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What Is Life?

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• • • • • • • • •

Characteristics of Living Things - Summary
Organization Evolution of populations DNA Reproduction Growth/development Response to environment Metabolism Homeostasis Contain one or more cells

Living things contain one or more Cells

Cell
-Basic unit of life - Smallest unit with the capacity to live and reproduce, independently or as part of a multicellular organism

How do we know this?
To STUDY and UNDERSTAND cells, we had to SEE them…

When were ‘cells’ first observed? a.Early 1400s b.Late 1500s c. Mid 1600s d.Early 1800s e.Early 1900s
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A Very Brief History of Cytology (Cell Biology)
Late 1500s (1595) • Zacharias Jansen invents the first microscope Mid 1600s • Leeuwenhoek greatly improves microscopes 1665 • “Micrographia”by Robert Hooke – Credited with coining the term ‘cell’ to describe the compartments he viewed in cork slices
• Hooke did not understand what his observations meant

Origin of the term 'cell‘. Robert Hooke is credited as the originator of the term ‘cell’, which he used in his description of the structure of cork. This illustration is taken from his book on microscopy, referred to as 'Micrographia'.

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A Very Brief History of Cytology (Cell Biology)
• After 1665…Not much happened in Cell Biology! • Scientists were limited by: 1) Optical instruments
• Limited resolving power of microscopes • Lack of detail
Rembrant. The Anatomy Lesson of Dr. Nicolaes Tulp. 1632

2) Their way of thinking
• 17th Century was an “Age of Observation” • Descriptive science
… “Ooohh! Look at this!!!”

• Not really interested in “WHY???”

• More than 150 years later…

A Very Brief History of Cytology (Cell Biology)
• 1830’s: Advances in Optics
– Lens quality improved – Development of the Compound Microscope
• Improved both Magnification & Resolution

• 1831: Robert Brown describes the ‘Nucleus’
– Scottish botanist, observed plant cells and plant fertilization – Noticed that every plant cell had a ‘rounded structure’ • Called it a ‘nucleus’…Latin for “kernel”
• Further implied the role of the nucleus in fertilization and development of the plant embryo
– FYI: Pollen grains suspended in water also led Brown to the discovery of ‘Brownian motion’

• Schleiden & Schwann…

A Very Brief History of Cytology (Cell Biology) 1838 • ‘Cell Theory’ put forth by Schleiden & Schwann – German scientists and colleagues – Schleiden was was a botanist
• Observed cell division in plants

– Schwann was a physiologist
• Observed cell division in animal tissues FYI: Legend has it:
• Shared their observations over dinner one evening • Schwann published these findings, omitting Schleiden as author • Thus, Schwann ‘scooped’ Schleiden!

A Very Brief History of Cytology (Cell Biology)
Cell Theory - Schwann (1839)
1) The cell is the unit of structure, physiology, and organization in living things.
2) The cell retains a dual existence as a distinct entity and a building block in the construction of organisms. 3) Cells form by free-cell formation, similar to the formation of crystals

Cell Theory - Interpreted
1) All organisms consist of one or more cells. 2) Cell is the basic unit of structure for all organisms. 3) Cells form by spontaneous generation.
WHAT’S WRONG WITH THIS THEORY?
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Cell Theory - Interpreted
1) All organisms consist of one or more cells. 2) Cell is the basic unit of structure for all organisms. 3) Cells form by spontaneous generation.

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“Omnis cellula e cellula”
1855 - Virchow
- Revised Schwann’s postulate - Based on:
- Brown’s discovery of the nucleus
RudolphVirchow

- Louis Pasteur’s discoveries of ‘germs’, refuting spontaneous generation

 All cells arise from only preexisting cells
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Cell Theory - Revised

1) All organisms consist of one or more cells.
2) Cell is the basic unit of structure for all organisms.
-Schlieden &Schwan (1839)

3) Cells form by spontaneous generation.
-Schwan (1839)

3) All cells arise only from preexisting cells.
-Virchow (1855)
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Founders of Cell Biology

RobertBrown RudolphVirchow LouisPasteur

• All provided important contribution that helped create a foundation for Cell Biology

Welcome to the World of the Cell!!!

Cells are just as diverse as Organisms…
Plant Cells: Protist Cells: Bacterial Cells:

Human Cells:

FYI: Estimated that the ‘average’ human body has ~30+ trillion cells!
Source: Bianconi et al. (2013) Annals of Human Biology.40, 463-471

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Images from Figure1-1

Phylogenetic Organization of Cells

There are ____different ‘Domains’ of living organisms recognized in the contemporary phylogenetic tree of life.
a) b) c) d) e) two three four five ten

Tree of Life
• Tree of Life is continually being redrawn
– Earlier classification systems based largely on morphological characteristics • Scala naturae (350 BC)
– Classification scheme outlined in Aristotle’s History of Animals » All matter organized as decreed by God » Definition of terms still used today: Vertebrates/Invertebrates

• 2 kingdoms (1700s)
– Classification pioneered by Carl Linneaus & his bionomial taxonomy – Everything was either a Plant or an Animal » Bacteria were considered plants

• 5 kingdoms (1960’s)
– Monera (prokaryotes), Protista, Plantae, Fungi, Animalia » FYI: Original paper published in Science:
Whittaker R.H. (1969). Science 163:150–160.
http://www.sciencemag.org/content/163/3863/150.full.pdf?ijkey=e364e760bafd3ddb27f995f0c1e9b08fdf28eb0f&keytype2=tf_ipsecsha

Tree of Life
• Current Phylogeny = 3 Domain system
Bacteria = Prokaryotes Archaea = Prokaryotes, many extremophiles Eukarya = Eukaryotes
• Group includes Protists, Plants, Fungi, Animals

• Pioneered by Carle Woese & George Fox in the late 1970’s
• Formally recognized in the 1990’s

• Redrawing of phylogenetic relationships based on analysis of rRNA sequences
• Why do you suppose rRNA was used for this purpose?

Redrawing of phylogenetic relationships based on analysis of rRNA sequences

• All cells require rRNA
Why?  Component of ribosomes

• rRNA sequences change slowly with time
– Reflects evolutionary history of life – Can be used to establish evolutionary relationships between all species

• Using such data, phylogenetic relationships were redrawn…

3 Domains of Life
‘Hypothetical’ Phylogenetic Tree of Life

Last Universal Common Ancestor

~3.5 BYA

*Note: Origins of LUCA and Eukarya lineage are hypothetical Reviews - Nature 440: 623-630; BioEssays 29: 74-84

Fig 26.21 24 from Campbell et al., 9th ed.

3 Domains of Life
• rRNA analysis revealed two separate groups of prokaryotes:

- Bacteria - Archaea
• Also suggests that eukaryotes and archaea are more closely related to each other than to bacteria
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What are Protists?
a) Diverse grouping of eukaryotic organisms b) A type of prokaryote
c) A single-celled organism d) A type of delicious pastry e) None of the above.

Protists
• Many diverse lineages of various eukaryotic organisms
• Can be unicellular
– e.g. dinoflagellates
Red tide dinoflagellate

• Can be multicellular
– e.g. some algae species
kelp

What is a ‘prokaryote’? What is a ‘eukaryote’?

