Dr. Ray was the only one who showed up. Here is my personal study sheet. Pray hard. You'll do fine.
Review sheet for Final in CMB
5 Senogles,1 Skapek, 2 Scott, 2 Zhang, 2 Ray questions on test
Senogles-Review points and sample questions
Senogles Review included
Know pedigrees. Recognize basic forms inheritance. Know about mapping methods and be able to distinguish. Know differences kinds of cloning. Know what’s on this review, and in her objectives. Proband is first individual Identified as having the disorder. Gene is not dominant or recessive. Phenotype is what you can see. Phenotype might be more than one trait. Affected person indicates a disease phenotype or something clinically observable.
I. Human genetics
a. Autosomal and pseudoautosomal
b. X linked
c. Y linked
Sample question:
What kind of inheritance is the following? Justify your answer.
For pedigrees, explain your answers. Use the questions from her lecture 1 for each type of inheritance. EMAIL HER YOUR QUESTIONS.
II. Mapping of the genome by cytogenetic, genetic and physical methods
Sample question:
Explain how one generates a physical vs. a genetic map of a human chromosome?.
III. Positional vs traditional cloning techniques
a. Chromosome walking and jumping
b. exon identification
Sample question: Explain the difference between functional and positional cloning.
IV. Shermans paradox and resolution
V. Genetic anticipation, somatic mosaicism
Skapek
Concepts to cover:
Developmental processes
Determination
Migration
Differentiation
Cell biology of myogenic differentiation
Molecular biology of myogenic differentiation
Key myogenic transcription factors
How myogenic differentiation is controlled
Ray
2 lectures on apoptosis. He put 8 or 9 questions on Blackboard.
Look at how questions can be answered differently under different contexts.
Be able to tell the difference between necrosis and apoptosis. How is apoptosis relevant to development and disease (remember examples to justify- What happens when apoptosis increases and decreases?)
Remember BCl2 protein domains and how they work. Remember caspases and how they work.
Remember CIAPs that inhibit caspase 3 activity. Do not remember the number of amino acids, remember general mechanisms of activation and inhibition of caspases.
Look at the sample questions. Remember that Each question on the test has one compulsory part, one choice part. You will have to use creativity and your understanding to answer questions. They will not be direct.
For example, in a system you induce apoptosis with TNF-CHX (inhibits anti-apoptotic proteins, which have short half lives), one system with caspase inhibitors. Look at beginning and after certain time point. What would happen? Some cell systems, do not need CHX. TNFa induces both pathways (apoptotic and anti-apoptotic) at same time. One overrrides the other. In the papers, proteins may have different names. Gamma irradiation and chemical induction of apoptosis do not go through receptor
Remember slide on p53 and irradiated mice. Classical example of p53 and BCl2 and apoptosis. Remember the slides. Put them in your memory. He will try to find out what we know, not what we do not know.
Q.1 How would you use the understanding of the mechanisms of cell death for the treatment strategies for the diseases involving apoptosis
Q.2 How would you differentiate a cell undergoing apoptosis from necrosis ?
Q.3 Giving suitable examples discuss in brief the significance of apoptosis during development and pathogenesis
Q.4 Discuss in brief the mechanism by which TNF-α and FAS induces apoptosis.
Q.5 Describe the role of Bcl-2 family proteins in the regulation of
Apoptosis.
Q.6 Discuss in brief the mechanisms by which γ-irradiation and chemical agents induce apoptosis.
Q.7 Giving suitable example discuss the role of apoptosis in the development of cancer.
Q.8 Discuss the role of mitochondria in the regulation of apoptosis
Zhang
Review Points
1. For cell-cell adhesion lecture
a. How is cadherin connected to cytoskeleton?
b. What are the structural and functional differences between gap junction and gap junction.
c. Compare the compositions of adherent junction and desmosome
d. List the calcium-independent cell-cell adhesion molecules.
e. What are cadherin-mediated functions?
f. Illustrate the roles of E-cadherin and β-catenin in tumorigenesis and tumor progression.
2. For cell-extracellular matrix adhesion lecture
a. What are the cellular receptors of fibronectin, laminin, and collagen?
b. What are the major components of the stromal environment in connective tissue?
c. Give an example that integrin functions as a cell-cell adhesion molecule.
d. Is the RGD sequence the binding site in collagen for integrin?
e. What are the components of basement membrane?
f. Why is integrin called bi-directional signaling machinery?
Scott
Know the 6-part definition of differentiation.
Know fundamental definitions of other terms: dedifferentiation, transdifferentiation, metaplasia, terms from his cancer lectures. Be able to describe the controversy surrounding transdifferentiation and dedifferentiation. Do stem cells grow in adult tissues into which they are implanted? Do they merely fuse with the cells in the tissue? (There is an article in Science about this from several years back that marked chromosomes in the injected cells with fluorescent dye. They showed cell fusion in heart tissue). Does it depend on the tissue? Be able to state evidence to support arguments either way.
This is what I have, folks. If you need more detail, contact the professors. Have fun studying.
Saturday, February 17, 2007
Friday, February 16, 2007
Senogles 6
Senogles 6 Huntington’s Disease
1
Huntington’s is a trinucleotide repeat expansion disease, but has unique characteristics. Dementia is due to severe effects on basal ganglia of brain. 2 forms: adult onset and juvenile form. Juvenile form gave insights into inheritance
2
Emphasis on juvenile is because of insights.
3
RFLP marker assigned to chromosome 4 in 1983. One of 1st testable markers for a disease.
Marker led to cloning of disease gene. Linkage disequilibrium- indicates ancestor gene close to marker.
4
Huge pedigrees back to 1800s from families around Lake Maracaibo in Venezuela.
5
Assigned to end of chromosome 4- 5 million base pair fragment.
6
Segregation with HD: Markers land close to disease gene. Diamonds indicate blind study- did not want to give identifying information about individuals.
7
Restriction map and theoretical locus: Knew that disease mapped to this region.Wanted to clone gene.
8
Problems: big region. Instead of chromosome walking and jumping and cros-hybridization, they used exon amplification.
Make family of fragments, generate probes, screen library.
9
Take vector with known sequences on either side of multiple cloning site (commonly use a beta globulin). Clone in DNA. Get family of fragments. Transfect cell. Isolate RNA, amplify by PCR using primers for known sequence. PCR product is exon flanked by known sequence. If it has an exon, will be transcribed and can be identified. Idea of trapping- if vector has exonic sequence- RNA will result. Can sequence PCR product. Now have probe to screen CDNA library and Northern blots.
10
Using strategy, cloned a number of CDNAs. Composite is 340 KB protein. Found that in giant clone had large ORF with 2 repeats in middle of coding sequence. Poly Q, and poly P. Poly P is not related to disease. Poly Q is.
12
Repeat size is larger, but not horrendously larger. This is more subtle. Disease state centers around 45 repeats.
13
Juvenile Huntington’s: imprinting phenomenon. Repeat size and age of onset correlate.
14
Huntington’s is autosomal dominant, so affected male has an allele in the normal range, and an expanded one. For autosomal dominant disease, one parent must be affected, shows up in every generation.
Some individuals have larger repeats than parent . 8 and 10 do. Age of onset correlates to size of expanded allele.
15
In Venezuela you can get huntington’s disease homozygous. About 25% of kids are homozygous dominant. Some are heterozygous. Age onset homozygous is 6-7 yrs.
16
Sporadic mutation occurs. PCR shown. Huntington’s can be caused by sporadic increase in repeats.
17
No evidence of Huntington’s until mutation. Expansions in fragile X were in UTR. These are in code for a protein. Changes in poly Q yield changes in protein function.
18
Genetic anticipation- bigger expanded allele, earlier child diagnosed with disease. Not Mendelian phenomenon. Through mother- repeat sizes do not change all that much. Changes dramatically in gene passed down from father.
19
Scattergram of juvenile age onset vs number of repeats.
20
Age of onset is a function of repeat length- no correlation with maternal descent- is correlated with paternal in juvenile Huntington’s.
21
Large repeats have paternal origin, in contrast to Fragile X.
22
Expansion may occur during male gametogenesis. Somatic mosaicism- different numbers of repeats in different somatic cells from same patient. Different tissues are different.
23
Protein product- you get a functional protein and can observe multiple species. Proteins are made with larger repeat. Heterogeneity detectable at protein level.
24
Lots of expression in basal ganglia.
25
From a given individual, samples from cortex and cerebellum are different. Individuals cannot efficiently replicate repeats. Different size proteins are reflected.
26
Result:
Basis of Huntington’s is undefined. Cellular inclusions form in cells and cause cell death. This may be a gain-of-function mutation- as protein expands, interacts with new things and causes bad effects.
Transgenic mice- can get Huntington’s phenotype.
1
Huntington’s is a trinucleotide repeat expansion disease, but has unique characteristics. Dementia is due to severe effects on basal ganglia of brain. 2 forms: adult onset and juvenile form. Juvenile form gave insights into inheritance
2
Emphasis on juvenile is because of insights.
3
RFLP marker assigned to chromosome 4 in 1983. One of 1st testable markers for a disease.
Marker led to cloning of disease gene. Linkage disequilibrium- indicates ancestor gene close to marker.
4
Huge pedigrees back to 1800s from families around Lake Maracaibo in Venezuela.
5
Assigned to end of chromosome 4- 5 million base pair fragment.
6
Segregation with HD: Markers land close to disease gene. Diamonds indicate blind study- did not want to give identifying information about individuals.
7
Restriction map and theoretical locus: Knew that disease mapped to this region.Wanted to clone gene.
8
Problems: big region. Instead of chromosome walking and jumping and cros-hybridization, they used exon amplification.
Make family of fragments, generate probes, screen library.
9
Take vector with known sequences on either side of multiple cloning site (commonly use a beta globulin). Clone in DNA. Get family of fragments. Transfect cell. Isolate RNA, amplify by PCR using primers for known sequence. PCR product is exon flanked by known sequence. If it has an exon, will be transcribed and can be identified. Idea of trapping- if vector has exonic sequence- RNA will result. Can sequence PCR product. Now have probe to screen CDNA library and Northern blots.
10
Using strategy, cloned a number of CDNAs. Composite is 340 KB protein. Found that in giant clone had large ORF with 2 repeats in middle of coding sequence. Poly Q, and poly P. Poly P is not related to disease. Poly Q is.
12
Repeat size is larger, but not horrendously larger. This is more subtle. Disease state centers around 45 repeats.
13
Juvenile Huntington’s: imprinting phenomenon. Repeat size and age of onset correlate.
14
Huntington’s is autosomal dominant, so affected male has an allele in the normal range, and an expanded one. For autosomal dominant disease, one parent must be affected, shows up in every generation.
Some individuals have larger repeats than parent . 8 and 10 do. Age of onset correlates to size of expanded allele.
15
In Venezuela you can get huntington’s disease homozygous. About 25% of kids are homozygous dominant. Some are heterozygous. Age onset homozygous is 6-7 yrs.
16
Sporadic mutation occurs. PCR shown. Huntington’s can be caused by sporadic increase in repeats.
17
No evidence of Huntington’s until mutation. Expansions in fragile X were in UTR. These are in code for a protein. Changes in poly Q yield changes in protein function.
18
Genetic anticipation- bigger expanded allele, earlier child diagnosed with disease. Not Mendelian phenomenon. Through mother- repeat sizes do not change all that much. Changes dramatically in gene passed down from father.
19
Scattergram of juvenile age onset vs number of repeats.
20
Age of onset is a function of repeat length- no correlation with maternal descent- is correlated with paternal in juvenile Huntington’s.
21
Large repeats have paternal origin, in contrast to Fragile X.
22
Expansion may occur during male gametogenesis. Somatic mosaicism- different numbers of repeats in different somatic cells from same patient. Different tissues are different.
23
Protein product- you get a functional protein and can observe multiple species. Proteins are made with larger repeat. Heterogeneity detectable at protein level.
24
Lots of expression in basal ganglia.
25
From a given individual, samples from cortex and cerebellum are different. Individuals cannot efficiently replicate repeats. Different size proteins are reflected.
26
Result:
Basis of Huntington’s is undefined. Cellular inclusions form in cells and cause cell death. This may be a gain-of-function mutation- as protein expands, interacts with new things and causes bad effects.
Transgenic mice- can get Huntington’s phenotype.
Wednesday, February 14, 2007
Senogles 5
Senogles 5
Fragile X syndrome
Review questions are posted on Blackboard under Assignments with bullets for important points. We will review after lecture tomorrow-short. You can also email her.
Tomorrow –Huntingdon’s disease.
Today-fragile X. These are trinucleotide expansion diseases (the only new development in genetics in a long time- dseases in which a repeat of three nucleotides is generated too many times, separating a gene from its promoter, and a disease results). This mechanism explains some diseases with weird inheritance.
Decreased penetrance can occur with an X-linked dominant disease. The phenotype is not manifested the same in each individual. More severe in males. Oddball is the normal transmitting male. Flag: carrier is not affected, but subsequent generations are severely affected.
2
Sherman’s paradox will be on the exam. Understand basic evidence for #2.
3
Sherman was a clinical psychologist who discovered aberrations in pedigrees. For X-linked dominant disorder, risk should not change in pedigree for succeeding generations. Something was wrong here. This is unusual genetics.
4
X-linked dominant disorders- all female children of affected males are carriers.
5
Review slide of normal X-linked transmission.
6
Sherman’s paradox pedigree
Risk gets higher with succeeding generations. Grandchildren at higher risk than siblings of the normal transmitting male (T).
7
Look at karyotype of patient- deprive cells of folate in vitro- X chromosome fragments at fragile sites. Name of the disease comes from karyotype.
8
Used perturbation to clone by somatic cell hybrids. Breakage is always at same place on chromosome. Take panel of rodent cells and fuse human cells to them. Used fragile X patient cells. X breaks under thymidine stress and reforms with rodent cells.
9
Heterokaryon is fused cell.
Mouse tumor cell is immortal and clonal. Form heterokaryons, select, then grow fused cells on permissive medium that selects only for hybrid cells. Wind up with hybrid cells that are clonal- each group grows from single cell. Can use RT-PCR to see what human chromosomes are carried in the heterokaryons.
10
Get a rodent chromosome with a bit of a human chromosome at the end.
11
When mapped out, get this diagram. The fragile X site is near telomeric end. B is a higher resolution map. CpG islands are normally unmethylated regions 5’ to a mammalian gene. Probes used are shown in D. Hypothesis was that something odd was going on near CpG island.
12
Southern analysis of 2 fragile X pedigrees. Filled in squares are affected individuals. Females indicate various degrees of clinical manifestation. Took blood, looked at DNA. Normal individual- Eag1 should cut, indicating a non-methylated CpG island.
