucine, isoleucine, and valine D. Transketolation reactions
found in the pentose phosphate pathway *
Riboflavin deficiency: A. Frequently accompanies other vitamin 1 point

deficiencies B. Genetic defect in tryptophan membrane transport C. Not
associated with a major human disease D. Tryptophan is used for excessive
5-hydroxytryptamine production *
D
Menaquinone: A. Found in plants B. In intestinal bacterial flora C. Synthetic 1 point
derivative D. Found in mammalian liver *
Critical functions of bound Ionic Ca++: A. Ion transport B. Muscle 1 point

contractions C. Nerve impulse transmission D. Signal transmission across
membranes *
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Biochemistry 2nd Sem 21-22 Midterm Quiz 2
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Biochemistry 2nd Sem 21-22 Midterm Quiz 2
Select the SINGLE BEST ANSWER.
Take note of your time.
The exam closes at 8:20 PM google time.
Codes for amino acids in proteins *
TAA
Exons
TGA
Introns
Is produced by RNA polymerase I. *
hnRNA
mRNA
tRNA
rRNA
The mechanism for termination of protein synthesis in eukaryotes requires *
release factors
nuclease cleavage of mRNA
a peptidyl-tRNA that cannot bind at the P site
the codon UGA, UAG, or AUG in the A site
The enzyme that joins segments D and E is (Use the figure to answer the
questions.) *
the replicative DNA polymerase
an endonuclease
a repair DNA polymerase
polynucleotide ligase
RNA polymerase
The sequence of part of a DNA strand is -ATTCGATTGCCCACGT-. When this

strand is used as a template for DNA synthesis, the product will be which one of
the following? *
TAAGCTAACGGGTGCA
ACGUGGGCAAUCGAAU
UAAGCUAACGGGUGCA
TGCACCCGTTAGCTTA
ACGTGGGCAATCGAAT
Eukaryotic genes that produce mRNA *
do not contain intervening sequences or introns
may contain a CAAT box in the 5' flanking region
contain a TATA box downstream from the start site of transcription
are transcribed by RNA polymerase III
contain long stretches of thymine nucleotides that produce the poly(A) tail of mRNA
Which of the following steps in the conversion of genetic information into a final
processed gene product is the most important mode for control of eukaryotic
gene expression? *
Initiation of transcription
Translation
Post-translational processing
Transport of transcript to cytoplasm
Transcript processing
When synthesis of segment C begins, which other segment is also being
synthesized? (Use the figure to answer the questions.) *
A
Analysis of a cell line that rapidly transforms into a tumor cell line demonstrated
an increased mutation rate within the cell. Further analysis indicated that there
was a mutation in the DNA polymerase enzyme that synthesizes the leading
strand. This inactivating mutation is likely to be in which of the following activities
of this DNA polymerase? *
Ligase activity
5′-3′ exonuclease activity
Phosphodiester bond making capability
3′-5′ exonuclease activity
Uracil-DNA glycosylase activity
Inhibits translocation. *
5-Fluorouracil
Tetracycline
Streptomycin
Erythromycin
Rifampicin
Which of the following statements about methionine is true? *
It is generally found at the N-terminus of proteins isolated from cells

It is formylated when it is bound to tRNA in eukaryotic cells
It requires a codon other than AUG to be added to growing polypeptide chains
It is the amino acid used for initiation of the synthesis of proteins
Contains introns and a cap at the 5' end. *
rRNA
tRNA
hnRNA
mRNA
Which of the following is made of several enzymes with helicase and nuclease
activity? *
RNAi
Short interfering RNA
Micro-RNAs
RNA-induced silencing complex
Which of the following sequences is complementary to the DNA sequence 5'-

AAGTCCGA-3'? *
3'-TCGGACTT-5'
5'-TTCAGGCT-3'
5'-AAGUCCGA-3'
3'-TTCAGGCT-5'
A bacterial mutant grows normally at 32°C but at 42°C accumulates short
segments of newly synthesized DNA. Which of the following enzymes is most
likely to be defective in this mutant? *
Polynucleotide ligase
An unwinding enzyme
An exonuclease
An endonuclease
DNA polymerase
Used by retroviruses to copy their RNA genome. *
Reverse transcriptase
Polynucleotide ligase
RNA polymerase
DNA polymerase
A man is suffering from chills, vomiting, and cramping and was rushed to the
emergency department. He had eaten wild mushrooms for dinner that he had
picked earlier in the day. His symptoms are due to an inhibition of which of the
following enzymes? *
DNA primase
RNA polymerase III