Prokaryotic Cells vs. Eukaryotic Cells
Ave. Size of Prokaryotic Cell ~1-5 um diameter

Figure 4-3

Ave. Size of Eukaryotic Cell ~10 – 100 um diameter
Figure 4-5
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Prokaryotes vs. Eukaryotes
• Prokaryotic cells (Bacteria & Archaea domains)
– “pro” (before) + “karyon” (nucleus) – Lack internal complexity seen in Eukaryotes – Average size ~1-5 μm diameter

• Eukaryotic cells (Eukarya domain)
– – – –
“eu” (true) + “karyon” (nucleus) Nucleus Extensive membrane-bound organelles Average size ~10-100 μm diameter

• Plant and animal cells
- Share many of the same subcellular structure - We’ll point out a few major differences

Regardless of their diversity, all cells share some common features…

Basic Features of All Cells:
1) Surrounded by Lipid-Based Plasma Membrane 2) Metabolic Machinery

3) DNA as Hereditary Information
4) Ribosomes as ProteinSynthesizing Machinery

Inner Life of a Cell
• http://multimedia.mcb.harvard.edu/media.html

Cells are teeny-tiny!
http://learn.genetics.utah.edu/content/begin/cells/scale/

Sizes of Cells and Subcellular Structures
“The World of the Micrometer”

Micrometer (m) is the most useful unit for expressing the size of cells and larger organelles

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Fig 1A-1

Sizes of More Subcellular Structures
“The World of the Nanometer”

Nanometer (nm) -orAngstrom (Å) are the units used to express size of molecules and smaller subcellular structures
= 70 Å

= 70-80 Å

= 20 Å

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Fig 1A-2

Prefixes for SI/Metric Units

* * *

Base Unit

* * * * * * *

u

* = important for Cell Biology

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Other Common Units
Angstrom (Å) = 0.1 nm or 10-10 m
• Commonly used to express dimension of molecules
20 Å

Ex. DNA helix diameter = 2 nm
 Convert to Å

2 nm x 1 Å/0.1 nm = 20 Å
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Other Common Units
Dalton (Da) = unified atomic mass unit = 1/12 12C = 1.66x10-24 g
• Commonly used to express size of proteins

Ex. Hemoglobin (Human) = 68000 Da – OR – = 68 kilodaltons (kD)
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Units Worksheet
- Posted on Blackboard (along with KEY) under ‘Course Documents’ - This is for your own practice. - This will not be handed in for assessment, but you are responsible for this material

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Cell = Basic Unit of Life

- Smallest unit with the capacity to live and reproduce, independently or as part of a multicellular organism

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Cell Division Pattern in Saccharomyces cerevisiae

Cell Size

growth

mitosis

0

1

2

3 Time

4

5

6

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Q: Why don’t the yeast cells just keep growing and growing? Why aren’t we just one giant cell?

Limits to cell size

Cell Size

growth

mitosis

0

1

2

3 Time

4

5

6

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What limits cell size?
1) Resource Availability (e.g. nutrients, space)
Let’s assume resources are plentiful. Cell size is then primarily limited by…

2) *Surface Area to Volume ratio
This in turn affects…

3) Rate of molecule diffusion within cell 4) Maintenance of adequate local concentrations of molecules within cell
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Surface Area to Volume ratio
– limits cells size To demonstrate this concept… Which has a greater surface area to volume ratio?
(provide calculations to support your answer) a) Cell 1: 1µm x 1µm x 1µm b) Cell 2: 5µm x 5µm x 5µm c) Multicellular Organism 1: 125(1µm x 1µm x 1µm)

Cell 1

Cell 2

Multicellular Organism 1

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Limits to Cell Size: Surface to Volume Ratios

*Similar to Figure 4-1

Limits to cell size

Cell Size

growth

mitosis

0

1

2

3 Time

4

5

6

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Surface Area to Volume ratio
– limits cells size To demonstrate this concept… Which has a greater surface area to volume ratio?
(provide calculations to support your answer) a) Cell 1: 1µm x 1µm x 1µm b) Cell 2: 5µm x 5µm x 5µm c) Multicellular Organism 1: 125(1µm x 1µm x 1µm) d) More than one of the above

Cell 1

Cell 2

Multicellular Organism 1

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Limits to Cell Size: Surface to Volume Ratios

*Similar to Figure 4-1

Surface Area to Volume ratio
- limits cells size
SA to V ratio is critical to cell metabolism SA (x2) = plasma membrane V (x3) = cell contents

…As cells increase in size…
• V (x3) grows proportionately more than SA (x2)
…leads to…

• Lower SA to V ratio
…leads to…

• Problematic exchange of substances between cell & environment
…affects...

• Localized [molecule] • Diffusion rate of molecules in cell
 Affects RATES of chemical reactions …Slow is not good!

How do cells cope with SA:V constraints?

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Strategies to cope with SA:V constraints
Cells divide
mitosis

Cell Size

0

1

2

3

4

5

6

7

Time

Strategies to cope with SA:V constraints
Growth Stops
- Cell enters G0phase
• • Withdraw from cell cycle No growth/proliferation

- Cell may become:
• Quiescent - Phase is reversible
i.e. some cells can re-enter Cell cycle to begin division e.g. some stem cells

Terminally differentiated - Non-reversible
e.g. cardiac muscle cells

Fig 19-32

Strategies to Increase Surface Area
Membrane Folding: e.g. brush border cells of intestinal epithelium

Figure 4-2

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Strategies to Increase Surface Area
Membrane Folding – also seen in some prokaryotes
e.g. Anabaena azollae (type of Cyanobacteria)

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Figure 11-4

Strategies to Increase Surface Area
Membrane Folding – not limited to the plasma membrane in eukaryotes
e.g. endoplasmic reticulum e.g. thylakoid membrane in chloroplast

Other Strategies Cells use to Cope with SA:V Constraints?

Which has the greatest surface area to volume ratio? a) Cell 1: 1µm x 1µm x 1µm

b) Cell 2: 5µm x 5µm x 5µm
c) Organism 3: 125(1µmxµm1xµm1) d) Cell 4: 0.125µm x 5µm x 200µm
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e) More than one of the above

200

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0.125

Strategies to Increase Surface Area Long, thin cells – greater surface area:volume

Muscle fiber

Neuron
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What about egg cells? Do egg cells break the SA:V rule?

Figure 1. Comparisonof egg sizes. Ostrich egg (right), compared to chicken egg (lower left) and quail eggs (upper left).
Photo by Rainer Zenz.

Do egg cells break the SA:V rule?
• Although some egg cells can be quite large…
– Ostrich eggs are several centimeters long!

• …Metabolically inactive • …Mostly lipids (fats), storage materials • …Actual viable ‘cell’ component is very small

Strategies to cope with SA:V constraints: Active Transport
• Transportation of ‘cargo’ by specialized carrier proteins

• May involve:
• Cytoskeleton
- Motor proteins + filamentous tracks

• Carrier Proteins
- Transport across membranes

• ACTIVE process = Expenditure of E by the cell

Strategies to cope with SA:V constraints: Cytoplasmic Streaming
• Bulk movement of cytoplasm
– Involves microfilaments

• Active process (E expenditure)

http://bio1151.nicerweb.com/med/Vid/Campbell7e/CytoplasmicStream-V.swf

Strategies to cope with SA:V constraints: Multicellularity
• SA:V sets an upper limit on cell size • The only way to get larger is for cells to cooperate • Driving force behind evolution of multicellularity?
*Similar to Figure 4-1
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- Volume stays the same - Surface Area increases

Question to ponder: What factors make it possible for eukaryotic cells to be so much larger than prokaryotic cells?

Ave. Size of Prokaryotic Cell ~1-5 um diameter Ave. Size of Eukaryotic Cell ~10 – 100 um diameter
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“Tour of the Cell”

‘Typical’ Prokaryotic cell
• Ave. Size of Prokaryotic Cell = ~1-5 um diameter

• Relatively ‘Simple’ Organization:
- Cell wall: protective layer of carbohydrate surrounding plasma membrane - Plasma membrane

Motility structure (e.g. flagella)

- DNA (not enclosed) – nucleoid region
-Some species have plasmids

- May/not have motility structure

Electron micrograph of cross section throughE. coli.