13
Different pedigree. Affected males and carrier females have higher fragment size from CpG islands being methylated. Region is larger in some affected males and carrier females. Two things are happening (methylation of the CpG islands and increased numbers of repeats).
14
Track through pedigree analysis and look at size of insertions- size on Southern blot gets larger (higher in gel). Gets substantially larger in terms of base pairs. The insertion is at the fragile X locus.
15
Cloned region. Circled area is CpG island. Repeat of CCG is shown by arrows.
Repeat could be basis of increase of fragment size, or a nearby gene could be changing.
17
Flanking regions did not change size. Repeat region changes, not flanking regions.
19
Examine repeat sequence in more detail. Is it polymorphic in normal individuals, or just in fragile X families?
20
Is polymorphic in normal population. Run sequencing gel to determine size of repeats. PCR through- get normal repeat size (29 repeats on average).
21
Look at fragile X pedigrees- centers on several hundred repeats.
22
Studied number of pedigrees. Found risk of expansion to full mutation was correlated through premutation of mother . Expansion occurs in carrier female. Graph shows repeat size in mother. Does not correlate with paternal allele size. Imprinting- differential expression depending on parent allele comes from.
23
This is consistent with Sherman’s observations. Expansions go through females, but only when allele is transmitted through female does risk increase. NTM (normal transmitting male) has expanded allele, but not fragile X. NTM is key. He has premutation repeat size. Size of allele correlates well with retardation in males, but X inactivation in females complicates things, producing diverse phenotypes.
24
3 is at 53/29 copies (females have 2 X chromosomes, so for a female, there can be two sets of repeat values). Daughter 4 is at 29/83 copies. 29 size does not change. Other one changes. Sons relevant DNA will not enter gel- off scale in terms of number of repeats.
Pedigree 2:
1 has 83 and 29 repeats. 3 has a reduction in size, which is possible. Repeat sizes are dynamic. Nobody knows how the expansion and reduction occur. Curent thinking is that repeats form odd structures so they can’t copy accurately.
25
premutation size is also dynamic. Normal family shown. Premutation size shown. There is flux even in premutation alleles.
26
Affected gene was cloned. Original start site in the literature was wrong. Originally reported repeat in coding region. It is actually in the 5’UTR. Called Fragile X Mental Retardation Gene (FMR).
27
Methylated CpG island and repeat size change in fragile X patients. How are they linked? Look at methylation status. By Southern blot analysis we see complete cleavage (unmethylated DNA) in normal individuals. Carrier females- methylated and unmethylated. Affected males- not digested by BssHII. They have methylated CpG island close to gene. Carrier females- half methylated. They have 2 copies. Fragile X males- CpG is completely methylated.
28
Expression or transcription of gene: there is a correlation between methylation and gene expression- inactivated by methylation.Silences DNA. Transcription of FMR1 was measured as follows: Isolated RNA, reverse transcriptased it,Used RT-PCR. No message- no band on PCR. All had HPRT housekeeping gene. N are normal individuals. Asterisks are fragile X patients- no transcript.Carriers also lack the transcription of the gene.
29
Scan gel with densitometer-2 blips for normal individual. In patient 411- no FMR1 product. Patient 412- reduced over normal population. 410 carrier female also has reduced quantities.
30
Correlates expression and methylation status.
31
Normal repeat size puts AUG close to CpG island unmethylated, allowing transcription. As repeat size gets larger, still get transcription.
Full mutation size, cell methylates CpG island and shuts down gene.
32
Resolved Sherman’s paradox with a mechanistic explanation to some degree. Repeat size gets large through mitotic expansion in the females. There is a threshold size of repeat.
Fragile X syndrome
Review questions are posted on Blackboard under Assignments with bullets for important points. We will review after lecture tomorrow-short. You can also email her.
Tomorrow –Huntingdon’s disease.
Today-fragile X. These are trinucleotide expansion diseases (the only new development in genetics in a long time- dseases in which a repeat of three nucleotides is generated too many times, separating a gene from its promoter, and a disease results). This mechanism explains some diseases with weird inheritance.
Decreased penetrance can occur with an X-linked dominant disease. The phenotype is not manifested the same in each individual. More severe in males. Oddball is the normal transmitting male. Flag: carrier is not affected, but subsequent generations are severely affected.
2
Sherman’s paradox will be on the exam. Understand basic evidence for #2.
3
Sherman was a clinical psychologist who discovered aberrations in pedigrees. For X-linked dominant disorder, risk should not change in pedigree for succeeding generations. Something was wrong here. This is unusual genetics.
4
X-linked dominant disorders- all female children of affected males are carriers.
5
Review slide of normal X-linked transmission.
6
Sherman’s paradox pedigree
Risk gets higher with succeeding generations. Grandchildren at higher risk than siblings of the normal transmitting male (T).
7
Look at karyotype of patient- deprive cells of folate in vitro- X chromosome fragments at fragile sites. Name of the disease comes from karyotype.
8
Used perturbation to clone by somatic cell hybrids. Breakage is always at same place on chromosome. Take panel of rodent cells and fuse human cells to them. Used fragile X patient cells. X breaks under thymidine stress and reforms with rodent cells.
9
Heterokaryon is fused cell.
Mouse tumor cell is immortal and clonal. Form heterokaryons, select, then grow fused cells on permissive medium that selects only for hybrid cells. Wind up with hybrid cells that are clonal- each group grows from single cell. Can use RT-PCR to see what human chromosomes are carried in the heterokaryons.
10
Get a rodent chromosome with a bit of a human chromosome at the end.
11
When mapped out, get this diagram. The fragile X site is near telomeric end. B is a higher resolution map. CpG islands are normally unmethylated regions 5’ to a mammalian gene. Probes used are shown in D. Hypothesis was that something odd was going on near CpG island.
12
Southern analysis of 2 fragile X pedigrees. Filled in squares are affected individuals. Females indicate various degrees of clinical manifestation. Took blood, looked at DNA. Normal individual- Eag1 should cut, indicating a non-methylated CpG island.
13
Different pedigree. Affected males and carrier females have higher fragment size from CpG islands being methylated. Region is larger in some affected males and carrier females. Two things are happening (methylation of the CpG islands and increased numbers of repeats).
14
Track through pedigree analysis and look at size of insertions- size on Southern blot gets larger (higher in gel). Gets substantially larger in terms of base pairs. The insertion is at the fragile X locus.
15
Cloned region. Circled area is CpG island. Repeat of CCG is shown by arrows.
Repeat could be basis of increase of fragment size, or a nearby gene could be changing.
17
Flanking regions did not change size. Repeat region changes, not flanking regions.
19
Examine repeat sequence in more detail. Is it polymorphic in normal individuals, or just in fragile X families?
20
Is polymorphic in normal population. Run sequencing gel to determine size of repeats. PCR through- get normal repeat size (29 repeats on average).
21
Look at fragile X pedigrees- centers on several hundred repeats.
22
Studied number of pedigrees. Found risk of expansion to full mutation was correlated through premutation of mother . Expansion occurs in carrier female. Graph shows repeat size in mother. Does not correlate with paternal allele size. Imprinting- differential expression depending on parent allele comes from.
23
This is consistent with Sherman’s observations. Expansions go through females, but only when allele is transmitted through female does risk increase. NTM (normal transmitting male) has expanded allele, but not fragile X. NTM is key. He has premutation repeat size. Size of allele correlates well with retardation in males, but X inactivation in females complicates things, producing diverse phenotypes.
24
3 is at 53/29 copies (females have 2 X chromosomes, so for a female, there can be two sets of repeat values). Daughter 4 is at 29/83 copies. 29 size does not change. Other one changes. Sons relevant DNA will not enter gel- off scale in terms of number of repeats.
Pedigree 2:
1 has 83 and 29 repeats. 3 has a reduction in size, which is possible. Repeat sizes are dynamic. Nobody knows how the expansion and reduction occur. Curent thinking is that repeats form odd structures so they can’t copy accurately.
25
premutation size is also dynamic. Normal family shown. Premutation size shown. There is flux even in premutation alleles.
26
Affected gene was cloned. Original start site in the literature was wrong. Originally reported repeat in coding region. It is actually in the 5’UTR. Called Fragile X Mental Retardation Gene (FMR).
27
Methylated CpG island and repeat size change in fragile X patients. How are they linked? Look at methylation status. By Southern blot analysis we see complete cleavage (unmethylated DNA) in normal individuals. Carrier females- methylated and unmethylated. Affected males- not digested by BssHII. They have methylated CpG island close to gene. Carrier females- half methylated. They have 2 copies. Fragile X males- CpG is completely methylated.
28
Expression or transcription of gene: there is a correlation between methylation and gene expression- inactivated by methylation.Silences DNA. Transcription of FMR1 was measured as follows: Isolated RNA, reverse transcriptased it,Used RT-PCR. No message- no band on PCR. All had HPRT housekeeping gene. N are normal individuals. Asterisks are fragile X patients- no transcript.Carriers also lack the transcription of the gene.
29
Scan gel with densitometer-2 blips for normal individual. In patient 411- no FMR1 product. Patient 412- reduced over normal population. 410 carrier female also has reduced quantities.
30
Correlates expression and methylation status.
31
Normal repeat size puts AUG close to CpG island unmethylated, allowing transcription. As repeat size gets larger, still get transcription.
Full mutation size, cell methylates CpG island and shuts down gene.
32
Resolved Sherman’s paradox with a mechanistic explanation to some degree. Repeat size gets large through mitotic expansion in the females. There is a threshold size of repeat.
Tuesday, February 13, 2007
Senogles 4
Senogles 4: Color Blindness
She expects us to know her learning objectives, and her tests are very straightforward. She shows references in her lectures, but we DO NOT have to read them.
1
Next 3 lectures are about the molecular basis of 3 different diseases. Humans are trichromats, and our brains mix the colors to produce what we see.
2
Opsins function as a tandem array.
Ancestor opsin duplicated and changed to produce color opsins.
6
Green deficiency-muddy brown. Cats and dogs are dichromatic.
7
Number cannot be perceived by colorblind person.
8
Rhodopsin allows dim-light vision. Cones are for color. Photoreceptors are named for color opsins they carry.
10
Bottom graph- different opsins are sensitive to different wavelengths.
11
Eye perceives light by rods and cones. Rods mediate light intensity. Cones carry distinct opsins for color receptors.
12
Rhodopsin is a G-protein-coupled receptor with 7 transmembrane spanning domains. Light is agonist causing isomerization of retinal. Cis-trans conformational change is the activation signal for the receptor.
13
Nathan Used rhodopsin as probe in eye library. Little squares indicate 6 exons for each protein he found.
14
He cloned them out and looked at protein similarity. Did stepwise comparison of proteins sequences for amino acid similarity. Amino acid identity shown in white. Differences are shown in black. Highly related proteins.
15
Probably result of gene duplication event.
16
3 reasons for tandem arrays in the human genome:
1. Histone tandem array (lots of copies of the genes for histone subunits arranged end to end) on genome because you need a lot of histones at certain points in growth. Get a lot made in a short period from master promoter.
2. BetaGlobin locus gene family: this protein has different fetus, embryo, adult forms. Region can be under coordinate regulation- region under master switch.
3. diversity in organisms. Evolutionary pressure to increase color vision for viability.
Unequal crossing over event creates 2 genes on one chromosome, none on other. The one with two may have a survival advantage.
17
Selection pressure leads to gene being maintained in original form if it is necessary for viability. If selection pressure is not so great, mutations may result in a gene with a related function or a pseudogene. There are lots of human pseudogenes. Slow and rapid are still millions of years. What was thought to happen in opsins was that rhodopsin maintained its original function and color opsins came later (18). Know this is how proteins with closely related functions are thought to be generated.
19
Photopigments underly both normal and color-deficient vision.
20
XLR=x-linked recessive.
AD=autosomal dominant
AR= autosomal recessive
Blue opsin is on chromosome 6, red and green on X.
21
Remember that affected males are related through mother- and which X male gets determines whether his vision is normal or not.
23
Nathan says take color normal individual and look a red and green opsins. He made probes for green and red opsins (at bottom). Used shorthand nomenclature as way to express data. Arrow is entire locus. Probes are A,B,C,D. Ag is the A probe for green.
He normalized DNA on the Southern blot. Intensity of bands is different. That means more copies of G on blot than R (For example, look at blot A. The bands for Ag are much darker than those for Ar. Another clue is that some color normal individuals are missing Dr. This is a key to the arrangement of the tandem array. They have to be in order on the chromosome. Dr was lost during recombination events that duplicated the green opsin gene. R gene is 5’. If it were at 3’ end you would sometimes lose parts of green.
24
Nathan speculated . Did densitometry scan on Southern blot and found it was common to have multiple copies of green. Had one red, 1 to 3 copies green.
A is color normal males. B shows mechanism for changing number of copies of green.
25
Looking at dichromats, genotypes shown across top of blot. Genomic probes shown upper left. This is a more complex analysis. Some individuals have complete loss of parts of opsins probed. You do ratio of green to red.
26
G-R+ had 2 phenotypes: missing entire Green opsin, or missing Dg. Just missing 3’ end probe.
G+R- more complex- variety phenotypes listed. All have Dg. 14 is color normal. Compare the others in this graph to him. These are “blown out” slides, so you cannot tell intensity easily.
Conclusions are based on Southern-blot data.
27
remember arrow represents entire gene region. Colored in to represent what is present.
A- color normal individual.
B- Recombination resulting in G+R-
C- Examples of how recombination results in unequal crossing over events with unusual arrays.
The data is complicated- go back and compare the slides and data. Slide 27 is a model to explain what is sitting in the genome to produce the Southern blot data. He hypothesized these structures to account for dichromatic vision. Look at analysis and blots to get it to make sense. She will not ask details of this. Understnd how got idea of 1 red to multiple copies of green and how to look at pathology of cases. How do you get the model from the data?
28
Achromatopsia is rare. No red or green.
30
X-linked trait.
31
genomic map red-green opsins. Added Z probe upstream of red locus.
32
Southern analysis again. 9 and 10 are color normal. Z missing or shortened in people with BCM. They have intact fragments for the opsins mostly, but missing 5’Z region (See data in G). Z region appears to be essential. Major control region just upstream is necessary.
Go back and look. It will make sense.
She expects us to know her learning objectives, and her tests are very straightforward. She shows references in her lectures, but we DO NOT have to read them.
1
Next 3 lectures are about the molecular basis of 3 different diseases. Humans are trichromats, and our brains mix the colors to produce what we see.
2
Opsins function as a tandem array.
Ancestor opsin duplicated and changed to produce color opsins.
