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Cytoskeleton

Cytoskeleton

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Published by mcwnotes

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Published by: mcwnotes on Jan 26, 2009
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06/29/2012

 
Method of Cell and Tissue Biology
: to understand biochemical reactions and physiology in the context of the cell, tissue and organ structure.Method of tissue preparation:
Tissue Fixation
(formaldehyde)
Dehydration (
H2O for EtOH)
 
Clearing
(replace Etoh w/ solventmiscible w/embedment)
 
Staining
(improves contrast of cells)
 
Sectioning
(microtome used)
Embedding (in hard medium plastic/paraffin)
Light Microscopy Methods:
Tissue Preparation:
Staining (
acidophillia
-
Eosin
- Red cytoplasm,
basophillia-hematoxylin
 blue nuclei)Special Stains:
Schiff Reagent
- DNA/glycogen
Sudan Stain
- lipophillic
Histochemistry
: detect specific protien based on enzymatic activity
Immunochemistry
- (detect specific proteins) antibodies are used as a stain (fluorescent tags)Indirect technique- 2
nd
antibody directed against the 1
st
is conjugated with a reporter group(fluorescein/Rhoda mine-ferritin/colloidal gold) that can be made visible
Antigen-IgG-IgG(tag-fluorophore)Autoradiography
- radio autography- Objective: detect a specific cellular activity (DNA synthesis, cellscycling, protein synthesis). H-thymidine (tritiated thymidine) is administered to cells that are later fixed.Developed Xray file B electron-interact w/Silver Bromide crystals
Intestinal crypts
- are the site of stems cells that replenish the cells lining the intestine. (demonstrated withtritiated thymidine). Soluble radioactive molecules are washed out during tissue processing while thoseincorparated into DNA are fixed in the tissues. Thus cells localizing DNA during the experiment arelocalized because they will have black silver grains over their nuclei.
In Situ Hybridization
- (detect specific RNA) nucleic acid hybridization based on complementary base- paring permits identification of specific DNA or RNA sequences, based on the ability to bind to a labeledcomplementary single strand of known sequence.
Confocal Microscopy
- detect high resolution; 3D image in thick section light from laser to excitefluorescent probes. Computer based assembly of multiple points = Confocal Image
Electron Microscopy
: detect high resolution, high magnification images= ultra structure. Fixationw/osmium
Freeze-Fracture
- 3-D TEM Image- in which cells are frozen in liquid nitrogen and then cracked open.Metallic replicas of fractured surfaces are imaged in EM. Cracks open hydrophobic interior of  phospholipids bilayers.
E-face
- half bilayer associated w/ outside of the cell.
P-Face
- half bilayer associated with the cytoplasm.
Hutchinson-Gilford-Progeria Syndrome
- LMNA gene/
NO lamin A
 protein deleted sequence
Nucleolus
- site of ribosomal RNA production + assembly ; comprised of portions of 10 chromosomes(each chrom contributes 40 rRNA genes for a total of 400 genes) Transcription of rRNAs via
PolymeraseI
.
NO MEMBRANE1)Light Fibrillar Center
- 10 chrmosomes2)
Dense Fibrillar Component
- synthesizing rRNA3)
Granular component
- maturing ribonucleoprotie subunits
Ribosome
- one large/one small subunit that independently migrate to cytoplasm to assemble the ribosome(cancer cells have prominent ribosome’s)1)
RNA polymerase I
transcribes 400 rRNA genes
 
5.8s, 18s, 28s
 
2) RNA polymerase III
generates
5s
rRNA3)
18s
 packages into
small ribosomal subunit
4)
5, 5.8, 28s
are packaged into
large subunit
5)
small
and
large
assemble in cytoplasm as
mature 80s ribosome
 