Eukaryotic Cell
e.g. ‘typical’ Animal cell
ENDOPLASMIC RETICULUM (ER)

Nuclear envelope Nucleolus Chromatin NUCLEUS

Rough ER Flagelium Centrosome
(with centrioles)

Smooth ER

Plasma membrane

CYTOSKELETON
Microfilaments Intermediatefilaments Microtubules

Ribosomes

Microvilli
Golgi apparatus Peroxisome Mitochondrion Lysosome Similar to Fig 4-5
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Eukaryotic Cell
e.g. typical Plant cell
Nuclear envelope NUCLEUS

Nucleolus Chromatin
Centrosome

Rough endoplasmic reticulum

Smooth endoplasmic reticulum

Ribosomes (small brwon dots)

Central vacuole

Golgi apparatus

Tonoplast Microfilaments Intermediate filaments Microtubules CYTOSKELETON

Mitochondrion Peroxisome Plasma membrane Cell wall Plasmodesmata Wall of adjacentcell Chloroplast

Similar to Fig 4-6

In plant cells but notanimal cells: Chloroplasts Central vacuole and tonoplast Cell wall Plasmodesmata

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Before we discuss cells… a few cell/molecular biology techniques…

Cell Fractionation
• Separation of a cell structures/components • Two phases 1. Homogenization
• lysing cells open • chemicals, enzymes, or sound waves

2. Centrifugation
Homogenize tissue
Mouse liver

Centrifugation
• Use of centrifugal force to separate mixture(s)
– Fixed-angle – Swinging-bucket

• Separation based on density & size
– “Pellet” = Substance(s) that sediment at bottom – “Supernatant” or “Super” = remaining liquid – Sometimes have an “Interface” – region between two phases
Figure 12A-1

Sedimentation coefficient (S)
= Measure of how rapidly a sample sediments when subjected to centrifugal force

Similar to Figure12A-2

S values ____________ from left to right.
a)Increase b)Decrease

S values ____________ from left to right.
a)Increase b)Decrease

• Larger/denser objects sediment more quickly (large S value) • Smaller/ less dense objects sediment less quickly (smaller S value)

Sedimentation coefficient (S)

Figure 12A-3

Tour of the Cell…

Keep in mind as we take our tour…

• Cells are DYNAMIC SYSTEMS!!!
Cells can adjust structures to meet changing needs
– e.g. adjust # of mitochondria to meet metabolic needs

Subcellular structures interact with one another
– i.e. they are not isolated entities

Cytoplasm
- Internal contents of cell - Contains: • Cytosol
~semi-fluid material •Organelles (eukaryotes)

• Subcellular structures
e.g. ribosomes

Plasma Membrane
• 4-8nm (40-80 Å) thick layer of lipids + protein •Boundary between cell and external environment
- defines ‘cell’ - regulates movement in/out - mediates communication with external environment (e.g. receptors)

•Fluid Mosaic
- many components (mosaic) - flexible, dynamic structure; not static

*More about this later (Ch. 7)…

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Extracellular Matrix (ECM)
• Region outside of cell
(“extra” cellular)

• Made of various proteins/polysaccharides (varies by species)
– e.g. collagen (fibrous protein); e.g. cellulose (polysaccharide)

• Linked to cell via Plasma membrane components
– e.g. integrins (intermembrane proteins)

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Functions of the ECM:
Include: – Support/Structure
• e.g. Cell Walls in plants (…more on this in a bit…) Bone matrix Protection against osmotic pressure changes

– Adhesion/anchorage to surrounding medium
• e.g. tissue formation

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ECM: Plant Cell Walls
– ECM of plant cells – Made of cellulose fibers embedded in other polysaccharides and protein
Cell Wall

– Functions
• mechanical strength • growth/development • protection

Fig 17-24

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ECM: Prokaryotic Cell Walls
Bacteria
Peptidoglycan:
• NAM-NAG polysaccharide • Cross-linked with peptides
(short amino acid chains)

Archaea
4 known variations: a. Sulfated polysaccharides b. Glycoproteins stabilized by Na+ c. S-layers – protein chain mail d. Pseudomurein (shown below)
• NAG – NAT polysaccharide • Cross-linked with peptides

N- Acetylmuramic acid (NAM) N- Acetylglucosamine (NAG)

N-Acetyltalosaminuronic acid (NAT)

Gram stain
• Tool to ID bacteria based on cell wall characteristics
 Cells stained with violet dye  Rinse with alcohol  Stain again with counter-stain (usually red dye)
Lipopolysaccharide Peptidoglycan layer Plasma membrane Protein Grampositive bacteria 20 m Gramnegative bacteria

Cell wall

Outer membrane Cell wall Peptidoglycan layer Plasma membrane
Protein

(a)

Gram-positive: -Simple cell walls - cell walls have large amount of peptidoglycan - traps violet dye in the cytoplasm - alcohol rinse does not remove the violet dye, which masks the added red dye. - Cells appear violet after counter-stain is added

(b)

Gram-negative: -Complex cell walls with less peptidoglycan - Cell wall located in layer between -PM and an outer membrane - outer membrane also has lipopolysaccharides -violet dye is easily rinsed from the cytoplasm, - cells appears pink/red after counter-stain is added

Pathogenic Prokaryotic Species
• Can be Gram+or Gram- ; also Gram variable
• Examples of Pathogenic Gram+
• Clostridium botulinum (Botulism) • Bacillus anthracis (Anthrax) • Streptococcus pneumoniae (bacterial pneumonia, bacterial meningitis)

• Examples of Pathogenic Gram• Yersinia pestis (Plague) • Bordetella pertussis (Whooping cough) • Chlamydia tachomatis (STD)

• Examples of Pathogenic Gram variable
• Mycobacterium tuberculosis (Tuberculosis) • M. leprae (Leprosy)

Gram Stain
• Used as a quick diagnostic tool • Examples:
– Classification of new species
• Based on CW characteristics • Also preserves shape (e.g. spiral, rods)

– To confirm/rule out bacterial infection
• Swabs taken from wounds, joint fluids, CSF, etc.
(see recent example on next slide)

– Assessing bacterial contamination of tissue cultures – Environmental/Industrial contamination
• Natural disasters • Milk production

Predictive value of superficial cultures to anticipate bloodstream infection.
Bouza E1, Rojas L2, Guembe M3, Marín M2, Anaya F4, Luño J4, López JM4, Muñoz P5; on behalf of the COCADI Study Group.

Abstract We performed a prospective study in patients with tunneled catheters to assess the validity of Gram stain and superficial culture for anticipating catheter exit-site infection and hemodialysis catheterrelated bloodstream infection. The sensitivity and negative predictive value were high, and we succeeded in identifying a subpopulation at low risk of infection. Diagn Microbiol Infect Dis. 2013 Dec 17. pii: S0732-8893(13)00650-0. doi: 10.1016/j.diagmicrobio.2013.12.008. [Epub ahead of print] Copyright © 2013 Elsevier Inc. All rights reserved.

Prokaryotic Cell Wall-Synthesis Inhibitors
• Certain antibiotics work by inhibiting cell wall biosynthesis • Compromise integrity of the CW Makes bacteria susceptible to osmotic pressures With weakened CWs, they will generally lyse • Examples:

- Penicillin
• Interferes with peptide synthesis and peptidoglycan cross-linking
Penicillium fungus (DIC image)

– Fosfomycin
• Interferes with peptidoglycan biosynthesis
Brightfieldimage of Streptomyces fradiae

Continue our “Tour of the Cell”…

Nucleus
• Stores DNA = CONTROL CENTER • Large organelle
~5-6um diameter ~10% total cell volume

• Surrounded by Nuclear Envelope (NE)
- Double membrane layer - Supported by Nuclear Lamina (…more in a bit…) - Punctured at intervals by Nuclear Pores (…more in a bit…)
Regulated openings through nuclear envelope - Control movement of substances in/out of nucleus

Nuclear Pores
• Regulated openings through Nuclear Envelope
• Openings controlled by Nuclear Pore Complex (NPC) • NPC controls movement of substances in/out of Nucleus
Q: What sorts of molecules are moving in/out of nucleus?