6
Green deficiency-muddy brown. Cats and dogs are dichromatic.
7
Number cannot be perceived by colorblind person.
8
Rhodopsin allows dim-light vision. Cones are for color. Photoreceptors are named for color opsins they carry.
10
Bottom graph- different opsins are sensitive to different wavelengths.
11
Eye perceives light by rods and cones. Rods mediate light intensity. Cones carry distinct opsins for color receptors.
12
Rhodopsin is a G-protein-coupled receptor with 7 transmembrane spanning domains. Light is agonist causing isomerization of retinal. Cis-trans conformational change is the activation signal for the receptor.
13
Nathan Used rhodopsin as probe in eye library. Little squares indicate 6 exons for each protein he found.
14
He cloned them out and looked at protein similarity. Did stepwise comparison of proteins sequences for amino acid similarity. Amino acid identity shown in white. Differences are shown in black. Highly related proteins.
15
Probably result of gene duplication event.
16
3 reasons for tandem arrays in the human genome:
1. Histone tandem array (lots of copies of the genes for histone subunits arranged end to end) on genome because you need a lot of histones at certain points in growth. Get a lot made in a short period from master promoter.
2. BetaGlobin locus gene family: this protein has different fetus, embryo, adult forms. Region can be under coordinate regulation- region under master switch.
3. diversity in organisms. Evolutionary pressure to increase color vision for viability.
Unequal crossing over event creates 2 genes on one chromosome, none on other. The one with two may have a survival advantage.
17
Selection pressure leads to gene being maintained in original form if it is necessary for viability. If selection pressure is not so great, mutations may result in a gene with a related function or a pseudogene. There are lots of human pseudogenes. Slow and rapid are still millions of years. What was thought to happen in opsins was that rhodopsin maintained its original function and color opsins came later (18). Know this is how proteins with closely related functions are thought to be generated.
19
Photopigments underly both normal and color-deficient vision.
20
XLR=x-linked recessive.
AD=autosomal dominant
AR= autosomal recessive
Blue opsin is on chromosome 6, red and green on X.
21
Remember that affected males are related through mother- and which X male gets determines whether his vision is normal or not.
23
Nathan says take color normal individual and look a red and green opsins. He made probes for green and red opsins (at bottom). Used shorthand nomenclature as way to express data. Arrow is entire locus. Probes are A,B,C,D. Ag is the A probe for green.
He normalized DNA on the Southern blot. Intensity of bands is different. That means more copies of G on blot than R (For example, look at blot A. The bands for Ag are much darker than those for Ar. Another clue is that some color normal individuals are missing Dr. This is a key to the arrangement of the tandem array. They have to be in order on the chromosome. Dr was lost during recombination events that duplicated the green opsin gene. R gene is 5’. If it were at 3’ end you would sometimes lose parts of green.
24
Nathan speculated . Did densitometry scan on Southern blot and found it was common to have multiple copies of green. Had one red, 1 to 3 copies green.
A is color normal males. B shows mechanism for changing number of copies of green.
25
Looking at dichromats, genotypes shown across top of blot. Genomic probes shown upper left. This is a more complex analysis. Some individuals have complete loss of parts of opsins probed. You do ratio of green to red.
26
G-R+ had 2 phenotypes: missing entire Green opsin, or missing Dg. Just missing 3’ end probe.
G+R- more complex- variety phenotypes listed. All have Dg. 14 is color normal. Compare the others in this graph to him. These are “blown out” slides, so you cannot tell intensity easily.
Conclusions are based on Southern-blot data.
27
remember arrow represents entire gene region. Colored in to represent what is present.
A- color normal individual.
B- Recombination resulting in G+R-
C- Examples of how recombination results in unequal crossing over events with unusual arrays.
The data is complicated- go back and compare the slides and data. Slide 27 is a model to explain what is sitting in the genome to produce the Southern blot data. He hypothesized these structures to account for dichromatic vision. Look at analysis and blots to get it to make sense. She will not ask details of this. Understnd how got idea of 1 red to multiple copies of green and how to look at pathology of cases. How do you get the model from the data?
28
Achromatopsia is rare. No red or green.
30
X-linked trait.
31
genomic map red-green opsins. Added Z probe upstream of red locus.
32
Southern analysis again. 9 and 10 are color normal. Z missing or shortened in people with BCM. They have intact fragments for the opsins mostly, but missing 5’Z region (See data in G). Z region appears to be essential. Major control region just upstream is necessary.
Go back and look. It will make sense.
Sunday, February 11, 2007
Senogles 2 and 3
Senogles 2
Correction for Yesterday:
Pseudoautosomal Inheritance
Erratum for yesterday’s lecture: pseudoautsomal domains include only the ends
Y linked is also holandric inheritance. No daughter carriers. Pseudoautosomal has carrier daughters.
Ways to map genome:
Complementary and tell you different things. Cytogenetic mapping today, then genetic mapping and physical mapping. Genetic is mapping by inheritance. Physical map is the sequence itself. Cytogenetic map is chromosome.
2
Get this. Be able to understand what mapping CF by RFLP means.
3
Chromosomes as spread here have 2 arms. Nomenclature is standard and based on Giemsa staining.
4
R banding is reversed.
5
position is defined unequivocally in international nomenclature.
Ideograms are based on G banding of actual chromosomes.
7
Pattern FYI
8
female pattern. These gross analyses are used for a 1st test for a lot of chromosomal abnormalities.
9
Know that abnormalities exist. She does not care if you know details. Know they can be seen using cytogenetic methods.
10
Aneuploidy
Nondisjunction can occur – chromosomes do not separate in first round- one cell gets none, one gets both chromosomes. Gametes still function and can fertilize or be fertilized. If nondisjunction at second round, normal complement for 2 cells,a diploid, and a zero complement. Know that aneuploidy happens because separation does not occur properly.
Outcome- trisomy 13,18,21 are viable, but bad. Many other trisomies are lethal. XXX not as devastating. Monosomy X is Turner’s syndrome- one of few monosomies to survive.
11
Rearrangements occur with DS breaks. Repair system usually correct these breaks. Balanced- swapping pieces of DNA- no removal or deletion.
Unbalanced- duplicate or loss- serious potential effects.
12
Possible changes. Squashes or FISH can detect these changes in order. Most of the time these are silent.
13
Reciprocal translocations- no net loss or gain, just change in location. This is not recombination. This is translocation between different chromosomes.
May disrupt gene function if break is between promoter and gene. May be silent.
14
Nonreciprocal translocation- adds chromosomal material to another chromosome.
16
Tandem duplication results in repitition of segment.
17
Have knowledge of FISH analysis. #1 for cytogenetic analysis. Can “paint” whole chromosome or other things listed. Human chromosome has lots of repeat sequence. SSRs are inherited markers.
18
FISH probe is complementary only to a region of the chromosome. Family of probes can be made to entire chromosome.
22
Whole chromosome painting- probe each chromosome with a different dye emitting at different wavelength for different color. Can see deletions, increase in chromosome size.
24
Chromosome 19 has unbalanced rearrangement to attach to another chromosome.
She likes FISH analysis.
29
Genetic mapping can cause problems with understanding.
Linkage analysis- tendency for alleles close together on chromosome to be transmitted together. Can map through pedigree analysis. Close together- stay together in recombination. RFLP not used much anymore. Markers are not necessarily genes- they can be followed in pedigrees. Markers allow us to map genes to chromosomes.
30
1. relative to each other.
2. Polymorphic means difference among individuals.
RFLP and SSR are markers.
31
cM is a genetic unit. Crossing over happens a lot, so if genes are together 1% of the time, they are close together. cM and base pairs are not equivalent. To map a disease to a chromosome, must map several markers on each chromosome for inheritance.
33
Allele view. D and M could be separated by recombination. Randomly distributed- not linked.
34
Look at progeny or gametes that result. Some linkage- intermediate.
35
Marker A is not linked to disease phenotype, but marker B is.
Marker is not a predictor of disease per se, but happens to be linked. Not predictor. To be a predictor of disease, if you find marker very very close, can be used as diagnostic event. It may be linked to the disease gene, not associated with the disease itself.
36
RFLP- anything can be used as a marker. Restriction site can be used. 1bp change creates an ECOR1 cut site. RFLP site is inherited. See it a lot in literature, but not used much anymore. Get tons of fragments. Lots of work.
37
How is it done now? SSR or SRS (simple repeat sequence). Microsatellites are the repeat sequence. Repeats det by PCR and electrophoresis. Each chromosome has a different repeat number.Each individual 2 copies with different repeat sequence. Useful way to map. Marker and easier to follow than RFLPs.
38
SSRs in chromosome 1 Markers are under linkage map.
39 Physical mapping
We know the sequence, but we need to be able to navigate around.
40
Take part of human chromosome and fuse into rodent cell line- panel of hybrids with different parts of chromosome.PCR for gene of interest. Clonal lines .
STS are physical sites that are not simple repeat sequence. These are not polymorphic. These do not change and are the same in every human being. Cannot follow in genetic map because everyone has the same ones. One copy.
Fri- clone CF gene. Tues- inherited color vision, fragile X, Huntingdon’s disease.
Senogles 3
Assignment is posted.
Today we talk about positional cloning. Text reference is to cystic fibrosis (CF). Specific references are not required reading. Presented because it is good science and illustrative of how you do the technique.
3
Functional common until 1990s. Purified protein, followed activity as you purified, got to homogeneity, sequenced, got protein sequence, made oligonucleotide, screened library, got gene. You knew what you were looking for and what the activity was.
Now we look on the basis of where it maps on the chromosome with very little protein information. Positional cloning- map by genetics, then clone.
4
Knowing the DNA sequence does not help because you do not know what you are looking for.
5
2 types of mapping: see slide
6
10% codes for exons. Small part of genome.
7
Localize by linkage analysis position of disease gene you are interested in. Want to localize to greatest extent we can. Yacs and BACs can handle large pieces of DNA by cloning.
If you go to human genome project, is good resource. Can click on a region and see all the known genes in the region.
The two markers were well known.
Linkage disequilibrium- had a mutation in a chromosome. As goes through successive generations, some markers closely segregate with it. B1 stays with CF. It is in linkage disequilibrium. Suggests a founder chromosome with that marker.
11
High resolution map. Things with D are markers, polymorphic, inherited. Closer you can narrow it, less DNA you have to sequence.
12
Trying to minimize brute-force sequencing.
CpG islands tend to be islands of methylated CG that are close to promoters in coding regions. If it cross hybridizes, it is conserved and may be the candidate gene.
13
Want to isolate large fragments first. Large fragment- Mb.
14 Question: Disease- how to clone it out?
Chromosome walking- series overlapping clones. Cut up DNA. Make series of restriction fragments. Pull out and sequence along. Laborious.
15
Chromosome jumping- maximize potential hits by jumping to places from which you can sequence. Covers maximum amount of distance and bypasses unsequenceable stretches of DNA.
Circularized- A and B become close. Can sequence short segment between A and B to find the sequence of site B. Can jump, do not plan to go anywhere. You don’t really care what was between A and B to begin. Can make jumping library, and make a library of sites to walk from. Jumping randomly, then sequence from positions in both directions. Increases chances you will hit something interesting.
16
Series of jumps and where sequence from are shown.
18
Arcs are jumps, horizontal lines are sequence. Roman numerals indicate exons.
20
How do we know we have sequenced something interesting? Look for an ORF.
2 types of candidate genes.
21
Used 2 criteria for candidate genes:
Conserved in phylogenetic tree if important for viability. Used strategy for trying to clone further. Looked for CpG islands at 5’end of genes.
22
CpGs tend to fall close to beginning of genes. Housekeeping genes do not often have unmethylated CpG islands, but class 2 proteins do.
23 Found cross-hybridized exons. They probed a zooblot.
24
Found exon1 with ORF. Screened library and got 24 exons. He had sequence of something.
26
Found 12 TM domains.
30
Evidence-Does mutation correlate with disease? Is function disrupted?
31
Sequencing ladder shown. CTT was deleted. Was an in-frame deletion. Segregated with disease.
Did Southern blot. Most of homozygotes had mutation.
33
Ahead 10 years- CFTR protein can have lots of mutations. II most common. Protein never gets to cell surface.
35
Point of exercise is to understand positional cloning (need a method to identify candidate exons- looking for kinase-can look for homology with other kinase sequence, or can express in culture and look for activity). Once you have CDNA, you have to give evidence that your putative candidate gene does in fact cause the disease. Do not have to know names of markers.
Correction for Yesterday:
Pseudoautosomal Inheritance
Erratum for yesterday’s lecture: pseudoautsomal domains include only the ends
Y linked is also holandric inheritance. No daughter carriers. Pseudoautosomal has carrier daughters.
Ways to map genome:
Complementary and tell you different things. Cytogenetic mapping today, then genetic mapping and physical mapping. Genetic is mapping by inheritance. Physical map is the sequence itself. Cytogenetic map is chromosome.
2
Get this. Be able to understand what mapping CF by RFLP means.
3
Chromosomes as spread here have 2 arms. Nomenclature is standard and based on Giemsa staining.
4
R banding is reversed.
5
position is defined unequivocally in international nomenclature.
Ideograms are based on G banding of actual chromosomes.
7
Pattern FYI
8
female pattern. These gross analyses are used for a 1st test for a lot of chromosomal abnormalities.
9
Know that abnormalities exist. She does not care if you know details. Know they can be seen using cytogenetic methods.
10
Aneuploidy
Nondisjunction can occur – chromosomes do not separate in first round- one cell gets none, one gets both chromosomes. Gametes still function and can fertilize or be fertilized. If nondisjunction at second round, normal complement for 2 cells,a diploid, and a zero complement. Know that aneuploidy happens because separation does not occur properly.
Outcome- trisomy 13,18,21 are viable, but bad. Many other trisomies are lethal. XXX not as devastating. Monosomy X is Turner’s syndrome- one of few monosomies to survive.
11
Rearrangements occur with DS breaks. Repair system usually correct these breaks. Balanced- swapping pieces of DNA- no removal or deletion.
Unbalanced- duplicate or loss- serious potential effects.
12
Possible changes. Squashes or FISH can detect these changes in order. Most of the time these are silent.
13
Reciprocal translocations- no net loss or gain, just change in location. This is not recombination. This is translocation between different chromosomes.
May disrupt gene function if break is between promoter and gene. May be silent.
14
Nonreciprocal translocation- adds chromosomal material to another chromosome.
16
Tandem duplication results in repitition of segment.
17
Have knowledge of FISH analysis. #1 for cytogenetic analysis. Can “paint” whole chromosome or other things listed. Human chromosome has lots of repeat sequence. SSRs are inherited markers.
18
FISH probe is complementary only to a region of the chromosome. Family of probes can be made to entire chromosome.