Internal Nuclear Matrix
- 99% nuclear matrix.
NON-RANDOM ORDER 
order must be maintained toensure normal duplication and transcription of genes. Matrix proteins tissue specific. Nuclear MatrixFunctions in DNA replication/transcription. Mostly
Chromatin
(
DNA/
attendant protiens (
Histones H1,H2A, H2B, H3, H4
;
Non-histones
transcription factors, RNA/DNA polymerases)
Euchromatin
- Active
Heterochromatin
- Inactive1)
Chromosomes are non randomly situated in interphase nucleus
(Centromere stuck to nuclear envelope at opposite side of nucleus) occupy same site of interphase nucleus (Barr body qalways in thesame position along the inner nuclear envelope, chromosomes occupy discrete territories, chromosomesmove within its territory)2)
Nuclear matrix proteins are tissue specific -
matrix proteins in normal and cancer cells differ 
.
3)
The nuclear matrix organizes the transcription, and replication of DNA
- nuclear matrix preparationsare enriches in transcriptionally active genes (mRNA is transcribed in nuclear matrix)
DNA replication utilizes machinery that is located in the nuclear matrix
- replicons (site of DNAsynthesis) are in nuclear matrix; DNA synthesis occurs in the matrix _____________________________________________________________________________________ 
Nuclear Matrix
- what remains after extracting more than 95% of the nuclear material with detergent,nuclease enzymes and salt-
framework of the nucleus
-
organizes the genomic DNA
into domains toregulate gene expression and cell replication
 PARTS:
Nuclear Envelope
- only in eukaryotes
double membrane
separates cytoplasm from chromatin,continuous with lumen ER 
Outer nuclear membrane
- faces cytoplasm contains
ribosomes
for protein translation
Inner nuclear membrane
-
integral proteins
that bind to
nuclear lamina
(inside inner that bindheterochromatin (
marginal heterochromatin
)
Pore Complex- holes 10nm, regulate Protiens IN and Protiens and RNA OUT,
situated whereintter/outter meet protiens smaller that
40kD
can diffuse through (larger protiens, complex regulation-understanding how drugs and other therapeutic agents might be delivered to the nucleus)
PORE
contain 3strata (octomeric rings cytoplasm, nuclear, middle)
NUCLEOPORINS-
serve as docking sites to which proteins from cytoplasm that contain
nuclear localization signals
encoded in their primary amino acidstructure can attach
Protein import
: larger must contain
NLS Nuclear Localization Sequence
(contain positively basic amino acids Lysine, Arginine) INCREASE NLS/INCREASE TRANSPORT.
Importin
-
NLS receptor
reside in cytoplasm
Importin A
 binds to NLS,
Importin B
 binds to nucleoporin receptor -
Energy dependant
movement through pore
Signal transduction pathway
- phosphorylation regulates entry into nucleus- transcription factorsmust be modified to permit nuclear entry (dephosphorylation, phosphorylation release from cytoplasmmasking protien which enables recognition by importin)
Protein and RNA Export
:
NES Nuclear Export Sequences
(leucine rich).
Exportins
 protiensRNA are exported based RNA binding proteinsProtein and Rna transport are not pore specificImport/Exports energized by GTP- binding protien
Ran-GTP (
 binding, transport, release
)RanGDP (hydrolysis release of impotin recycling) cytoplasm, Ran GTP in nucleus (release)Nuclear Lamina-
subjacent to inner nuclear envelope- interphase maintains nucleus as sphere , mitosis breaks down
Lamins A + Lamins
C = same gene interact w/ marginal heterochromatin
Lamins B
- bind to inner nuclear membrane via lamin B receptor - stays attached during mitosis,reforms inner nuclear after mitosis MAINTAIN INTEGRITY OF INTERPHASE NUCLEUS
Laminopathies
: mutations of lamin A; cardiac, skeletal muscular dystrophies, preaging(progeria)
 
NucleolusInternal Nuclear Matrix
 _____________________________________________________________________________________ Cell Cycle:
Tg generation Time
- duration of each revolution of the cell cycle = four cell cycle phases
G1
- Senescence, Differentiation, Apoptosis, Proliferation Time varies
S
-Phase- DNA synthesis (2N
4N) Histones synthesized
G2
- Centrosome duplicated, Hyperphosphorylation of histones/ non histone protiens
M
 phase- cells round up, nuclear membrane disintegrates, chromatin condenses, segregation
Cell Cycle Check Points:External factors
:
growth factors
(activate FGF, IGF, Wnt inhibit: TGF), cytokines, hormones
Internal Factors
:
early response genes
:
myc, fos
delayed response
cdk/ cyclins
1) G1 phase Cyclin D-CDK4, phosporylates
retinoblastoma
Rb releases
E2F1 (activate E/A)
2) S phase cyclin E-CDK2 breachs
Restriction check point
, DNA duplicated ,A- CDK2 - sustains S phase3) M phase cyclin B- CDK1 cdc25 dephosphorylates Bcdk1--> nucleus
mega protein phosphory4) Anaphase by
Anaphase Promoter Complex APC
contains
ubiquitinCancer:
caused by inherited mutations/ environmental insults/ aging malignancyDecrease in Differentiation, Increase in Proliferation/ invasiveness of cells
1)Proto-oncogenes
: Abnormal cell cycle stimulation
Tumor : Mutations activate protiens encoded by proto-oncogenes:
oncogenes
..overly expressed/protiens intensified activity:
Ras, src, cyclin D, EGFR 2)Tumor Supressor Genes
: Mutations inactivate tumor suppressors (
p21, p53, Rb, BRCA
)
3)Genes that Regulate Apoptosis: Programmed Cell Death
: normal and necessary, cause tumors toregress
Macrophages
release
TNF
(tumor necrosis factor) binds to TNFR (receptor); balance between pro-apoptotic/anti-apoptotic factors; pro induces leakyness of outter cell membrane of mitochondria;
Cytochrome C
escapes to cytoplasm
Activate
Caspase
(destroy cell organelles)
Apoptosis Ex:
bcl-24)Genes that Induce Cellular Immortality: Telomerase
: shortened telomere signals the cell to becomesenescent (Stem cells, germ cells contain telomerase to restore ends of chromosomes after division)Telomerase is inhibited in somatic cells if its mutated
carcinogenesis5)
Genes that repair DNA
: mutations in DNA repair enzymes
cancer Cancer Therapy:Treatment is currently non-specific. Must develop
specific
treatments.*drugs that inhibit proteases (block metastasis)*drugs that bind and activate mutates
p53
, bind and inactivate mutated
EGFR 
*neutralize antibodies (ritual
CD20)*drugs that inhibit angiogenesis*RNA interferens vi SiRNA1)
Targeting Metastasis
- inhibit
proteases
via active site, metalloproteinases facilitate metastastic spread by enabling the primary cancer cell to eat through the ECM -
TIMPS tissue inhibitors of metalloproteinases
2)
Targeting Angiogenesis
: Prevent blood supply3) Targeting Specific, Sick Molecules:
monoclonal antibodies
or 
small inhibitory RNA (siRNA
) targetand destroy pathogenic RNAs and virusesA.Target cells that are transfected w/dsRNA complementary to mRNAB. enzyme dicer fragmens this dsRNA into 21-28 bp siRNAsC. siRNA complex w/ enzyme
RISC
D. RISC/siRNA complex base-pairs with the complementary pathogenic RNAE. RISC cleaves the pathogenic RNA _____________________________________________________________________________________ 
Cytoskeletal Filaments:

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