Nuclear Lamina
• Network of intermediate filaments (components of the cytoskeleton)

CLSM of nuclear lamina (green) & DNA (red) from mousenucleus
Source:http://medicalphysicsweb.org/cw s/article/opinion/34548/1/SIM_1006

• Lie just beneath inner layer of Nuclear Envelope
• Scaffolding that supports nuclear structure

• Thought to also play a role in chromatin organization

Diagram source: http://www.goldmanlab.northwestern.edu/index.htm

Nucleolus
• Region within the Nucleus • Clustered regions of ribosomal RNA genes surrounded by specific RNAs & proteins • Site of ribosomal subunit synthesis • Q: How do ribosomal subunits exit the nucleus? Q: What happens after they leave the nucleus?

Nucleus

(SEM)

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Ribosome
• Found in all cell types (pro/eukaryote),and certain organelles
• Not an organelle

25 – 30 nm

Type of microscopy used?

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The process by which genetic information coded in messenger RNA directs the formation of a polypeptide sequence is called____________.
a) b) c) d) e) Transcription Transformation Translation Transgression None of the above

Prokaryotic vs. Eukaryotic Ribosomes • Ribosomes are RNP (ribonucleoprotein) complexes
Q: What is an RNP complex?? A: Complex of RNA and protein
Prokaryote

• Sizes of ribosomes differ between prokaryotes & eukaryotes

Eukaryote

Announcements: Reminder - Exam 1
• Exam 1 will take place on Wednesday, 2/5/14, from 12:30 pm-1:45pm. Please go to the following exam location based on the spelling of your last name: A-J: Schmitt; K -Z: Ford Auditorium...*2 pt deduction on exam for going to the wrong room* :(
• Everything through lec 6 will be covered. • Please bring a No. 2 pencil. • You must also show your student ID when handing in your exam. • Only 4-function calculators are permitted. • See syllabus for more details regarding the exam, approved calculators, and secure testing.

Announcements: MasteringBiology Assignments

• MasteringBiology Assignments #1 and #2 were due today at 12:30pm • Assignments #3 and #4 are now available. –due at 12:30pm on 2/3/14 –i.e. ONE week from the availability date

Protocol Describing Fractionation/Centrifugation
Nuclei extraction from brain tissue Total nuclei were extracted via sucrose gradient ultracentrifugation. In the work presented here (mouse), each sample was derived from a single forebrain (adult males, 8–12 weeks of age); (human) 1000 mg of cerebral cortex. All the reagents used were pre-chilled and the entire procedure was performed on ice. Fresh or frozen samples were homogenized by douncing 50 times in 5 mL NEB with 0.1% Triton X-100, or alternatively, 0.1% NP-40. Triton X-100 was preferable if nuclei require immunotagging (with NeuN, for example), while NP-40 as a milder detergent left more nuclei intact and sufficient when working with nuclei expressing GFP. After douncing, brain homogenates were transferred into 14 mL ultracentrifuge tubes (Beckman, 14 × 95 mm, 344061), and 9 mL of Sucrose Cushion was carefully loaded directly to the bottom of the ultracentrifuge tube. Ultracentrifugation was performed at 24400 rpm for 2.5 hrs at 4°C (Beckman, L8-70M, SW28 rotor). After centrifugation, a nuclei pellet – thin and typically with a light yellow taint – was formed on the bottom of the tube. The sticky white tissue debris was restrained in the middle interface of the two sucrose layers. Supernatant, including debris, was carefully removed.

Source: Jiang et al. (2008). Isolation of neuronal chromatic from brain tissue. BMC Neuroscience 9, 42.

Ribosome
• Found in all cell types (pro/eukaryote),and certain organelles
• Not an organelle

25 – 30 nm

Type of microscopy used?

4

Prokaryotic vs. Eukaryotic Ribosomes • Ribosomes are RNP (ribonucleoprotein) complexes
Q: What is an RNP complex?? A: Complex of RNA and protein
Prokaryote

• Sizes of ribosomes differ between prokaryotes & eukaryotes

Eukaryote

Ribosome

Fig. 17-16 Campbell and Reece, 8 ed.

Translation

E

P

A

Fig. 17-14 Campbell and Reece, 9 ed.

MasteringBiology® animation on Translation
• View ‘Protein Synthesis’ (BioFlix tutorial) in Assignment #4

Overview of Translation in Ch 22

Overview of Translation
AA1

tRNA carrying first amino acid UAC Anticodon 5 AUG Start codon

Large

Ribosomal subunits Small INITIATION 3 5

AA1

AUG

mRNA

UAG Stop codon

During initiation, the components of the translational apparatus come together with an mRNA, and a tRNA carrying the first amino acid (AA1) binds to the start codon (AUG).

1

© 2012 Pearson Education, Inc.

Figure 22-7

Overview of Translation
AA1

tRNA carrying first amino acid: INITIATOR tRNA UAC Anticodon 5 AUG Start codon

Large

Ribosomal subunits Small INITIATION 3 5

AA1

AUG ELONGATION 2
During elongation, amino acids are brought to the mRNA by tRNAs and are added, one by one, to a growing polypeptide chain.
AA2 AA3 AA4 AA5

mRNA

UAG Stop codon

During initiation, the components of the translational apparatus come together with an mRNA, and a tRNA carrying the first amino acid (AA1) binds to the start codon (AUG).

1

AA1

5

© 2012 Pearson Education, Inc.

Figure 22-7

Overview of Translation
AA1

tRNA carrying first amino acid UAC Anticodon 5 AUG Start codon

Large

Ribosomal subunits Small INITIATION 3 5

AA1

AUG ELONGATION 2
During elongation, amino acids are brought to the mRNA by tRNAs and are added, one by one, to a growing polypeptide chain.
AA2 AA3 AA4 AA5

mRNA

UAG Stop codon

During initiation, the components of the translational apparatus come together with an mRNA, and a tRNA carrying the first amino acid (AA1) binds to the start codon (AUG).

1

AA1

Recycling of translational components 5

Completed polypeptide

Release factor
TERMINATION

5 UAG Stop codon

3

3 During termination, a stop codon in the mRNA is recognized by a protein release factor, and the translational apparatus comes apart, releasing a completed polypeptide.

© 2012 Pearson Education, Inc.

Figure 22-7

Please note: If these topics seem totally foreign to you, please review transcription/translation (Ch. 21 & 22)

Prokaryotic Protein-Synthesis Inhibitors
• Certain antibiotics work by inhibiting ribosome activity

• Examples: - Tetracyclin
• irreversibly binds to 30S subunit; • prevents tRNA from entering A site on 70S ribosome

– Spectinomycin
• interferes with mRNA interaction with the 30S ribosome

Endomembrane System
Nuclear envelope

ER

Golgi Transport Vesicles Vacuoles (not shown) Lysosomes Plasma Membrane
16

Endomembrane System
• System of membranes and internal spaces

• Can be directly connected or connected via transport vesicles
• Includes:
– Nuclear envelope – ER – Golgi – Lysosomes – Vacuoles – Plasma Membrane – Transport Vesicles

The Endoplasmic Reticulum (ER)
• Accounts for ~½ the membranes in eukaryotic cell
– ~10% total cell volume
Smooth ER Nuclear envelope Rough ER

ER lumen
Cisternae Ribosomes Transportvesicle Smooth ER Rough ER
200 µm

• Contiguous with outer nuclear membrane • Two distinct regions of ER
- Smooth ER, lacks ribosomes - Rough ER, contains ribosomes

18

Rough ER
– Large, flattened sheets – Ribosomes temporarily bound to cytosolic side
= “rough” appearance

– Produces proteins, glycoproteins – Products are distributed throughout cell by transport vesicles
• Other endomembrane components • Secreted products
19

Smooth ER
• Lack ribosome
– “smooth” appearance

• Functions:
– Lipid synthesis
• e.g. cholesterol, steroids

– Carbohydrate metabolism – Stores Calcium – Detoxifies poison

20

The Endoplasmic Reticulum (ER)
Smooth ER

Two distinct regions of ER
– Smooth ER, lacks ribosomes – Rough ER, contains ribosomes
Rough ER

Nuclear envelope

ER lumen

Functionally SEPARATE compartments

Cisternae Ribosomes Transportvesicle Smooth ER Rough ER
200 µm

e.g. A Secreted Protein
 would go through the RER  would NOT go through the SER

ER Adaptations
• Like most subcellular structures, the ER is dynamic • Cells can adjust relative amounts in response to changing conditions

Abundant smooth ER in a hepatocyte. TEM of a hepatocyte of chronic alcoholic.
23

The Golgi Apparatus
• The Golgi – System of flattened membranous sacs – Receives many of the transport vesicles produced in the ER - Cis Golgi network (CGN): close to ER - Trans (TGN): other side • Functions of the Golgi: - Modification of ER products
e.g. glycoproteins

- Manufacture of complex carbohydrates - Sorts and packages molecules for transport to final destinations

24

Transport through Endomembrane System

25

Figure 12-1

Recall from Biol 214…
Ribosome

mRNA
Signal peptide
Signalrecognition particle (SRP)

Signal peptide removed
Translocation complex

ER membrane Protein

CYTOSOL ER LUMEN
SRP receptor protein

Fig. 17-21 from ‘Biology’ by Campbell et al.