22
Whole chromosome painting- probe each chromosome with a different dye emitting at different wavelength for different color. Can see deletions, increase in chromosome size.
24
Chromosome 19 has unbalanced rearrangement to attach to another chromosome.
She likes FISH analysis.
29
Genetic mapping can cause problems with understanding.
Linkage analysis- tendency for alleles close together on chromosome to be transmitted together. Can map through pedigree analysis. Close together- stay together in recombination. RFLP not used much anymore. Markers are not necessarily genes- they can be followed in pedigrees. Markers allow us to map genes to chromosomes.
30
1. relative to each other.
2. Polymorphic means difference among individuals.
RFLP and SSR are markers.
31
cM is a genetic unit. Crossing over happens a lot, so if genes are together 1% of the time, they are close together. cM and base pairs are not equivalent. To map a disease to a chromosome, must map several markers on each chromosome for inheritance.
33
Allele view. D and M could be separated by recombination. Randomly distributed- not linked.
34
Look at progeny or gametes that result. Some linkage- intermediate.
35
Marker A is not linked to disease phenotype, but marker B is.
Marker is not a predictor of disease per se, but happens to be linked. Not predictor. To be a predictor of disease, if you find marker very very close, can be used as diagnostic event. It may be linked to the disease gene, not associated with the disease itself.
36
RFLP- anything can be used as a marker. Restriction site can be used. 1bp change creates an ECOR1 cut site. RFLP site is inherited. See it a lot in literature, but not used much anymore. Get tons of fragments. Lots of work.
37
How is it done now? SSR or SRS (simple repeat sequence). Microsatellites are the repeat sequence. Repeats det by PCR and electrophoresis. Each chromosome has a different repeat number.Each individual 2 copies with different repeat sequence. Useful way to map. Marker and easier to follow than RFLPs.
38
SSRs in chromosome 1 Markers are under linkage map.
39 Physical mapping
We know the sequence, but we need to be able to navigate around.
40
Take part of human chromosome and fuse into rodent cell line- panel of hybrids with different parts of chromosome.PCR for gene of interest. Clonal lines .
STS are physical sites that are not simple repeat sequence. These are not polymorphic. These do not change and are the same in every human being. Cannot follow in genetic map because everyone has the same ones. One copy.
Fri- clone CF gene. Tues- inherited color vision, fragile X, Huntingdon’s disease.
Senogles 3
Assignment is posted.
Today we talk about positional cloning. Text reference is to cystic fibrosis (CF). Specific references are not required reading. Presented because it is good science and illustrative of how you do the technique.
3
Functional common until 1990s. Purified protein, followed activity as you purified, got to homogeneity, sequenced, got protein sequence, made oligonucleotide, screened library, got gene. You knew what you were looking for and what the activity was.
Now we look on the basis of where it maps on the chromosome with very little protein information. Positional cloning- map by genetics, then clone.
4
Knowing the DNA sequence does not help because you do not know what you are looking for.
5
2 types of mapping: see slide
6
10% codes for exons. Small part of genome.
7
Localize by linkage analysis position of disease gene you are interested in. Want to localize to greatest extent we can. Yacs and BACs can handle large pieces of DNA by cloning.
If you go to human genome project, is good resource. Can click on a region and see all the known genes in the region.
The two markers were well known.
Linkage disequilibrium- had a mutation in a chromosome. As goes through successive generations, some markers closely segregate with it. B1 stays with CF. It is in linkage disequilibrium. Suggests a founder chromosome with that marker.
11
High resolution map. Things with D are markers, polymorphic, inherited. Closer you can narrow it, less DNA you have to sequence.
12
Trying to minimize brute-force sequencing.
CpG islands tend to be islands of methylated CG that are close to promoters in coding regions. If it cross hybridizes, it is conserved and may be the candidate gene.
13
Want to isolate large fragments first. Large fragment- Mb.
14 Question: Disease- how to clone it out?
Chromosome walking- series overlapping clones. Cut up DNA. Make series of restriction fragments. Pull out and sequence along. Laborious.
15
Chromosome jumping- maximize potential hits by jumping to places from which you can sequence. Covers maximum amount of distance and bypasses unsequenceable stretches of DNA.
Circularized- A and B become close. Can sequence short segment between A and B to find the sequence of site B. Can jump, do not plan to go anywhere. You don’t really care what was between A and B to begin. Can make jumping library, and make a library of sites to walk from. Jumping randomly, then sequence from positions in both directions. Increases chances you will hit something interesting.
16
Series of jumps and where sequence from are shown.
18
Arcs are jumps, horizontal lines are sequence. Roman numerals indicate exons.
20
How do we know we have sequenced something interesting? Look for an ORF.
2 types of candidate genes.
21
Used 2 criteria for candidate genes:
Conserved in phylogenetic tree if important for viability. Used strategy for trying to clone further. Looked for CpG islands at 5’end of genes.
22
CpGs tend to fall close to beginning of genes. Housekeeping genes do not often have unmethylated CpG islands, but class 2 proteins do.
23 Found cross-hybridized exons. They probed a zooblot.
24
Found exon1 with ORF. Screened library and got 24 exons. He had sequence of something.
26
Found 12 TM domains.
30
Evidence-Does mutation correlate with disease? Is function disrupted?
31
Sequencing ladder shown. CTT was deleted. Was an in-frame deletion. Segregated with disease.
Did Southern blot. Most of homozygotes had mutation.
33
Ahead 10 years- CFTR protein can have lots of mutations. II most common. Protein never gets to cell surface.
35
Point of exercise is to understand positional cloning (need a method to identify candidate exons- looking for kinase-can look for homology with other kinase sequence, or can express in culture and look for activity). Once you have CDNA, you have to give evidence that your putative candidate gene does in fact cause the disease. Do not have to know names of markers.
Wednesday, February 7, 2007
Senogles 1
Senogles 1
Why is genetics in curriculum?
Design of course- human and Mendelian genetics and basis of disease. We need to be able to read a pedigree for following lectures. Friday- looking at positional cloning of genetic disease genes. She means her learning objectives. Know what she says.
Also know pseudoautosomal or Y-linked.
3.
Definitions she will use. Email her if you have questions.
Marker is known DNA sequence- not a gene enecessarily. Locus usually refers to a gene.
Dominant and recessive refers to traits, Not genes.
5
44 of our chromosomes are autosomes. 2 sex chromosomes.
Somatic cell is anything nongonadal. Germ cells are haploid.
6.
G bands are reproducible.
Upper arm is P, lower Q. Centromere in middle. Number corresponds to nomenclature shown here when you look up a genetic mutation or deletion for a disease.
8
Mitosis- how somatic cells reproduce. Same complement of chromosomes results in the end.
9
Meiosis- haploid complement.
10
Recombination between nonsister chromaids occurs at metaphase 1.
2 points: reduces to haploid state, and recombination in metaphase 1 scrambles information between maternal and paternal alleles.
13
Standard
Open symbol- person not affected by disease. Fillled in affected.
Proband- where pedigree begins.
16
Principle of independent segregation- each offspring independently has probability of inheriting a gene.
Each offspring independent.
17
Autosomal dominant- can be caused by new mutation, but otherwise appears every generation. No gender bias.
18 Sometimes normal mates are not shown. Proband is first person identified.
Pedigree may not show exact 50/50 segregation.
19
Phenotype may not show up until later.
22
Autosomal recessive option often left to end. Often sproadic
23
X-linked is trickier. Females compensate for gene dosage by inactivating one X chromosome in somatic tissue. Females are X mosaics.
26
X-linked dominant- affected male- affected daughters, not sons. No male to male transmission. Follow pattern- connected through female. No male to male.
29X-linked recessive- transmitted from affected male through daughters. No male to male transmission. Carrier females show in pedigree.
30
Half males affected, 25% of females carrier.
32
Obligate heterozygote is carrier. (synonym)
33
Y linked traits do occur. X and Y line up- only recombine on end portions. Not a gene dense region. Rest of chromosome cannot recombine X-Y.
34 Only males affected. See it in every generation, but only males affected. Rare pedigrees.
35
Atypical- genomic imprinting is difference in gene expression between allele from mom and one from Dad. Only a few of these. Mice have more than we do. Not completely understood- thought to be differential methylation of DNA. Imprinting- which parent the chromosome is from determines outcome.
Why is genetics in curriculum?
Design of course- human and Mendelian genetics and basis of disease. We need to be able to read a pedigree for following lectures. Friday- looking at positional cloning of genetic disease genes. She means her learning objectives. Know what she says.
Also know pseudoautosomal or Y-linked.
3.
Definitions she will use. Email her if you have questions.
Marker is known DNA sequence- not a gene enecessarily. Locus usually refers to a gene.
Dominant and recessive refers to traits, Not genes.
5
44 of our chromosomes are autosomes. 2 sex chromosomes.
Somatic cell is anything nongonadal. Germ cells are haploid.
6.
G bands are reproducible.
Upper arm is P, lower Q. Centromere in middle. Number corresponds to nomenclature shown here when you look up a genetic mutation or deletion for a disease.
8
Mitosis- how somatic cells reproduce. Same complement of chromosomes results in the end.
9
Meiosis- haploid complement.
10
Recombination between nonsister chromaids occurs at metaphase 1.
2 points: reduces to haploid state, and recombination in metaphase 1 scrambles information between maternal and paternal alleles.
13
Standard
Open symbol- person not affected by disease. Fillled in affected.
Proband- where pedigree begins.
16
Principle of independent segregation- each offspring independently has probability of inheriting a gene.
Each offspring independent.
17
Autosomal dominant- can be caused by new mutation, but otherwise appears every generation. No gender bias.
18 Sometimes normal mates are not shown. Proband is first person identified.
Pedigree may not show exact 50/50 segregation.
19
Phenotype may not show up until later.
22
Autosomal recessive option often left to end. Often sproadic
23
X-linked is trickier. Females compensate for gene dosage by inactivating one X chromosome in somatic tissue. Females are X mosaics.
26
X-linked dominant- affected male- affected daughters, not sons. No male to male transmission. Follow pattern- connected through female. No male to male.
29X-linked recessive- transmitted from affected male through daughters. No male to male transmission. Carrier females show in pedigree.
30
Half males affected, 25% of females carrier.
32
Obligate heterozygote is carrier. (synonym)
33
Y linked traits do occur. X and Y line up- only recombine on end portions. Not a gene dense region. Rest of chromosome cannot recombine X-Y.
34 Only males affected. See it in every generation, but only males affected. Rare pedigrees.
35
Atypical- genomic imprinting is difference in gene expression between allele from mom and one from Dad. Only a few of these. Mice have more than we do. Not completely understood- thought to be differential methylation of DNA. Imprinting- which parent the chromosome is from determines outcome.
Zhang notes, both lectures
Recommended reading is from Lodish. He recommends Albert’s molecular biology of the cell. Everything we will discuss is in handout. It should be good enough for the exam.
Cells are assembled into functionally coordinated assemblies called tissue in multicellular organisms. They are connected by cell-cell adhesions. Beside contacting with other cells, cells can also interact with the extracellular matrix.
Interaction categories:
1. cell-cell adhesion
2. cell-matrix adhesion
19-1:
Epithelium is full of cell-cell contact. Also interact with underlying connective tissue. Large molecules are secreted by fibroblasts. They synth and org the ECM.
Today- mechanism to make cells stick together.Epithelium has 3 sections: apical surface, baso-lateral surface, basal. Basal- interact through cell junction and non-cell junction. Interact with basal membrane through ECM adhesion . Junction functions listed in handout. Junction also active in signalling.
Skip a few pages- classification cell adhesion
Junctional mechanism means adhesion based on adhesive structures (cell-cell junction or cell-matrix junction) . Many adhesion mechanisms- nothing visible under EM (Ex: T cell interaction with APC) . When neuron extends neurites, extension based on cell-cell adhesion. Junction mechanisms listed in picture; you can tell functions from name. Tight junction also called occluding junction. Desmosome sticks cells together. Gap junction makes pores between cells for communication.
Cell-matrix adhesion: desmosome and hemidesmosomes are in skin. Desmosome connected to intermediate filaments, thicker than cytoskeleton to resist mechanical stress. Cadherin junction found in many tissues. Non junction adhesion mechanisms- no structural base to visualize under EM.
Tight junction also called occluding junction. Present in most epithelium as well as endothelium. Present at apical end of the basolateral membrane. Network sealing strands account for cell-cell adhesion. EM shows them in the intestinal lumen. The thin strands form the tight junction to seal apical end. Functions- next page.
If you are starving- tight junction is regulated to allow glucose into circulation under extreme conditions. Otherwise it does not allow glucose in.
Tight junction regulated by pore.
Regultion by assembly or disassembly- tight junction comes and goes in previous slide. For individual cells, junction subdivides plasma membrane into different compartments. Basolateral proteins cannot be freely translocated to apical surface or vice versa. Apical membrane has different functions from basolateral. Cancer cells lose tight junction and behave aberrantly due to loss of distinction.
Compsition: Every cell adhesion mechanism can be divided into 3 parts:
1. TM linker proteins called CAMs
2. cytoplasmic adaptor proteins
3. cytoskeleton.
Engage environmental elements and internal elements to create adhesion. All 3 are essential. Adaptor proteins used for regulation and bridging.
Occludin and claudin are 4 transmembrane proteins. Desmosome interacts with intermediate filaments, tight junction with actin filaments.
Extracellular: claudins and occludins. Called type III proteins (N and C terminus intracellular). Matches up with corresponding portions of protein in adjacent cells. Intracellular interact with adaptor proteins. There are many adaptor proteins. Through ZO1 and ZO2 interact with cytoskeleton. Essential components are still controversial.
GAP junction- communicating junctions. Found even in fibroblasts, epithelium, endothelium. Is channel for small molecules between 2 intracellular environments. Is nonspecific. See handout. 10 Amino acids=1 kD. Can label ubiquitin, but it will not pass, which is <1 kD. Can try injecting dye molecules of different sizes into cells and see if they pass.
Difference between gap junction and synapse- chemical in synapse has delay passing from upstream to downstream cell. No delay in gap junction. Fish escape fast not based on synapse, but on diffusion of messages through gap junction. Heart beat transduced through gap junction mechanism.
EM junction is next.
Molecular base: Gap junction formd by connexins with 4 TM domains. 6 of them form oligomer from one cell. One connexon engages another in extracellular domain.
Back to p.5
Families of CAMs here.
Cadherin is important CAM. Engages homophilic interaction. Counterreceptor is itself. Cadherin engages cadherin from another cell. Depends on Ca or Mg- divalent cation. Only cell adhesion molecule not dependent on Ca or Mg is Ig family.