RNA polymerase III
Telomerase
RNA polymerase I
RNA polymerase II
It inhibits the binding of aminoacyl-tRNA in the A-site of the ribosomal complex. *
Lincosamides
Kanamycin
Aminoglycosides
Neomycin
Erythromycin
Chloramphenicol
Tetracycline
Puromycin
Gentamicin
Streptomycin
A man is diagnosed with a typical pneumonia. He was prescribed erythromycin,

which is specific for prokaryotic cells. Which of the following best explains the
mechanism of prokaryotic specificity? *
The drug binds to the 50S ribosomal subunit of bacteria and inhibits f-met-tRNAi
binding
The drug binds to the 30S ribosomal subunit of bacteria and blocks peptide bond
formation
The drug binds to the 50S ribosomal subunit of bacteria and blocks translocation
The drug binds to the 30S ribosomal subunit of bacteria and blocks initiation of
protein synthesis
protein synthesis
The drug binds to both ribosomal subunits and prevents bacterial ribosome assembly
Replication of a particular DNA sequence is noted to be under inhibitory control

usually. However, when substance “A” is added, it binds to a repressor, rendering
the repressor inactive and allowing transcription to occur. Which of the following
terms describes agent “A”? *
Transcriber
Inducer
Polymerase
Histone
Operon
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BIOCHEMISTRY

[BIOCHEM] LEC 1- BASIC CONCEPTS OF METABOLISM

LECTURE OUTLINE 2. Anabolic (synthetic)

Fig.1. Catabolism vs. Anabolism

- Contains the important central pathways of energy

UNP CMED 1F Page 1 of 6

- Building blocks are further degraded to acetyl coenzyme A

3. Oxidation of acetyl CoA:

- The tricarboxylic acid (TCA) cycle is the final common

- Generates larger amount of ATP

IV. ANABOLIC PATHWAYS

• Combine small molecules, such as amino acids, to form

Fig.2. Metabolic Map

III. CATABOLIC PATHWAYS

• Catabolic reactions capture chemical energy in the form of

A. Three stages of Catabolism

-Complex molecules are broken down into component

UNP CMED 1F Page 2 of 6

• Catabolism and anabolism pathways must be regulated.

Fig.6. Intracellular communication

The ligand binds to the receptor causing change in receptor’s

B.Communication between cells (Intercellular)

Fig.4. Some commonly used mechanisms for transmission of

3 Ways to Regulate Metabolism

A. Signals from within the cell (Intracellular)

UNP CMED 1F Page 3 of 6

Fig.9. Structure of a typical I G protein-coupled receptor

a. GTP- dependent regulatory protein

• The effect of the activated, occupied GPCR on second

UNP CMED 1F Page 4 of 6

• Protein kinase A can also phosphorylate proteins that bind

Fig.11. Actions of cAMP

2 Ways of Protein Kinase Regulation

UNP CMED 1F Page 5 of 6

• Phosphodiesterase is inhibited by methyl xanthine 4.__________Signal that provides long-range integration of

a) All statements are true

7. True or False: Gi stimulates adenylyl cyclase while the Gs

8. Regulatory signals that inform an individual cell of the

UNP CMED 1F Page 6 of 6

o Type II Diabetes Mellitus - you have enough insulin

Figure 4. Structure of Insulin.

3 stimulants for insulin secretion:

Figure 10. Effects of insulin on glucose and lipid metabolism.

B. EFFECTS ON LIPID METABOLISM

C. EFFECTS ON PROTEIN SYNTHESIS A. MEMBRANE EFFECTS OF INSULIN

*See larger image in appendices

VII. GLUCAGON - In these situations, regardless of the concentration of

Figure 14. Opposing actions of insulin and glucagon plus

kinase molecules. This causes the production of many active

*See larger image in appendices

UNP CMED 1F Page 7 of 15

hypoglycemia. The most important hormone that combats A. SYMPTOMS OF HYPOGLYCEMIA

UNP CMED 1F Page 8 of 15

IX. TYPES OF HYPOGLYCEMIA 2. Postprandial Hypoglycemia:

Figure 20. Reversal of insulin-induced hypoglycemia by

UNP CMED 1F Page 9 of 15

4. Hypoglycemia and Alcohol Intoxication: X. ALCOHOL INTOXICATION

*See larger image in appendices

UNP CMED 1F Page 10 of 15

UNP CMED 1F Page 11 of 15

Figure 17. Stimulation and inhibition of glycogen degradation.

UNP CMED 1F Page 12 of 15

UNP CMED 1F Page 13 of 15

KEY CONCEPT MAP