Hydrolases are enzymes (biological catalysts) which catalyze the hydrolysis of macromolecules. What exactly does a hydrolase do?
a) Adds a water molecule to a bond, causing it to break b) Adds a water molecule in order to form a bond c) Removes a water water molecule from a bond, causing it to break d) Removes a water molecule in order to form a bond e) I have no idea!
27

Lysosomes: Digestive Compartments
Lysosome – membranous sac of hydrolytic enzymes – Function is digestion
Nucleus

Rough ER

Lysosomes are part of endomembrane system - bud off from Golgi

Smooth ER Nuclear envelop

cis Golgi

trans Golgi

Plasma membrane

Lysosome

28

Intracellular ‘Digestion’ by Lysosomes e.g. Phagocytosis
- Ingestion of large particles (>0.5um diamter)

- Breakdown (digestion) of particles carried out by Hydrolases inside of lysosomes

29

Intracellular ‘Digestion’ by Lysosomes e.g. Autophagy
- Process of organelle degradation that takes place inside the cell
“auto” = self “phagy” = to eat

- Used to remove old/damaged cell structures

30

Homework for next class:
Based on what we have discussed, describe, step by step, the pathway taken by hydrolase enzymes in order to localize them to the lumen of the lysosome. Start with transcription of the hydrolase gene.
Lysosome
Hydrolase ?

Vacuoles: Diverse Maintenance Compartments
• Cells may have one or several vacuoles • Function is cell-specific
Examples: – Central vacuole in plants
(…more in a bit…)

Amoeba proteusstained with pH-dependent dye. The dye becomes dark red in the acidic environment of the food vacuoles.

– Food vacuoles (phagosomes)
• formed by phagocytosis

– Contractile vacuoles
• osmoregulation
32

Central Vacuoles
– Found in plant cells – Hold reserves of important organic compounds and water – Maintain fluid balance/ turgor
Central vacuole
Cytosol Tonoplast

Nucleus Cell wall Chloroplast

Central vacuole

5 µm

33

Vacuoles in Animal Cells
e.g. vacuoles in Paramecium (unicellular protist)

Food Vacuoles

34

Vacuoles in Animal Cells

35

http://www.linkpublishing.com/video-transport.htm#Paramecium_-_Contractile_Vacuoles

Vacuoles in Animal Cells
• Vary in number (depending on cell type)
– Few to many

• Much smaller than Central Vacuole found in plants

Energy-converting Organelles

Mitochondrion Chloroplast
37

Mitochondria and Chloroplasts
• Change energy from one form to another • Mitochondria
– Sites of cellular respiration – Found in most eukaryotes, including photosynthetic organisms

• Chloroplasts
– Found only in photosynthetic eukaryotes – Sites of photosynthesis
38

Evolution of Chloroplasts & Mitochondria: Endosymbiotic Theory
• Idea championed by Lynn Margullis (Umass) in the late 1960’s

• Hypothesis: Mitochondria & chloroplasts originated as freeliving prokaryotes
• Mitochondria: proteobacteria • Chloroplasts: cyanobacteria
– *Proteobacteria & cyanobacteria are types of Bacteria

 Smaller cel taken inside another cell (larger prokaryote) Symbiotic relationship developed  Dependency increased over time such that cells became ONE

Evolution of Eukaryotic Cells

What evidence might support the Endosymbiotic Theory for the Origin of Mitochondria & Chloroplasts?

Evidence Supporting the Endosymbiotic Theory for the Origin of Mitochondria & Chloroplasts
1) 2) 3) Mit/cp Similar size to prokaryotes (~1 um) Replicate by binary fission Double membrane
– Inner membrane similar to prokaryotes, outer membrane may have been derived from host ‘phagosome’

4) 5) 6)

70S ribosomes
– Sensitive to some antibiotics

Circular genome
– Prokaryotic promoters, no histones

Genome sequence similarity
– Cp: cyanobacteria;Mit: proteobacteria

7) Reduction of organellar genomes
Gene transfer to nucleus Many genes needed by mit/cp are nuclear encoded Sequence similarity b/w genes in nucleus and cyanobacteria/proteobacteria

Peroxisomes
• Similar in shape & size to lysosome
However, NOT part of endomembrane system

Main function: Compartmentalize hydrogen peroxide (H2O2)-producing reactions -RH2 + O2  R + H2O2
Note: ‘R’ generically refers to an organic molecule

~Figure 4-19

-H2O2is toxic to cells -H2O2 is then degraded into H2O + O2
 degradation catalyzed by the enzyme catalase
44

Peroxisomes
Other functions: • Breakdown of long-chain fatty acids
- via β-oxidation pathway (…more on this later…)

Detoxification of oxidizeable substrates
- e.g. alcohols
Chloroplast Peroxisome Mitochondrion

• Photorespiration in Plants
- Strategy to recover carbon that would be otherwise lost (…more
on this later…)
45

Figure 4-20

1 µm

Cytoskeleton
• Network of protein fibers and associated proteins • Network extends throughout the cytoplasm and underlie nuclear envelope • Organizes structures and activities in the cell
Microtubule

0.25 µm

Microfilaments

47

Structure of Cytoskeletal Elements

Table 15-1

Cytoskeletal-Associated Motor Proteins

49

Cell Locomotion

50

Neutrophil chasing Bacterium
• http://www.dnatube.com/video/4330/Neutrophil-Chases-Bacteria

Neutrophil chasing Bacterium
How does the Eukaryotic cell perceive the bacterium?

signal

52

Neutrophil chasing Bacterium
How is movement towards to bacterium generated?

signal

53

Neutrophil chasing Bacterium
Outline the steps involved in engulfment and digestion of the bacterium.

signal

54

Announcements: Reminder - Exam 1
• Exam 1 will take place on Wednesday, 2/5/14, from 12:30 pm-1:45pm. Please go to the following exam location based on the spelling of your last name: A-J: Schmitt; K -Z: Ford Auditorium...*2 pt deduction on exam for going to the wrong room* :(
• Everything through lec 6 will be covered. • Please bring a No. 2 pencil. • You must also show your student ID when handing in your exam. • Only 4-function calculators are permitted. • See syllabus for more details regarding the exam, approved calculators, and secure testing.

Evolution of Eukaryotic Cells

Evolution of Eukaryotic Cells
Archaea

Proteobacteria

Homework:
Based on what we have discussed, describe, step by step, the pathway taken by hydrolase enzymes in order to localize them to the lumen of the lysosome. Start with transcription of the hydrolase gene.
Lysosome
Hydrolase ?