Cmadh- integrins. Integrin can also engage cell-cell adhesion.
Cadherin is a type 1 TM protein. Functions dependent on Ca. Se handout.Classical form adherens junction.
How do cadherins engage homophilic interaction?Cadherin has affinity for Ca. Conformational change in absence- can be chipped off by protease. Trypsin and EDTA (chelates Ca)used to make cells confluent. Cadherin floppy without Ca. Some cells can be detached only with EDTA iif cell-cell adhesion not well developed (as in cancer cells) Prtottype is E cadherin. Extracellullarly has 5 domains. Each domain contains Ca binding seq. Membrane-proximal has higher Ca affinity than distal. Low [Ca]- binds to proximal dom first. Triggers conformational change resulting in stiffness of subdomain. Higher Ca- distal stiff. Cis interaction must occur first. Dimerization on same cell is intermediated by seq between C1 and C2. Once homodimerization accomplished. Conformation is ready for trans interaction with another pair from adjacent cell to accomplish homophilic interaction.
Functions: see handout.Cadherins serve as sorting mechanism for cells in embryogenesis. Only functions ID cells come together. Prevents intrusion from other tissues. How would you demonstrate interaction homophilically? Simple experiment. Have to find system that does not express E cadherin, express it, and observe. Fibroblasts do not express E-cadherin or other cadherins. Transfected fibroblasts and observed. Fibroblasts with E cadherin came together. Were important for cell-cell adhesion and only cells with E cadherin could be sorted to specific place.
Neural tube development from epithelial cells. E-cadherin is major one experessed in epithelial cells. Neural tube cells stopped expressing E and started expressing N cadherin. Formed 2 organs based on sorting.
Neural crest cells migrate and form lot of organs. When they migrate lose N cadherin expression. Aggregate wih expression of N cadherin again at the end.
Cadherins also play role in pathologic conditions.
When cancer loses E cadherin, can undergo epithelial-mesenchymal transition. Transfection in cancer cells to increase cadherin expression can stop cancer migration.
How cadherin connected to intracellular cytoskeleton- Slide shows classical cadherins interaction with actin. Catenins are adaptor proteins. Beta and gamma engage cytoplasmic domains of cadherins. Alpha interacts with cytoskeleton as a bridge. In some pathological conditions, level of one or more catenins is altered.
Well known signalling pathway-wnt pathway. Ligand engages frizzled receptor. Activates disheveled (names from Drosophila mutation) Dsh inactivates GSk3beta (glycogen synthase kinase) Once GSK3B activated, associates with B-catenin, binds APC (adenomatous polyposis coli protein)GSK phosphorylates N terminal B catenin and targets by APC through ubiquitination to be degraded.
If APC is mutated, B catenin accumulates in cytoplasm. Wnt signalling activation results in inactivated GSK. No B-catenin gotten rid of- accumulates in cytoplasm. Consequent accumulation of B-catenin- saturates cadherin junction. Extra will translocate interaction to nucleus. Can bind a variety of TFs to activate transcription. This is necessary during embryogenesis, but once development is finished, wnt pathway should be deactivated.
Desmosome- homophilic, Ca dependent interaction. Difference between desmosome and cadherin junction is cytoskeletal part. Linked to intermediate filaments. Plakoglobins are gamma catenins. Mutation in desmosome results in disease.Mutation in desmoplakins- blister easily.
Hemidesmosome is not cell-cell adhesion molecule. It is a is cell matrix adhesion molecule. Connects to keratins in skin.
Some molecules have no cytoskeletal structural base. Cell-cell adhesion mediated by Ig family of molecules.Ca independent interaction. Structural Ig subdomains is similar to antibody subdomains. VCAM is Ig superfamily proteins.
Zhanglecture 2
This talk about extracellular matrix and adhesion molecules necessary for ECM adhesion. Complex matrix of large molecules is the extracellular matrix. Produced by fibroblasts. Fibroblasts are migratory and use the fibers they produce to migrate around. Basal lamina is produced by epithelial layer above it.
What are the functions of the ECM?
Fill and provide order to extracellular space.
ECM has ligands for cell adhesion proteins and can communicate signals to cell about survival and differentiation and motility through these molecules. Also reservoirs for growth factors, chemokines, cytokines. Prevent growth factor effects from being too transient or abrupt- buffer them and prolong effects through persistent release. ECM is important for educating stem cells to commit for different lineages.
Provides way for cells to move within tissues. Cells migrate on collagen molecules like a railroad track.
Components ECM?
Complicated. Only going through major components. 2 parts: proteins and polysaccharides or proteoglycans.
Large space in ECM filled by proteoglycans- combination of peptides with polysaccharides. Difference between proteoglycan and glycoprotein- glycoprotein mostly from AAs. Proteoglycan- opposite. Cell culture used to be on the flask, but this is not physiological. In vivo, growth factors come from below, not above. More experiments are done these days on 3-D collagen gel to closely mimic in vivo conditions. Lots of proteoglycans included.
Structure is simple for proteoglycan- GAGs are building blocks of them.
Major component connective tissue- collagen.Basic biochem feature is Gly-X-Y triplet repetitive sequence. Y is typically proline or hydroxyproline. Enzyme prolyl hydroxylase is important for maturation of collagen. Hydroxylation required for lateral association. Each collagen- 3 chains. Form a triplex. Multiple helices for collagen fibers. Hydroxyproline negatively charged- can form H bonds between chains to provide strong bonds in helix. Enzyme to hydroxylate proline require vitamin C.
Fibrillar collagen- connective tissues.
Fibril-associated collagens have more flexibility.
Network form networks instead of fibers.
Fibril assoc collagen attach to fibrillar collagens. Triplets are interrupted by non-triplet sequence- increases flexibility.
Type IV is different- network in basement membrane.Laminin, entactin, proteoglycans, other proteins are also there. C terminal interaction is covalent. Lateral interactions are mediated by H bonds.
Goodpasture’s syndrome- affects glomerulus and lung because endothelium and epithelium interact at basement membrane with blood vessel on one side and lumen on other. Patient develops autoantibody to Alpha 3 chain of collagen IV. Antibody precipitates in lung and kidney.
Different forms basement membrane occur. The muscle form is thin and less well developed than that in other locations, but it is still important for health. It surrounds individual muscle cells. Muscular dystrophy can be caused by many factors- one is when the muscle cell cannot attach securely to basement membrane. Mutation in an integrin keeps muscle from attaching to membrane and cannot retract firmly.
Stereotype is that of epithelium and endothelium. The basement membrane in kidney is fusion of basement membrane of epithelium and endothelium.
Matrigel- constructed from basement membrane to use as 3-D culture system- from mouse tumor thans secretes lots of basement membrane components.
Called recombinant basement membrane- used to culture cancer cells, stem cells, epithelium.
Laminin- isolated from lamina. Large protein formed by 3 beta chains.
Fibronectin is a major non-collagen ECM protein. Typically basic unit is single chain. Dimerizes at C terminal through disulfide bond.Type III fibronectin subdomain is one of the most observed ones in the body. Found in lots of proteins. 3AAs- RGD form a loop projecting from Type III domain- is bonding site for integrin 5 beta1, which is fibronectin receptor.Cell binding region is Type III subdomain where it binds to integrins listed. Leukocytes use Cell Binding Site #2 to migrate into tissues.
Integrins- affinity ECM and proteins is lower than intracellular interactions. Could use affinity chromatography to isolate. Ligand to column and pass cell lysate through. Put fibronectin on column and found the cellular receptor called integrin.
Integrins function as cell-cell adhesion molecules as well.Function as dimer of alpha and beta chains. Relatively large EC domain and short cytoplasmic chains..
Collagen receptors are alpha1 and alpha2 (major ones)See sheet.
RGD binding integrins bind to fibronectins and other RGD-containing proteins. Slide is a headache- have rough concept. Do memorize the ones for collagen and laminin.
Integrins can also be used as receptor to invade cells by bacteria and viruses.
Cytoplasmic view of integrins- has transmembrane linker proteins and intracellular skeletal protein and adapter proteins. Most integrins bind to actin cytoskeleton. Actinin and talin are well-established bridging proteins.
Special case in spleen- intermediate filaments interact with alpha 6 beta 4 integrin in spleen.
Integrins are often physiologically inactive. Dangerous to be active all the time. Only want to activate in neutrophils or platelets under special circumstances. Only under wound condition do you want platelets to aggregate.
Most cells do not survive unless attached because of integrin signalling. Cancer cells have aberrant integrin signalling to avoid apoptosis.
Cells are assembled into functionally coordinated assemblies called tissue in multicellular organisms. They are connected by cell-cell adhesions. Beside contacting with other cells, cells can also interact with the extracellular matrix.
Interaction categories:
1. cell-cell adhesion
2. cell-matrix adhesion
19-1:
Epithelium is full of cell-cell contact. Also interact with underlying connective tissue. Large molecules are secreted by fibroblasts. They synth and org the ECM.
Today- mechanism to make cells stick together.Epithelium has 3 sections: apical surface, baso-lateral surface, basal. Basal- interact through cell junction and non-cell junction. Interact with basal membrane through ECM adhesion . Junction functions listed in handout. Junction also active in signalling.
Skip a few pages- classification cell adhesion
Junctional mechanism means adhesion based on adhesive structures (cell-cell junction or cell-matrix junction) . Many adhesion mechanisms- nothing visible under EM (Ex: T cell interaction with APC) . When neuron extends neurites, extension based on cell-cell adhesion. Junction mechanisms listed in picture; you can tell functions from name. Tight junction also called occluding junction. Desmosome sticks cells together. Gap junction makes pores between cells for communication.
Cell-matrix adhesion: desmosome and hemidesmosomes are in skin. Desmosome connected to intermediate filaments, thicker than cytoskeleton to resist mechanical stress. Cadherin junction found in many tissues. Non junction adhesion mechanisms- no structural base to visualize under EM.
Tight junction also called occluding junction. Present in most epithelium as well as endothelium. Present at apical end of the basolateral membrane. Network sealing strands account for cell-cell adhesion. EM shows them in the intestinal lumen. The thin strands form the tight junction to seal apical end. Functions- next page.
If you are starving- tight junction is regulated to allow glucose into circulation under extreme conditions. Otherwise it does not allow glucose in.
Tight junction regulated by pore.
Regultion by assembly or disassembly- tight junction comes and goes in previous slide. For individual cells, junction subdivides plasma membrane into different compartments. Basolateral proteins cannot be freely translocated to apical surface or vice versa. Apical membrane has different functions from basolateral. Cancer cells lose tight junction and behave aberrantly due to loss of distinction.
Compsition: Every cell adhesion mechanism can be divided into 3 parts:
1. TM linker proteins called CAMs
2. cytoplasmic adaptor proteins
3. cytoskeleton.
Engage environmental elements and internal elements to create adhesion. All 3 are essential. Adaptor proteins used for regulation and bridging.
Occludin and claudin are 4 transmembrane proteins. Desmosome interacts with intermediate filaments, tight junction with actin filaments.
Extracellular: claudins and occludins. Called type III proteins (N and C terminus intracellular). Matches up with corresponding portions of protein in adjacent cells. Intracellular interact with adaptor proteins. There are many adaptor proteins. Through ZO1 and ZO2 interact with cytoskeleton. Essential components are still controversial.
GAP junction- communicating junctions. Found even in fibroblasts, epithelium, endothelium. Is channel for small molecules between 2 intracellular environments. Is nonspecific. See handout. 10 Amino acids=1 kD. Can label ubiquitin, but it will not pass, which is <1 kD. Can try injecting dye molecules of different sizes into cells and see if they pass.
Difference between gap junction and synapse- chemical in synapse has delay passing from upstream to downstream cell. No delay in gap junction. Fish escape fast not based on synapse, but on diffusion of messages through gap junction. Heart beat transduced through gap junction mechanism.
EM junction is next.
Molecular base: Gap junction formd by connexins with 4 TM domains. 6 of them form oligomer from one cell. One connexon engages another in extracellular domain.
Back to p.5
Families of CAMs here.
Cadherin is important CAM. Engages homophilic interaction. Counterreceptor is itself. Cadherin engages cadherin from another cell. Depends on Ca or Mg- divalent cation. Only cell adhesion molecule not dependent on Ca or Mg is Ig family.
Cmadh- integrins. Integrin can also engage cell-cell adhesion.
Cadherin is a type 1 TM protein. Functions dependent on Ca. Se handout.Classical form adherens junction.
How do cadherins engage homophilic interaction?Cadherin has affinity for Ca. Conformational change in absence- can be chipped off by protease. Trypsin and EDTA (chelates Ca)used to make cells confluent. Cadherin floppy without Ca. Some cells can be detached only with EDTA iif cell-cell adhesion not well developed (as in cancer cells) Prtottype is E cadherin. Extracellullarly has 5 domains. Each domain contains Ca binding seq. Membrane-proximal has higher Ca affinity than distal. Low [Ca]- binds to proximal dom first. Triggers conformational change resulting in stiffness of subdomain. Higher Ca- distal stiff. Cis interaction must occur first. Dimerization on same cell is intermediated by seq between C1 and C2. Once homodimerization accomplished. Conformation is ready for trans interaction with another pair from adjacent cell to accomplish homophilic interaction.
Functions: see handout.Cadherins serve as sorting mechanism for cells in embryogenesis. Only functions ID cells come together. Prevents intrusion from other tissues. How would you demonstrate interaction homophilically? Simple experiment. Have to find system that does not express E cadherin, express it, and observe. Fibroblasts do not express E-cadherin or other cadherins. Transfected fibroblasts and observed. Fibroblasts with E cadherin came together. Were important for cell-cell adhesion and only cells with E cadherin could be sorted to specific place.
Neural tube development from epithelial cells. E-cadherin is major one experessed in epithelial cells. Neural tube cells stopped expressing E and started expressing N cadherin. Formed 2 organs based on sorting.
Neural crest cells migrate and form lot of organs. When they migrate lose N cadherin expression. Aggregate wih expression of N cadherin again at the end.
Cadherins also play role in pathologic conditions.
When cancer loses E cadherin, can undergo epithelial-mesenchymal transition. Transfection in cancer cells to increase cadherin expression can stop cancer migration.
How cadherin connected to intracellular cytoskeleton- Slide shows classical cadherins interaction with actin. Catenins are adaptor proteins. Beta and gamma engage cytoplasmic domains of cadherins. Alpha interacts with cytoskeleton as a bridge. In some pathological conditions, level of one or more catenins is altered.
Well known signalling pathway-wnt pathway. Ligand engages frizzled receptor. Activates disheveled (names from Drosophila mutation) Dsh inactivates GSk3beta (glycogen synthase kinase) Once GSK3B activated, associates with B-catenin, binds APC (adenomatous polyposis coli protein)GSK phosphorylates N terminal B catenin and targets by APC through ubiquitination to be degraded.
If APC is mutated, B catenin accumulates in cytoplasm. Wnt signalling activation results in inactivated GSK. No B-catenin gotten rid of- accumulates in cytoplasm. Consequent accumulation of B-catenin- saturates cadherin junction. Extra will translocate interaction to nucleus. Can bind a variety of TFs to activate transcription. This is necessary during embryogenesis, but once development is finished, wnt pathway should be deactivated.
Desmosome- homophilic, Ca dependent interaction. Difference between desmosome and cadherin junction is cytoskeletal part. Linked to intermediate filaments. Plakoglobins are gamma catenins. Mutation in desmosome results in disease.Mutation in desmoplakins- blister easily.
Hemidesmosome is not cell-cell adhesion molecule. It is a is cell matrix adhesion molecule. Connects to keratins in skin.
Some molecules have no cytoskeletal structural base. Cell-cell adhesion mediated by Ig family of molecules.Ca independent interaction. Structural Ig subdomains is similar to antibody subdomains. VCAM is Ig superfamily proteins.
Zhanglecture 2
This talk about extracellular matrix and adhesion molecules necessary for ECM adhesion. Complex matrix of large molecules is the extracellular matrix. Produced by fibroblasts. Fibroblasts are migratory and use the fibers they produce to migrate around. Basal lamina is produced by epithelial layer above it.
What are the functions of the ECM?
Fill and provide order to extracellular space.
ECM has ligands for cell adhesion proteins and can communicate signals to cell about survival and differentiation and motility through these molecules. Also reservoirs for growth factors, chemokines, cytokines. Prevent growth factor effects from being too transient or abrupt- buffer them and prolong effects through persistent release. ECM is important for educating stem cells to commit for different lineages.
Provides way for cells to move within tissues. Cells migrate on collagen molecules like a railroad track.
Components ECM?
Complicated. Only going through major components. 2 parts: proteins and polysaccharides or proteoglycans.
Large space in ECM filled by proteoglycans- combination of peptides with polysaccharides. Difference between proteoglycan and glycoprotein- glycoprotein mostly from AAs. Proteoglycan- opposite. Cell culture used to be on the flask, but this is not physiological. In vivo, growth factors come from below, not above. More experiments are done these days on 3-D collagen gel to closely mimic in vivo conditions. Lots of proteoglycans included.
Structure is simple for proteoglycan- GAGs are building blocks of them.
Major component connective tissue- collagen.Basic biochem feature is Gly-X-Y triplet repetitive sequence. Y is typically proline or hydroxyproline. Enzyme prolyl hydroxylase is important for maturation of collagen. Hydroxylation required for lateral association. Each collagen- 3 chains. Form a triplex. Multiple helices for collagen fibers. Hydroxyproline negatively charged- can form H bonds between chains to provide strong bonds in helix. Enzyme to hydroxylate proline require vitamin C.
Fibrillar collagen- connective tissues.
Fibril-associated collagens have more flexibility.
Network form networks instead of fibers.
Fibril assoc collagen attach to fibrillar collagens. Triplets are interrupted by non-triplet sequence- increases flexibility.
Type IV is different- network in basement membrane.Laminin, entactin, proteoglycans, other proteins are also there. C terminal interaction is covalent. Lateral interactions are mediated by H bonds.
Goodpasture’s syndrome- affects glomerulus and lung because endothelium and epithelium interact at basement membrane with blood vessel on one side and lumen on other. Patient develops autoantibody to Alpha 3 chain of collagen IV. Antibody precipitates in lung and kidney.
Different forms basement membrane occur. The muscle form is thin and less well developed than that in other locations, but it is still important for health. It surrounds individual muscle cells. Muscular dystrophy can be caused by many factors- one is when the muscle cell cannot attach securely to basement membrane. Mutation in an integrin keeps muscle from attaching to membrane and cannot retract firmly.
Stereotype is that of epithelium and endothelium. The basement membrane in kidney is fusion of basement membrane of epithelium and endothelium.
Matrigel- constructed from basement membrane to use as 3-D culture system- from mouse tumor thans secretes lots of basement membrane components.
Called recombinant basement membrane- used to culture cancer cells, stem cells, epithelium.
Laminin- isolated from lamina. Large protein formed by 3 beta chains.
Fibronectin is a major non-collagen ECM protein. Typically basic unit is single chain. Dimerizes at C terminal through disulfide bond.Type III fibronectin subdomain is one of the most observed ones in the body. Found in lots of proteins. 3AAs- RGD form a loop projecting from Type III domain- is bonding site for integrin 5 beta1, which is fibronectin receptor.Cell binding region is Type III subdomain where it binds to integrins listed. Leukocytes use Cell Binding Site #2 to migrate into tissues.
Integrins- affinity ECM and proteins is lower than intracellular interactions. Could use affinity chromatography to isolate. Ligand to column and pass cell lysate through. Put fibronectin on column and found the cellular receptor called integrin.
Integrins function as cell-cell adhesion molecules as well.Function as dimer of alpha and beta chains. Relatively large EC domain and short cytoplasmic chains..
Collagen receptors are alpha1 and alpha2 (major ones)See sheet.
RGD binding integrins bind to fibronectins and other RGD-containing proteins. Slide is a headache- have rough concept. Do memorize the ones for collagen and laminin.
Integrins can also be used as receptor to invade cells by bacteria and viruses.
Cytoplasmic view of integrins- has transmembrane linker proteins and intracellular skeletal protein and adapter proteins. Most integrins bind to actin cytoskeleton. Actinin and talin are well-established bridging proteins.
Special case in spleen- intermediate filaments interact with alpha 6 beta 4 integrin in spleen.
Integrins are often physiologically inactive. Dangerous to be active all the time. Only want to activate in neutrophils or platelets under special circumstances. Only under wound condition do you want platelets to aggregate.
Most cells do not survive unless attached because of integrin signalling. Cancer cells have aberrant integrin signalling to avoid apoptosis.
Wednesday, January 31, 2007
Skapek
Skapek- Muscle cell differentiation
Talking about general concepts in muscle that can be extended in principle to other types of differentiation.
2
important concepts.
3
Skeletal muscle develops from the mesoderm layer of the embryo. Mesodermal cells are pluripotent progenitors. Some stage determines cell to skeletal muscle lineage. Happens in somites (clusters of cells along developing notochord and neural axis in segments). Initially all look the same. Cells in dorsoventral aspect are dermal myotome. Get cues from epithelial cells and from medial notochord which set up gradients of diffusible proteins. Determined at that stage, but must migrate. Myogenic cells migrate to limbs or body wall. Differentiate after migration. While or after migration start undegoing morphological and biochem chnges. Become myotube or myofiber of myotubes. Can get multiple nuclei by failure of cytokinesis or fusion of cells. The latter causes multinucleation in skeletal muscle cells.
4
Some epigenetic events allow expression of transcription factors. Cell undergoes some changes due to autonomous factors (programming) or signalling from outside of cell.
5
Expression of a few key genes is necessary. MyoD was the first transcription factor discovered. Found exclusively in myoblasts. Myf5 has overlapping function. Expression MyoD enough to make cell muscle. Works with other transcription factors. Others are not muscle-specific. Environmental cues from notochord and ectoderm important as shown in mouse and chick models.
6
MyoD and Myf5 may guide lineage commitment to different types of skeletal muscle. Pax3 is not muscle- specific, but critical for induction of MyoD. Nature couples transcription factors promoting migration and differentiation. Myogenin and MRF4- transcription factors in positive feedback loop with prior transcription factor. They also induce transcription of proteins to make muscle cells. They can also induce cell cycle arrest. Knockout MyoD- normal skeletal muscle. Some functional redundancy or compensation. More to story was other transcription factors. Double knockout- no muscle embryonic lethal. Myogenin knockout- no differentiation of myocytes to myotubes. Pax3 is secreted from notochord due to Wnt signaling from ectoderm in chick. Myf5/Pax3 double knockout is lethal.
7
See papers. 5-azacytidine blocks DNA methylation. Could get some fibroblasts to become muscle cells. Later found myoD expression, and used it to turn cells into muscle cells.
8
Skeletal muscle differentiation is a one-way street. They do not de-differentiate.
10
What controls migration?
Met receptor is an RTK expressed on myoblasts when migrating. Activated by hepatocyte growth factor. Signalling critical for migration. Rhabdomyosarcomas express a lot of muscle proteins.
11
What is basis of differentiation?
Cell has to undergo biochemical and morphological change. What starts process?
12
whether is autonomous or not is not clear. Certain cues have to happen in vivo.
13
bHLH has domains you should know. Does not interact with RNA pol 2 directly, but withHATs, etc. No TAD, unable to make muscle cell. Transcription factors must bind DNA at E boxes. CANNTG genome- E box every 250 nt. MyoD does not bind every one. Basic region essential for specificity. 4 are myogenic, many other bHLH transcription factors are not myogenic. 3AAs in basic region are essential. What they do exactly is unknown.
15
E proteins interact with MyoD thrugh HLH domain. MyoD cannot bind DNA by itself- needs E box.
16 Carboxyterminus is large. Can be lopped off and still cells form muscle cells in vitro. May control stabilization, etc.
19
Myoblasts- MyoD is inactive. One critical way to control MyoD is heterodimer formation. MyoD can form homodimers.
21
Dominant negative inhibitors of differentiation- dominates another protein to turn it off. Id has no basic domain for DNA binding. Shuts off MyoD. Id means inhibitors of differentiation. There are 4 Id proteins.
22
MEF2 is involved in cardiac muscle and skeletal muscle. MyoD and Mef 2 cooperate functionally.E boxes have Mef2 binding sites adjacent to them.
23
Mef2 and MyoD attract CBP/p300 with HAT activity.
24
cultured cells- if you grow to confluence or add mitogens, they start to differentiate. Arrest causes differentiation.
25
E2F induces genes involved in DNA synthesis. Rb is complex. Guides lineage specification. Rb prevents G1/S transition. Phophorylated by cyclin/cdk. Proliferation signals induce cyclins. Cdk2 inhibitors also work.
28
Rb is essential for muscle cell development. Cells can become commited, but not become normal muscle cells without it. Without it, cells do not arrest and become post-mitotic.2 defects: lack of muscle specific genes and lack of cell cycle arrest.
31
3 D type cyclins expressed early in proliferating cells. Kinase is constant .
32
In the presence of cyclin D 1- MyoD phosphorylated.
34
In vivo differentiation signals unknown.
35
Transcriptional machinery regulated at multiple levels.
36
Twist encodes a bHLH like protein with properties like Id proteins. Blocks MyoD-E heterodimer formation and functional activity Mef 2.
39
Mapk involved. Positive feedback activates p38 MAPK.
43
How do we study this?
Take fibroblasts- cells undergo changes in medium with MyoD. Stain for muscle proteins and see them.
44
Reporter gene on plasmid driven by muscle-specific promoter can be manipulated quantitatively.
47-49
Review
Similar bHLH are essential for other developmental processes.
Email him questions- test from chapter and these slides. Make sure you understand molecular biology of these transcription factors and how they work . Challenging question about how to find this and what to do with results.
Talking about general concepts in muscle that can be extended in principle to other types of differentiation.
2
important concepts.
3
Skeletal muscle develops from the mesoderm layer of the embryo. Mesodermal cells are pluripotent progenitors. Some stage determines cell to skeletal muscle lineage. Happens in somites (clusters of cells along developing notochord and neural axis in segments). Initially all look the same. Cells in dorsoventral aspect are dermal myotome. Get cues from epithelial cells and from medial notochord which set up gradients of diffusible proteins. Determined at that stage, but must migrate. Myogenic cells migrate to limbs or body wall. Differentiate after migration. While or after migration start undegoing morphological and biochem chnges. Become myotube or myofiber of myotubes. Can get multiple nuclei by failure of cytokinesis or fusion of cells. The latter causes multinucleation in skeletal muscle cells.
4
Some epigenetic events allow expression of transcription factors. Cell undergoes some changes due to autonomous factors (programming) or signalling from outside of cell.
5
Expression of a few key genes is necessary. MyoD was the first transcription factor discovered. Found exclusively in myoblasts. Myf5 has overlapping function. Expression MyoD enough to make cell muscle. Works with other transcription factors. Others are not muscle-specific. Environmental cues from notochord and ectoderm important as shown in mouse and chick models.
6
MyoD and Myf5 may guide lineage commitment to different types of skeletal muscle. Pax3 is not muscle- specific, but critical for induction of MyoD. Nature couples transcription factors promoting migration and differentiation. Myogenin and MRF4- transcription factors in positive feedback loop with prior transcription factor. They also induce transcription of proteins to make muscle cells. They can also induce cell cycle arrest. Knockout MyoD- normal skeletal muscle. Some functional redundancy or compensation. More to story was other transcription factors. Double knockout- no muscle embryonic lethal. Myogenin knockout- no differentiation of myocytes to myotubes. Pax3 is secreted from notochord due to Wnt signaling from ectoderm in chick. Myf5/Pax3 double knockout is lethal.
7
See papers. 5-azacytidine blocks DNA methylation. Could get some fibroblasts to become muscle cells. Later found myoD expression, and used it to turn cells into muscle cells.
8
Skeletal muscle differentiation is a one-way street. They do not de-differentiate.
10
What controls migration?
Met receptor is an RTK expressed on myoblasts when migrating. Activated by hepatocyte growth factor. Signalling critical for migration. Rhabdomyosarcomas express a lot of muscle proteins.
11
What is basis of differentiation?
Cell has to undergo biochemical and morphological change. What starts process?
12
whether is autonomous or not is not clear. Certain cues have to happen in vivo.
13
bHLH has domains you should know. Does not interact with RNA pol 2 directly, but withHATs, etc. No TAD, unable to make muscle cell. Transcription factors must bind DNA at E boxes. CANNTG genome- E box every 250 nt. MyoD does not bind every one. Basic region essential for specificity. 4 are myogenic, many other bHLH transcription factors are not myogenic. 3AAs in basic region are essential. What they do exactly is unknown.
15
E proteins interact with MyoD thrugh HLH domain. MyoD cannot bind DNA by itself- needs E box.
16 Carboxyterminus is large. Can be lopped off and still cells form muscle cells in vitro. May control stabilization, etc.
19
Myoblasts- MyoD is inactive. One critical way to control MyoD is heterodimer formation. MyoD can form homodimers.
21
Dominant negative inhibitors of differentiation- dominates another protein to turn it off. Id has no basic domain for DNA binding. Shuts off MyoD. Id means inhibitors of differentiation. There are 4 Id proteins.