Inner Life of a Cell
• Segment describing co-translational import:
http://multimedia.mcb.harvard.edu/media.html

Transport through Endomembrane System

6

Figure 12-1

Protein Targeting to Lysosomal Lumen
At the RER: - Hydrolase is fully synthesized - Deposited in RER lumen - Carbohydrate ‘tag’ gets added (mannose) Vesicle containing hydrolase buds off of RER  Fuses to Golgi At the Golgi: - As glycosylated hydrolase moves through Golgi, mannose ‘tag’ is phosphorylated by Golgispecific enzymes Result: Hydrolase with a Mannose-6-Phosphate ‘tag’
Figure 12-9

Protein Targeting to Lysosomal Lumen
At the Golgi: Hydrolase with a M-6-P ‘tag’ - ‘Tag’ serves as a ‘Recognition System’… - Phosphate group binds to a receptor in Trans Golgi membrane - Receptor specifically recognizes the M-6-P ‘tag’ - Binding triggers packaging of hydrolase into a vesicle Vesicle fuses to acidified compartment: endosome - Low pH causes dissociation of Hydrolase from receptor  Endosome matures into a lysosome

Figure 12-9

Neutrophil chasing Bacterium
• http://www.dnatube.com/video/4330/Neutrophil-Chases-Bacteria

Neutrophil chasing Bacterium
How does the Eukaryotic cell perceive the bacterium?

signal

11

Neutrophil chasing Bacterium
How is movement towards to bacterium generated?

signal

12

Neutrophil chasing Bacterium
Outline the steps involved in engulfment and digestion of the bacterium.

signal

13

Tuberculosis
• Infectious disease caused by mycobacteria strains • WHO estimates 1/3 of the world population infected with mycobacteria • Commonly affects lungs
– Aerial transmission (e.g. coughing, sneezing)

CASE STUDY: Examination of white blood cells taken from a patient with tuberculosis reveals living bacteria in large vesicles. Continued observation over time shows that the bacteria remain alive for a long time.
In this case, which white blood cell process might be defective?

[EM image of] Mycobacterium tuberculosis phagosome in alveolar macrophage from an individual co-infected with HIV. Arrowhead, phagosome with M. tuberculosis. Arrows, virus-like particles suspected to be HIV buddinginside the macrophage.
Scale bar = 1 μm. Annu. Rev. Cell Dev. Biol. 2004. 20:367–94

Examination of white blood cells taken from a patient with tuberculosis reveals living bacteria in large vesicles. Continued observation over time shows that the bacteria remain alive for a long time. In this case, what white blood cell process might be responsible? a) Deficient receptors on the white blood cell for detecting bacteria b) Slow white blood cell motility c) Failure of the phagocytic vesicle and lysosome to fuse

d) Mitochondrial deficiency e) Chloroplast defect

What are cells made of?

What are cells made of?

What are cells made of?

~25 of all known elements are needed for life Of those, 5 are found in all living things…

H, C, N, O, P
• 5 elements found in all living things • Constitute 99% of an organism’s weight • Incomplete outer e- shells
– donate, accept or share electrons to form ions and molecules – i.e. they are REACTIVE

Which element do you think contributes to most of our body mass?

a) b) c) d) e)

Calcium Carbon Hydrogen Nitrogen Oxygen

Elemental Composition of the Human Body
• Percent by mass (approximate) Oxygen - 65 Carbon – 18.5 Hydrogen – 9.5 Nitrogen – 3.3 Calcium - 1.5 Phosphorous - 1.0 *these six elements make up ~99% of our bodies
Other ~1%: Potassium 0.2 Sulfur 0.2 Chlorine 0.2 Sodium 0.1 Magnesium 0.05 Cobalt, Copper, Zinc, Iodine < 0.05 each Selenium, Fluorine, Manganese, Molybdenum, Nickel < 0.01 each Iron 3.9 – 6.4 x 10 -5

Why so much Oxygen? • Cells are mostly water
– Average cell ~70% water – Others have less
• Example: bone cell ~20% water

• Most cellular reactions take place in solution (aq)

What makes an element REACTIVE?

Anatomy of an Atom
• Atom = basic unit of matter
– Nucleus surrounded by electrons

Electrons:
-ve charged particles

Nucleus: consists of protons
(+ve) and neutrons
(except H which has only 1 proton & no neutron)

Electrons
• Attracted to positively-charged nucleus
– have potential energy because of attraction to the nucleus

• Can have only certain amounts, or levels, of energy • E levels are called electron shells
– pictured as spherical regions around the nucleus

Valence Electrons
• Electrons in the last shell • Valence # = Group # (disregarding transition metals) • Important determinant of reactivity
– Can be gained or lost in a chemical reaction – Atoms tend to react in ways that result in filled outer shell

Electronegativity
• Affinity of an atom/molecule for electrons • Electronegativity Scale:

• The greater the EN value, the stronger the affinity for electrons

Which atom has a greater affinity for electrons?

a) Oxygen b) Carbon c) Neither. They are equal

Types of Bonds

Covalent bonds – sharing electrons
• Electrons are shared between atoms • Two different types of covalent bonds:
– Polar covalent – Nonpolar covalent

Covalent bonds – sharing electrons
• Polar molecules
– Electrons are unequally shared  One part of molecule is more negative than the another part of the molecule – Molecule thus has negative and positive ‘poles’
• Similar to a battery • e.g. Water molecule

– Hydrophilic (‘water loving’)

Covalent bonds – sharing electrons
• Nonpolar molecules
– Electrons are equally shared – No one part of molecule is distinctly negative or positive
• no ‘poles’

– Hydrophobic ‘water fearing’

Ionic bond
• Formed through electrostatic attraction between oppositely charged ions. • Due to the attraction between:
– an atom that has lost 1 or more electron (e.g. cation+) – and an atom that has gained one or more electrons (e.g. anion-)

• Electrons are NOT shared in an ionic bond

Ionic bond

Nonpolar ∂+

Polar ∂

-

Covalent bonds

Ionic bond

+

Hydrogen bond
• Forces between polar molecules
– Specifically involves hydrogen (H) bound to a more electronegative atom, such as N, O or F,) and an O, N or F of another molecule.

• Electrons are not shared
– Electrostatic interaction between dipoles

• Example: Interactions between water molecules:

What other type of force, found in all molecules, is important?

London Dispersion Forces
• Temporary attractive force resulting when electrons in two adjacent atoms occupy positions that make the atoms form temporary dipoles • Found in all molecules • Weakest of all forces

Macromolecules of the Cell

Which molecule is organic?
a) b) c) d) e) CO2 H2O CH3OH O2 More than one answer

Organic molecules
• Hydrocarbon based
– i.e Carbon with hydrogen – e.g. carbohydrates, CH3OH

• The presence of Carbon alone does not constitute an organic molecule
– e.g. CO2 = inorganic carbon
*Note: This is the definition of ‘organic chemistry’ that we will use in this class. Other sources may define ‘organic’ differently.

Carbon

What is so Special about Carbon?
• • • • Capable of forming four bonds Relatively neutral electronegativity Covalent bonds Forms stable molecules

HUGE variety of organic molecules
Straight chains

Rings Branched Chains

Single bonding

Double bonding

Triple bonding

47

Functional Groups
• Specific groups of atoms added to organic molecules • Lend specific chemical characteristics to molecule in which group occurs

What charge will the following molecule have in an aqueous environment (aq)?

a) Positive b) Negative c) Neutral

49

Functional Groups

50

Figure 2-5

Functional Groups

STRUCTURE

NAME OF COMPOUND

FUNCTIONAL PROPERTIES EXAMPLE

True or False?: In an aqueous environment, a hydroxyl group will be negatively charged.
a) True b) False

-OH functional group IS NOT the same as a hydroxide ion (OH-)
• In an aqueous environment, -OH (hydroxyl) functional groups are NOT charged. • -OH (hydroxyl) functional groups DO NOT deprotonate in an aqueous environment.

Functional Groups
NAME OF COMPOUND

STRUCTURE

FUNCTIONAL PROPERTIES

EXAMPLE

Functional Groups

Acetyl group (CH3C-) = Specific type of carbonyl

R

Functional Groups
Carboxyl
STRUCTURE Carboxylic acids, or organic acids NAME OF COMPOUND

EXAMPLE Has acidic properties because the covalent bond between oxygen and hydrogen is so polar; for example, Acetic acid, which gives vinegar its sour taste

FUNCTIONAL PROPERTIES

Acetic acid

Acetate ion

Found in cells in the ionized form with a charge of 1– and called a carboxylate ion (here, specifically, the acetate ion).