22
MEF2 is involved in cardiac muscle and skeletal muscle. MyoD and Mef 2 cooperate functionally.E boxes have Mef2 binding sites adjacent to them.
23
Mef2 and MyoD attract CBP/p300 with HAT activity.
24
cultured cells- if you grow to confluence or add mitogens, they start to differentiate. Arrest causes differentiation.
25
E2F induces genes involved in DNA synthesis. Rb is complex. Guides lineage specification. Rb prevents G1/S transition. Phophorylated by cyclin/cdk. Proliferation signals induce cyclins. Cdk2 inhibitors also work.
28
Rb is essential for muscle cell development. Cells can become commited, but not become normal muscle cells without it. Without it, cells do not arrest and become post-mitotic.2 defects: lack of muscle specific genes and lack of cell cycle arrest.
31
3 D type cyclins expressed early in proliferating cells. Kinase is constant .
32
In the presence of cyclin D 1- MyoD phosphorylated.
34
In vivo differentiation signals unknown.
35
Transcriptional machinery regulated at multiple levels.
36
Twist encodes a bHLH like protein with properties like Id proteins. Blocks MyoD-E heterodimer formation and functional activity Mef 2.
39
Mapk involved. Positive feedback activates p38 MAPK.
43
How do we study this?
Take fibroblasts- cells undergo changes in medium with MyoD. Stain for muscle proteins and see them.
44
Reporter gene on plasmid driven by muscle-specific promoter can be manipulated quantitatively.
47-49
Review
Similar bHLH are essential for other developmental processes.
Email him questions- test from chapter and these slides. Make sure you understand molecular biology of these transcription factors and how they work . Challenging question about how to find this and what to do with results.
Shanklin Last Lectures
Shanklin 3
Developmental disorders: General Aspects, Genetic Factors
Yesterday: Response to injury is also to preserve homeostatic metabolic status of individual. Energy is stored in glycogen and ATP in muscle. Has to be replenished.
Shock can be due to surge of endotoxin or blood loss. Vital functions dampen. ATP supplies discharge. Cardiac output drops, so the organism cannot restore ATP levels.
Genetics
Historical classifications human disease
1. aggressive- neoplasms
2. reactive – inflammation
3. submissive- atrophy
4. degenerative- calcification
5. development and maturation of tissues, organs, integrated functions
Developing organs respond to disease differently from mature ones. Most newsworthy diseases are infectious.
Measles can be theoretically eliminated by vaccination. Distemper in dogs is the same virus. Vaccine introduced in 1960s. Guam had outbreak 1994 Lowered vaccination age to 6 months. The graph is the number of cases they knew about. Vaccinated everyone- seemed to stop it. Graph says nothing about natural course of epidemic. Graph might represent epiphenomenon. Course of disease, or vaccination? Does not establish cause or effect.
There are pockets of measles virus around the world. There is a similar program for polio.
Abnrmalities of Early Development:
Malformations, teratomas, neoplasms occur in course of development. Can have malignant tumors in stillborns or newborns. If a tumor is in a fetus, because of growth potential of organism, tends to be large. Brain can be replaced by glioma.
Definitions:
Syndrome- set of symptoms that occur together. Pattern of malformations in genetics morphologically related.
Medical history as a database:
Anecdotal evidence is often derided, but used a lot. Doctors make decisions based on incomplete information. Anecdote becomes falsifiable hypothesis for testing for pathologists.
Children mostly die from accidents. 10% of childhood deaths are from cancer, a lot of those from leukemia.
Congenital malformations are important. 2059 auptopsies- 953 classifiable malformations. 403 clinically significant-death due to lesions or complications of attempts to correct lesions.
Abnormal fetoplacental hemodynamics. Placenta usually ovoid disk with cord from center. If it is off, circulation can flow one direction and one twin of two may fail to develop.
Ordinary blood grouping- RH- woman with Rh+ fetus will be sensitized to factor. These days vaccine can prevent problem. Double mismatch- (O woman, A fetus)-woman destroys fetal blood cells instead of sensitizing. B is more likely to cause problems.
Anemia of prematurity- most RBC in fetus made in liver. Little marrow- gradually expands. Prematurely born can become anemic quickly. Do not absorb iron well. ABO discovered 1930s.
Achondroplasic parents can have normal babies.
1960s Human chromosome count discovered to be 46 not 48. Number is not static. Ex: XXY or XXXY in Kleinfelter’s. Phenotypic presentation implies genetic abnormality causes phenotype. Does not mean we know how it works.
Down Syndrome- trisomy 21 or group G. Down babies have similarities. Broad face, eyes far apart, tranverse crease across hands (more common in first degree relatives).
Syndrome- low to low normal birth weight, brain small for body, microcephaly and brachycephaly, trainable, cheerful and pleasant, want to be around people.
Many have visceral disease- congenital heart disease, other problems. Have 150% SOD of normal. And gal-1-phosphate uridyl transferase.
There is repeat risk. Around age 20- can get translocation of chromosome for variant of Downs. Usual explanation- older ovary, abnormal disjunction more likely. Meiosis not studied as well as mitosis. Evidence is that it increases with age. Problem: Data from ovaries not available for running commentary. “Mongoloid” is old term.
Down Syndrome and Leukemia- myoblastic leukemia in stillborn.
Rate of birth: 1: 1087. Nondisjunction is descriptive name for abnormal separation of chromosomes. Colorado has twice the rate of Kansas. People compensate for the lack of oxygen at high altitude. Spaniards took over Peru- took 2 generations of adult adaptation to produce children at high altitude. Is it a matter of oxygen? No. Nutrition matters. Fewer Down’s babies with relatively high protein diet. Monosomy 21 can look like Downs. Mosaic Down Syndrome occurs with some cells normal and some abnormal.
Survival has improved with time. May be improved diagnosis. 50 years is about their life span. The chronic disease (heart, renal, diabetes) causes problems.
Adult polycystic renal disease- manifests genetic anticipation (nucleosides that run in triplets tend to multiply- above a critical number, disease occurs in next generation earlier.) Same thing in Huntingdon’s chorea.
Epigenetics- above and beyond genetics. Methylation of cytosine may be important. Body has 10^13 cells, 1:50,000 mosaic even in normal human.
Wed PM lecture
Objectives:
Systems biology
Healing after surgery requires AA intake. We do not have protein storage. Serum albumin is most mobile of structural poteins. Easily fungible. Hard tissue most easily fungible is lymphoid tissue. Prolonged borderline starvation leads to infection.
Pathology objectives:
Understand principles and steps of pathogenesis. Can study at molecular, cellular, tissue level. Pathogenesis of disease means an event that leads to a response that leads to a lesion with various consequences. Understand principles- can look up details. We focus on human. Career options: research biologist, paraclinical area (clinical pharm), ed research, hospital diagnostics.
No class tomorrow or Friday. As far as testing, take info from previous lecturers and be able to apply it to think up a solution to a problem. He wants to see imagination, logic, and careful organization. And don’t worry about getting copies of all his notes to memorize.
Developmental disorders: General Aspects, Genetic Factors
Yesterday: Response to injury is also to preserve homeostatic metabolic status of individual. Energy is stored in glycogen and ATP in muscle. Has to be replenished.
Shock can be due to surge of endotoxin or blood loss. Vital functions dampen. ATP supplies discharge. Cardiac output drops, so the organism cannot restore ATP levels.
Genetics
Historical classifications human disease
1. aggressive- neoplasms
2. reactive – inflammation
3. submissive- atrophy
4. degenerative- calcification
5. development and maturation of tissues, organs, integrated functions
Developing organs respond to disease differently from mature ones. Most newsworthy diseases are infectious.
Measles can be theoretically eliminated by vaccination. Distemper in dogs is the same virus. Vaccine introduced in 1960s. Guam had outbreak 1994 Lowered vaccination age to 6 months. The graph is the number of cases they knew about. Vaccinated everyone- seemed to stop it. Graph says nothing about natural course of epidemic. Graph might represent epiphenomenon. Course of disease, or vaccination? Does not establish cause or effect.
There are pockets of measles virus around the world. There is a similar program for polio.
Abnrmalities of Early Development:
Malformations, teratomas, neoplasms occur in course of development. Can have malignant tumors in stillborns or newborns. If a tumor is in a fetus, because of growth potential of organism, tends to be large. Brain can be replaced by glioma.
Definitions:
Syndrome- set of symptoms that occur together. Pattern of malformations in genetics morphologically related.
Medical history as a database:
Anecdotal evidence is often derided, but used a lot. Doctors make decisions based on incomplete information. Anecdote becomes falsifiable hypothesis for testing for pathologists.
Children mostly die from accidents. 10% of childhood deaths are from cancer, a lot of those from leukemia.
Congenital malformations are important. 2059 auptopsies- 953 classifiable malformations. 403 clinically significant-death due to lesions or complications of attempts to correct lesions.
Abnormal fetoplacental hemodynamics. Placenta usually ovoid disk with cord from center. If it is off, circulation can flow one direction and one twin of two may fail to develop.
Ordinary blood grouping- RH- woman with Rh+ fetus will be sensitized to factor. These days vaccine can prevent problem. Double mismatch- (O woman, A fetus)-woman destroys fetal blood cells instead of sensitizing. B is more likely to cause problems.
Anemia of prematurity- most RBC in fetus made in liver. Little marrow- gradually expands. Prematurely born can become anemic quickly. Do not absorb iron well. ABO discovered 1930s.
Achondroplasic parents can have normal babies.
1960s Human chromosome count discovered to be 46 not 48. Number is not static. Ex: XXY or XXXY in Kleinfelter’s. Phenotypic presentation implies genetic abnormality causes phenotype. Does not mean we know how it works.
Down Syndrome- trisomy 21 or group G. Down babies have similarities. Broad face, eyes far apart, tranverse crease across hands (more common in first degree relatives).
Syndrome- low to low normal birth weight, brain small for body, microcephaly and brachycephaly, trainable, cheerful and pleasant, want to be around people.
Many have visceral disease- congenital heart disease, other problems. Have 150% SOD of normal. And gal-1-phosphate uridyl transferase.
There is repeat risk. Around age 20- can get translocation of chromosome for variant of Downs. Usual explanation- older ovary, abnormal disjunction more likely. Meiosis not studied as well as mitosis. Evidence is that it increases with age. Problem: Data from ovaries not available for running commentary. “Mongoloid” is old term.
Down Syndrome and Leukemia- myoblastic leukemia in stillborn.
Rate of birth: 1: 1087. Nondisjunction is descriptive name for abnormal separation of chromosomes. Colorado has twice the rate of Kansas. People compensate for the lack of oxygen at high altitude. Spaniards took over Peru- took 2 generations of adult adaptation to produce children at high altitude. Is it a matter of oxygen? No. Nutrition matters. Fewer Down’s babies with relatively high protein diet. Monosomy 21 can look like Downs. Mosaic Down Syndrome occurs with some cells normal and some abnormal.
Survival has improved with time. May be improved diagnosis. 50 years is about their life span. The chronic disease (heart, renal, diabetes) causes problems.
Adult polycystic renal disease- manifests genetic anticipation (nucleosides that run in triplets tend to multiply- above a critical number, disease occurs in next generation earlier.) Same thing in Huntingdon’s chorea.
Epigenetics- above and beyond genetics. Methylation of cytosine may be important. Body has 10^13 cells, 1:50,000 mosaic even in normal human.
Wed PM lecture
Objectives:
Systems biology
Healing after surgery requires AA intake. We do not have protein storage. Serum albumin is most mobile of structural poteins. Easily fungible. Hard tissue most easily fungible is lymphoid tissue. Prolonged borderline starvation leads to infection.
Pathology objectives:
Understand principles and steps of pathogenesis. Can study at molecular, cellular, tissue level. Pathogenesis of disease means an event that leads to a response that leads to a lesion with various consequences. Understand principles- can look up details. We focus on human. Career options: research biologist, paraclinical area (clinical pharm), ed research, hospital diagnostics.
No class tomorrow or Friday. As far as testing, take info from previous lecturers and be able to apply it to think up a solution to a problem. He wants to see imagination, logic, and careful organization. And don’t worry about getting copies of all his notes to memorize.
Tuesday, January 30, 2007
Shanklin 2
Shanklin 2
Subjects on schedule mean nothing.
Today we will overview/review general pathological concepts.
Injury- in pathology means something that happens to cells or tissues. Injury is disturbance from balanced, normal state (homeostasis). Balancing mechanisms are built in to overcome this problem up to a point. Homeostasis means various forces are in balance. Normality is a functional range. Homeostasis happens within and without that range. Body chemistry- talking about a mixture of forces that maintian a certain pH, concentration, rate of secretion. For example, Cl- content of human saliva-abundant. Hypersalivate and don’t swallow- lose a lot of Cl. Body is set up in balance.
Circadian rhythms make a difference in cetain injuries. Corticosteroids low in the morning and high in the afternoon. Say someone has 2nd degree burns over 50% of body. MRSA makes this a real and risky problem. Person burned- steroids released. ACTH from anterior pituitary stimulated by hypothalamus → neuroendocrine reaction to injury. Surge overrides circadian rhythm. Level of corticosteroids goes down in anterior pituitary- extremely depleted- takes 72 hours to restore. Debridement at 72 hours would result in further depletion. Burns can become lethal if treatment pattern augments pattern of injury.
What about myocardial infarction?
Sequence of pathogenesis:
Functional imbalance between cardiac muscle need and oxygen supply. Obstruction in artery, or narrowing of artery plus blood loss from trauma or stomach ulcer. Area undergoes necrosis. Area near outer part has extra blood flow. Organ continues to function. Keel over dead- not an infarction. How does body respond to infarction? There is collateral supply in heart- if artery is completely blocked- boundaries of infarct speak of competency of collateral supply. Even after infarct some of collateral supply comes in to help with restoration process. Cells begin exuding potassium and magnesium. Potassium loss is important. Serum potassium high enough- functional range is narrow. Heart stops at either end. Volume of heart knocked out and it still work= volume of functional reserve. 50% of lung is necessary. Kidney- about 70% is extra. Liver can remove 90% rat, 75% human. It regenerates to some degree. Half of spleen can be lost. Location of infarct in heart is critical. Infarct in superior part of interventricular septum interferes with function. In a zone of left ventricle can have up to 80% destroyed. PMNs move in. Endothelium stimulated. Boundary created. Macrophages gobble debris. Fibroblasts invade. Granulation tisue forms. Fibrous scar laid down. Collagen begins to contract and pulls in muscle. Problem is in PMN leukocytes. Proteolytic enzymes of PMNs can weaken the fibrous network enough for cardiac rupture. More PMNs present 7-8 days after attack- defense mechanism can become pathological.