Functional Groups
Amino
STRUCTURE NAME OF COMPOUND Amines

EXAMPLE

Glycine Because it also has a carboxyl group, glycine is both an amine and a carboxylic acid; compounds with both groups are called amino acids.

Acts as a base; can pick up an H+ from the surrounding solution (water, in living organisms).

FUNCTIONAL PROPERTIES

(nonionized)

(ionized)

Ionized, with a charge of 1+, under cellular conditions.

Functional Groups
Amide

- Formed by reaction between an acid and an amine:
Carboxylic Acid Amine Amide

C

+
OH

H2N C

C

Example of Amide: Peptide bond that joins amino acids in a polypeptide as a result of a dehydration reaction

Note: Unlike amines, amides are uncharged in solution (aq).

O

O

+

H2O

N C
H

Functional Groups
Sulfhydryl
STRUCTURE Thiols NAME OF COMPOUND

(may be written HS—)

EXAMPLE Two sulfhydryl groups can react, forming a covalent bond. This “cross-linking” helps stabilize protein structure. Cross-linking of cysteines in hair proteins maintains the curliness or straightness of hair. Straight hair can be “permanently” curled by shaping it around curlers, then breaking and re-forming the cross-linking bonds.

FUNCTIONAL PROPERTIES

Cysteine Cysteine is an important sulfur-containing amino acid.

Functional Groups
Phosphate
STRUCTURE NAME OF COMPOUND

Organic phosphates

EXAMPLE

Glycerol phosphate In addition to taking part in many important chemical reactions in cells, glycerol phosphate provides the backbone for phospholipids, the most prevalent molecules in cell membranes.

Contributes negative charge to the molecule of which it is a part (2– when at the end of a molecule; 1– when located internally in a chain of phosphates).
Has the potential to react with water, releasing energy.

FUNCTIONAL PROPERTIES

Organic Macromolecules found in Cells

a) Lipids

b) Nucleic Acids (polynucleotides) - Biol 214
Polymers c) Carbohydrates (polysaccharides) d) Proteins (polypeptides)

Monomer subunits
• Carbohydrates
– Monosaccharides

• Nucleic Acids
– Nucleotides

• Proteins
– Amino Acids
62

Organic macromolecules are synthesized by ______________ reactions. a) b) c) d) e) condensation hydrogenation hydrolysis polyhydration transamination

63

Breakdown of organic macromolecules occurs by ______________ reactions. a) b) c) d) e) condensation hydrogenation hydrolysis polyhydration transamination

64

Synthesis of Polymers occurs by Dehydration Reaction (type of Condensation Reaction)

monomer

monomer

monomer

OH

H

OH

H

polymer

–H and –OH are removed (~water) subunits join into a polymer. i.e. components are ‘dehydrated’

Announcements: Reminder - Exam 1
• Exam 1 will take place on Wednesday, 2/5/14, from 12:30 pm-1:45pm. Please go to the following exam location based on the spelling of your last name: A-J: Schmitt; K -Z: Ford Auditorium...*2 pt deduction on exam for going to the wrong room* :(
• Everything through lec 6 will be covered. • Please bring a No. 2 pencil. • You must also show your student ID when handing in your exam. • Only 4-function calculators are permitted. • See syllabus for more details regarding the exam, approved calculators, and secure testing.

Why does a carboxylic acid (carboxyl) functional group behave differently in solution compared to a hydroxyl?
• • • • Has TWO electronegative oxygens The O’s pull electrons away from the H atom Weakens the bond between O and H H atoms tends to dissociate from the molecule as a hydrogen (H+) ion. • Because it donates hydrogen ions, this group is considered acidic • Molecules that contain these groups are known as carboxylic acids.

Functional Groups

3

Figure 2-5

Organic Macromolecules found in Cells

a) Lipids

b) Nucleic Acids (polynucleotides) - Biol 214
Polymers c) Carbohydrates (polysaccharides) d) Proteins (polypeptides)

Monomer subunits
• Carbohydrates
– Monosaccharides

• Nucleic Acids
– Nucleotides

• Proteins
– Amino Acids
5

Organic macromolecules are synthesized by ______________ reactions. a) b) c) d) e) condensation hydrogenation hydrolysis polyhydration transamination

6

Breakdown of organic macromolecules occurs by ______________ reactions. a) b) c) d) e) condensation hydrogenation hydrolysis polyhydration transamination

7

Synthesis of Polymers occurs by Dehydration Reaction (type of Condensation Reaction)

monomer

monomer

monomer

OH

H

OH

H

polymer

–H and –OH are removed (~water) subunits join into a polymer. i.e. components are ‘dehydrated’

Protein Synthesis via Amino Acid Polymerization Example of Dehydration Reaction
(aka Condendation reation)

Removal of H2O

water

amino acid 1

amino acid 2
polypeptide

Imagesource: http://en.wikipedia.org/wiki/Condensation_reaction

Breakdown of Polymers by hydrolysis reaction “hydro” = water “lysis” = break apart
polymer

monomer

monomer

monomer

Polymer is split into smaller subunits by adding –H and – OH (~water) i.e. polymer is hydrolyzed

Why is hydrolysis of macromolecules necessary?
• Some macromolecules are too large to get imported into cells
 e.g. starch

• Hydrolysis yields smaller subunits that can enter cells
 e.g. glucose

• Subunits can then be used in the cell
 e.g. assembled into polymers via dehydration reactions

Example of Hydrolysis Reaction: Starch Breakdown

Imagesource: http://www.biology-books.com/Standard/standard.html

Organic Macromolecules found in Cells

a) Lipids b) Nucleic Acids (polynucleotides) c) Carbohydrate (polysaccharides)

d) Proteins (polypeptides)
13

Lipids
• Heterogeneous group of molecules
– Defined in terms of solubility characteristics (not structural characteristics)

• All lipids are primarily Hydrophobic molecules
– Little affinity for water – Readily soluble in nonpolar solvents
• e.g. chloroform or ether

• Some lipids are amphipathic, having polar and nonpolar regions
– Primarily hydrophobic

Lipids
• Functions include:
- energy storage - membrane structure - signal transduction

• Different from other macromolecules
- not formed by the same type of linear polymerization as proteins, nucleic acids, and polysaccharides

• Still regarded as macromolecules due to:
• high molecular weight • importance in cellular structures
• particularly membranes

Classes of Lipids
• Various classes based structure
– Fatty acids – Triacylglycerols
• a.k.a. triglycerides

– Phospholipids – Glycolipids – Steroids
*Ignore Terpines

…More on this later (Ch 7)…

Figure 3-27

Organic Macromolecules found in Cells

a) Lipids b) Nucleic Acids (polynucleotides) - Biol 214 c) Carbohydrate (polysaccharides) d) Proteins (polypeptides)
17

Organic Macromolecules found in Cells

a) Lipids b) Nucleic Acids (polynucleotides)- Biol 214 c) Carbohydrates (polysaccharides)

d) Proteins (polypeptides)
18

Carbohydrates
• Most abundant organic molecules on Earth!
#1: Cellulose – produced by photosynthetic organisms #2: Chitin – fungal cell walls, exoskeletons of arthropods

• Main Functions in cells:
• Structural components • Storage = Major E source
1 2 3

Molecular Structure:
• Poly-hydroxyls ~one per carbon “carbo” “hydrate” = carbon with water • Aldehyde – or – ketone • Often have ‘-ose’ ending - e.g. glucose, sucrose

4

5 6

galactose

Carbohydrates

Aldehyde

– or –

Ketone
- carbonyl within molecule
Figure 3-20

- terminal carbonyl

Categories of Carbohydrates
• Monosaccharides “Mono” = one, “sacchar” = sugar • Disaccharides “Di” = two, “sacchar” = sugar • Polysaccharides “Poly” = many, “sacchar” = sugar

Important Monosaccharides
• Monosaccharides
“Mono” = one, “sacchar” = sugar - simple sugars - generally have a molecular formula that is some multiple of CH2O

Glucose:
- 6 Carbon aldose = aldohexose C6H12O6 -

Main E source for cells *central to cell metabolism

- Structural component of important disacharides and polysaccharides

Important Monosaccharides
Fructose:
- 6 Carbon ketose = ketohexose
• C6H12O6

- Referred to as “fruit sugar” - Metabolic intermediate - Important disaccharide component
• Combines with glucose to form sucrose (table sugar)

Galactose:
6 Carbon aldose = aldohexose

• •

C6H12O6
Combines with glucose to form lactose (milk sugar) Galactosemia - genetic disorder - afflicted individuals cannot metabolize galactose - high levels of galactose can lead to metal retardation

Important disaccharide component

Important Monosaccharides
Glyceraldehyde & Dihydroxyacetone (DHA): - 3 Carbon sugars = triose • Glyceraldehyde = trialdose • DHA = ketotriose - Metabolic intermediates • Intermediates of both glycolysis pathway and photosynthesis • Reversible interconversion (equilibrium!!!)

FYI: DHA is active ingredient in sunless tanning products • Combines with different amino acids to form melanoidins (brown polymers)

Disaccharides • Monosaccharides link together to form larger structures
– e.g. two monosaccharides = disaccharide “di” = two, “sacchar” = sugar

• Join together via condensation reaction to form glycosidic bond:

Important Disaccharides

Maltose - Dissacharide of glucose
(14 glycosidic linkage)

- Breakdown product of starch (glucose polymer)

26

Important Disaccharides

Sucrose - Dissacharide of glucose + fructose
(15 glycosidic linkage) - Table sugar - Transport carbohydrate in many photosynthetic organisms
27

Important Disaccharides

Lactose - Dissacharide of galactose + glucose
(14 glycosidic linkage) - Milk sugar - Important component of mammalian milk

Important Disaccharides • Lactose Intolerance:
– Inability to digest lactose – Caused by missing/defective lactase enzyme
• Lactase = enzyme that cleaves the (14) glycosidic bond between galactose and glucose

– Results in GI disturbances
• e.g. upset stomach, diarrhea

• Management options:
– Avoid dairy products – Lactase supplements
• Not on empty stomach as low stomach pH will denature the enzyme

– Treat dairy products with lactase

Important Polysaccharides
• “poly” = many, “sacchar” = sugar

• Polysaccharides are polymers of monosaccharides or disaccharides

Important Polysaccharides

- Short term E Storage

• α(14) glucose polymers • α(16) branch points (glycogen and amylopectin)
Starch : amylose (linear)or amylopectin (branched)

Glycogen – branched polymer

Similar to Fig 3-24

Important Polysaccharides

- Structural

Cellulose •β(14) glucose polymer

~2000 per microfibril

25nm

32

Figure 3-25

α-Glucose and β-Glucose
• Monosaccharides can exist as linear chains or rings • In aqueous environments (aq), ring structures are more energetically favorable (more stable)

Humans can digest ___________.
a) starch b) cellulose c) both starch and cellulose d) neither starch nor cellulose

Many animals (including humans) CAN digest starch but NOT cellulose
• Enzymes that digest starch (amylases) by hydrolyzing  linkages can’t hydrolyze  linkages in cellulose (requires cellulases) • Cellulose in human food passes through the digestive tract as fiber

• Some microorganisms use enzymes (cellulases) to hydrolyze  linkages in cellulose • Many herbivores (e.g. cows, termites) have symbiotic relationships with such microbes

Important Polysaccharides
- structural
Chitin • β(14) glucosidic linkages of N-acetylglucose amine (GlcNAc) residues

Figure 3-26

Found in: - Cell walls of fungi

- Exoskeleton of insects and arthropods (crunchy!)

36

Important Polysaccharides: Structural
Peptidoglycan • Repeating dimer of β(14) linked NAG (GlcNAc) and N-acetylmuramic acid (MurNAc)

Figure 3-26

Found in: - Cell walls of bacteria

N- Acetylmuramic acid (NAM)

N- Acetylglucosamine (NAG)

37

Important Polysaccharides

- Structural

Hyaluronic Acid (aka Hyaluronate) • Repeating dimer of glucuronic acid (glucuronate) and NAG, linked
via alternating β(13) and β(14) glucosidic linkages
• Prevalent in connective tissue and epithelial tissue

38

Hyaluronic Acid (aka Hyaluronate) • Cosmetic applications:
•Injectable filler for wrinkle remover •Trade Names: Restylane, Juvederm

Before

After!!!

Glucose modifications
• Replacement of a hydroxyl group with another functional group
• Examples:
– NAG = N-acetyl glucose amine
• Amine linked to Acetyl

– Glucuronic acid
• Carboxyl group

Infants suffering from galactosemia should avoid products with _________.
a) b) c) d) e) Starch Cellulose Sucrose Maltose Lactose

Organic Macromolecules found in Cells

a) Lipids b) Nucleic Acids (polynucleotides) c) Carbohydrate (polysaccharides)

d) Proteins (polypeptides)
42

Proteins
• Polymers of amino acids
• IMMENSELY diverse structures/functions • Structure ↔ Function

43

Protein Functions

*

44

Table 3-1

Amino Acid Structure

Figure 3-1
45

Classes of Amino Acids: Nonpolar

Glycine (Gly or G)

Alanine (Ala or A)

Valine (Val or V)

Leucine (Leu or L)

Isoleucine (Ile or )

Methionine (Met or M)

Phenylalanine (Phe or F)

Tryptophan (Trp or W)

Proline (Pro or P)
46

Figure 3-2

Nonpolar amino acids are also referred to as ________________, meaning they are water-_____________.

a) b) c) d)

hydrophilic; insoluble hydrophilic; soluble hydrophobic; insoluble hydrophobic; soluble

Classes of Amino Acids: Polar, uncharged

Serine (Ser or S)

Threonine (Thr or T)

Cysteine (Cys or C)

Tyrosine (Tyr or Y)

Asparagine (Asn or N)

Glutamine (Gln or Q)

Classes of Amino Acids: Polar, Charged
Negative Positive

Aspartate (Asp or D)
aka. Aspartic acid

Glutamate (Glu or E)
aka. Glutamic acid

Lysine (Lys or K)

Arginine (Arg or R)

Histidine (His or H)

When placed into solution (aq), the amino acids classified as ‘negatively charged’ behave as _______ and the amino acids classified as ‘positively charged’ amino acids behave as ________.

a) b) c) d)

acids; bases bases; acids acids; acids bases; bases

Negative

Positive

Aspartate (Asp or D)

Glutamate (Glu or E)

Lysine (Lys or K)

Arginine (Arg or R)

Histidine (His or H)

Classes of Amino Acids: Polar, Charged
Acidic Basic

Aspartate (Asp or D)

Glutamate (Glu or E)

Lysine (Lys or K)

Arginine (Arg or R)

Histidine (His or H)

Functional Groups
Carboxyl or Carboxylic Acid
STRUCTURE Carboxylic acids, or organic acids NAME OF COMPOUND

EXAMPLE Has acidic properties because the covalent bond between oxygen and hydrogen is so polar; for example, Acetic acid, which gives vinegar its sour taste

FUNCTIONAL PROPERTIES

Acetic acid

Acetate ion

Found in cells in the ionized form with a charge of 1– and called a carboxylate ion (here, specifically, the acetate ion).

Please note:
• Students are required to memorize the structures of the 20 amino acids • Students DO NOT need to memorize amino acid abbreviations
(Would be helpful for you to know, but you will not be required to memorize these for our class).

What type of amino acid is shown below?

a)Acidic b)Basic c)Nonpolar d)Polar, uncharged e)Hydrophobic

Is the amino acid shown below in solution?

a)Yes, it’s in solution. b)No, it’s NOT in solution. c)Impossible to tell.