Long term process of healing using the innate inflammation system is :
Injury, reaction, stabilization, restitution, healing. There may be restoration of effective function. Lenticulostriate artery of brain interferes with internal capsule of fibers that go from cortex to muscles in the event of a stroke. Brain-get polys and macrophages (microglia and outside ones). Loose fibrillar mass from astrocytes forms. Get space instead of collapse. If stroke hits voice center, functional problem results. Brain is plastic, though, and can be retrained.
Lung has built in collateral supply. Bronchial and pulmonary arteries. Bronchials bring in 10-15% of flow, consisting of oxygenated blood. Main stream of pulmonary artery is curve to right, right angle to left. Embolus causes infarct of lung. Get PMNs, macrophages, restorative process.
Adaptive side works differently. If did not have scar production through innate system, could not have surgery. More damage from retractors than scalpel.
Body will heal any injury if you give it time and do not disturb it farther. Reinjury is not good. Reinjury sets into motion everything we have talked about. If organism is immune challenged, adaptive system comes into play. Reason no immune reaction to simple cut, is that you prevent sensitization from bacteria or viruses. How long to develop lymphocyte ability to respond to bacteria or virus? 5 to 6 Days. Lymph nodes there- get response. Th, B, plasma cells. Secondary reaction is humoral through antibodies. Plasma cell life in tissue is a few days. Plasma cell infiltration is a sign of active chronic inflammation. When you stop seing them- inactive chronic inflammatory reaction. T cells are stimulated by IL-2. If stimulate lymphocyte clone with IL-2, get expansion. Add more- they die. Receptor density and receptor affinity are involved. Fill low affinity ones- get secondary decline. In vivo- not that simple. Lots of secondary feeds.
How does bone marrow “know” that you have a thorn in your thumb? Signal not through brain. Leukotaxin means “to move white” . Cells release peptides to lymphatics to marrow cells. Cells sense concentration gradient. Deep inside tissue- lymphatic has epithelial tissue. Tissue altered to HEV, with selectins on surface. Adult marrow cannot be weighed- roughly 1500g scattered all over. We do not know if nearest marrow responds or not. Dilution of message occurs, but it is sufficient. Couple of molecules leucotaxic peptide- get a bit of pus formation in wound. If immune injury occurs at same time, move into adaptive response. Sutures stimulate dense scar around them to preclude secondary response.
Tomorrow- genetic pathology.
Start with a cell called a leukoblast in marrow. Goes through steps and wind up with PMN, or called neutrophil. Appearance varies by preparation. Different in smear or in tissue. PMN has lobation of nuclear material in cell. Hypersegmented in pernicious anemia occurs in B-12 or folate deficiency. Normal life span 10 days -2 weeks. Activated- 1-2 days. Pelger-Huet anomaly- does not segment at all. Myelocyte band form (nucleoplasm is band across center of cell) is immature. Bad bacterial infection- band form released. P-H anomaly stops at band form. It is a genetically dominant trait. Sufficiently uncommon that it is unk whether it is one allele or 2. P-H relationship with TB is contributory to establishment of chronic infection. Hard to kill mycobacteria. Acquired P-H anomaly how to account for it? There could be cellular factors promoting hyposegmentation.
Subjects on schedule mean nothing.
Today we will overview/review general pathological concepts.
Injury- in pathology means something that happens to cells or tissues. Injury is disturbance from balanced, normal state (homeostasis). Balancing mechanisms are built in to overcome this problem up to a point. Homeostasis means various forces are in balance. Normality is a functional range. Homeostasis happens within and without that range. Body chemistry- talking about a mixture of forces that maintian a certain pH, concentration, rate of secretion. For example, Cl- content of human saliva-abundant. Hypersalivate and don’t swallow- lose a lot of Cl. Body is set up in balance.
Circadian rhythms make a difference in cetain injuries. Corticosteroids low in the morning and high in the afternoon. Say someone has 2nd degree burns over 50% of body. MRSA makes this a real and risky problem. Person burned- steroids released. ACTH from anterior pituitary stimulated by hypothalamus → neuroendocrine reaction to injury. Surge overrides circadian rhythm. Level of corticosteroids goes down in anterior pituitary- extremely depleted- takes 72 hours to restore. Debridement at 72 hours would result in further depletion. Burns can become lethal if treatment pattern augments pattern of injury.
What about myocardial infarction?
Sequence of pathogenesis:
Functional imbalance between cardiac muscle need and oxygen supply. Obstruction in artery, or narrowing of artery plus blood loss from trauma or stomach ulcer. Area undergoes necrosis. Area near outer part has extra blood flow. Organ continues to function. Keel over dead- not an infarction. How does body respond to infarction? There is collateral supply in heart- if artery is completely blocked- boundaries of infarct speak of competency of collateral supply. Even after infarct some of collateral supply comes in to help with restoration process. Cells begin exuding potassium and magnesium. Potassium loss is important. Serum potassium high enough- functional range is narrow. Heart stops at either end. Volume of heart knocked out and it still work= volume of functional reserve. 50% of lung is necessary. Kidney- about 70% is extra. Liver can remove 90% rat, 75% human. It regenerates to some degree. Half of spleen can be lost. Location of infarct in heart is critical. Infarct in superior part of interventricular septum interferes with function. In a zone of left ventricle can have up to 80% destroyed. PMNs move in. Endothelium stimulated. Boundary created. Macrophages gobble debris. Fibroblasts invade. Granulation tisue forms. Fibrous scar laid down. Collagen begins to contract and pulls in muscle. Problem is in PMN leukocytes. Proteolytic enzymes of PMNs can weaken the fibrous network enough for cardiac rupture. More PMNs present 7-8 days after attack- defense mechanism can become pathological.
Long term process of healing using the innate inflammation system is :
Injury, reaction, stabilization, restitution, healing. There may be restoration of effective function. Lenticulostriate artery of brain interferes with internal capsule of fibers that go from cortex to muscles in the event of a stroke. Brain-get polys and macrophages (microglia and outside ones). Loose fibrillar mass from astrocytes forms. Get space instead of collapse. If stroke hits voice center, functional problem results. Brain is plastic, though, and can be retrained.
Lung has built in collateral supply. Bronchial and pulmonary arteries. Bronchials bring in 10-15% of flow, consisting of oxygenated blood. Main stream of pulmonary artery is curve to right, right angle to left. Embolus causes infarct of lung. Get PMNs, macrophages, restorative process.
Adaptive side works differently. If did not have scar production through innate system, could not have surgery. More damage from retractors than scalpel.
Body will heal any injury if you give it time and do not disturb it farther. Reinjury is not good. Reinjury sets into motion everything we have talked about. If organism is immune challenged, adaptive system comes into play. Reason no immune reaction to simple cut, is that you prevent sensitization from bacteria or viruses. How long to develop lymphocyte ability to respond to bacteria or virus? 5 to 6 Days. Lymph nodes there- get response. Th, B, plasma cells. Secondary reaction is humoral through antibodies. Plasma cell life in tissue is a few days. Plasma cell infiltration is a sign of active chronic inflammation. When you stop seing them- inactive chronic inflammatory reaction. T cells are stimulated by IL-2. If stimulate lymphocyte clone with IL-2, get expansion. Add more- they die. Receptor density and receptor affinity are involved. Fill low affinity ones- get secondary decline. In vivo- not that simple. Lots of secondary feeds.
How does bone marrow “know” that you have a thorn in your thumb? Signal not through brain. Leukotaxin means “to move white” . Cells release peptides to lymphatics to marrow cells. Cells sense concentration gradient. Deep inside tissue- lymphatic has epithelial tissue. Tissue altered to HEV, with selectins on surface. Adult marrow cannot be weighed- roughly 1500g scattered all over. We do not know if nearest marrow responds or not. Dilution of message occurs, but it is sufficient. Couple of molecules leucotaxic peptide- get a bit of pus formation in wound. If immune injury occurs at same time, move into adaptive response. Sutures stimulate dense scar around them to preclude secondary response.
Tomorrow- genetic pathology.
Start with a cell called a leukoblast in marrow. Goes through steps and wind up with PMN, or called neutrophil. Appearance varies by preparation. Different in smear or in tissue. PMN has lobation of nuclear material in cell. Hypersegmented in pernicious anemia occurs in B-12 or folate deficiency. Normal life span 10 days -2 weeks. Activated- 1-2 days. Pelger-Huet anomaly- does not segment at all. Myelocyte band form (nucleoplasm is band across center of cell) is immature. Bad bacterial infection- band form released. P-H anomaly stops at band form. It is a genetically dominant trait. Sufficiently uncommon that it is unk whether it is one allele or 2. P-H relationship with TB is contributory to establishment of chronic infection. Hard to kill mycobacteria. Acquired P-H anomaly how to account for it? There could be cellular factors promoting hyposegmentation.
Saturday, January 27, 2007
Review for next-to-last CMB test
CMB Review Next to Last Exam
Most of this was not said in the review session. Only Pfeffer and Park were there. These are the notes I use to study. Pray hard. Everything will turn out fine.
Rao’s Review Points
Signal Transduction
1. Strategies in study of signaling pathways.
Briefly know the strategies used in study of signal transduction.
2. To recognize the features and cellular functions of intracellular signal transduction pathways.
Know the mechanisms involved in the conserved commonly existing signaling proteins
3. To understand the mechanism of cyclic AMP pathway of signal transduction.
Know the details of cyclic AMP pathway of signal transduction, including adenylyl cyclase and PKA
4. To understand the role of second messengers derived from phosphoinositide.
The details of signaling pathway involving PLC, PKC and calcium are required.
5. To understand the structure and activation of receptor tyrosine kinases (RTK) and activation of Ras by RTKs.
Know the mechanism involved in receptor activation and Ras activation
6. To understand the mechanism of activation of MAP kinase pathway.
Mechanism involved in activation of MAP kinase is important
7. To understand some of the features of integration and control of signals in tissues
Cytoskeleton
1. Structure of actin cytoskeleton
Structure of G-actin monomer and F-actin polymer
Polarity of actin filaments
Arrangement of actin filaments into bundles and networks
Cross linking proteins
2. Dynamics of actin polymerization
Different phases of polymerization
Critical concentration
Polarity of polymerization
Effects of toxins on actin dynamics
3. Actin binding proteins (when a name of the protein is given know what it does to microfilaments and how)
Actin polymerizing proteins
Actin severing proteins
Actin capping proteins
Nucleating proteins
4. Myosin-powered cellular movements
Different types of myosins and their structure and function
Movement of myosin heads along filaments
5. Role of actin and myosin in muscle contraction
Contractile apparatus - thick and thin filaments
6. Role of actin and myosin in cell migration
Four different steps in cell migration
Role of actin dynamics in different steps of cell migration
7. Microtubules
Know the similarities and differences between the structure and function of microfilaments and microtubules.
8. The structure of microtubules, which includes:
structure of tubulin subunits
organization of subunits into tubules
polarity of microtubules
microtubule organizing center
9. The dynamics of microtubules assembly and disassembly:
temperature dependence
kinetics of assembly
polarity of assembly
disruption of microtubules by drugs
dynamic instability of microtubules (know how to describe it, but you do not have to know the mechanism of it)
10. Role of microtubules and motor proteins in transport of vesicles:
vesicle transport in axons; role of microtubules, motor proteins and polarity of vesicle movement
microtubule motor proteins – kinesins - structure and function
11. The role of microtubules in the structure and function of mitotic apparatus:
different types of microtubules in mitotic apparatus and their functions
motor proteins in mitotic apparatus
kinetochore and attachment of chromosomes during prophase
stabilization of chromosomes at the equatorial plate of cell during metaphase
microtubules and motor proteins in separation of chromosomes during anaphase
Pfeffer
Pfeffer- questions straightforward. No bizarre answers. Read text and notes. Control of cell cycle, mitosis and meiosis. One question per lecture.
Lecture Objectives:
70. Overview of Cell Cycle
To understand:
General Cell Cycle control
Phases of cell cycle
Experimental systems used to study cell cycle
Regeneration potential of different cells
Role of checkpoints
How to determine length of cell cycle and its phases
Mitosis versus meiosis
71. Cell cycle control mechanisms
To understand:
Control of cell cycle in Xenopus laevis
Maturation promoting factor (MPF)
Role of cyclins in cell cycle
Control of cell cycle in yeast
Temperature sensitive mutants
Role of protein phosphorylation
Role of protein degradation
Mitotic index-what % of cells in mitosis. Labelling index- how many of mitotic cells are labelled.
Park
2 questions- one on nuclear receptors- structure function relationships.
Signalling- most emphasis on insulin and cAMP system. Focus on that. Remember CBP-brings in HAT activity./p300, steroid receptor coactivators, TRAP/DRIP/ARC complex, SRC-1
Learning objectives:
Thyroid and Glucocorticoid Receptors
To examine the regulation of gene expression by thyroid hormone
and glucocorticoids
common elements of receptor structure will be examined
intracellular localization will be examined
DNA binding properties of the NRs will be discussed
coactivator recruitment will be reviewed
classes of coactivators will be listed
actions of coactivators will be discussed
Common themes will be that the ligand will activate the receptor and increase gene expression
a nuclear localization signal (NLS) may be exposed
ligand binding will induce receptor activation
activated receptors will increase gene expression
activated receptors will recruit coactivators
To examine signaling pathways from the extracellular to the nucleus
Systems to be presented include:
cAMP and CREB activation (chapter 11/Fig 13-32)
Insulin and growth factors (chapter 14)
Cytokines and Jak/Stat (Chapter 14)
TNFalpha and NF-Kß (Chapter 14)
TGFß and Smads (chapter 13)
Steroid hormones and nuclear receptors
Common themes will be that an extracellular signal or ligand will activate a nuclear protein and increase gene expression
a nuclear localization signal (NLS) may be exposed
covalent modification of transcription factor may occur
ligand binding may induce factor activation
Kriwacki
Read and understand the Kirschner and Reed papers he handed out in class.
Understand Mechanisnms of cell cycle control including the cyclins, cyclin-dependent kinases, inhibitors.
Know how protein degradation pathways regulate the cell cycle.
Most of this was not said in the review session. Only Pfeffer and Park were there. These are the notes I use to study. Pray hard. Everything will turn out fine.
Rao’s Review Points
Signal Transduction
1. Strategies in study of signaling pathways.
Briefly know the strategies used in study of signal transduction.
2. To recognize the features and cellular functions of intracellular signal transduction pathways.
Know the mechanisms involved in the conserved commonly existing signaling proteins
3. To understand the mechanism of cyclic AMP pathway of signal transduction.
Know the details of cyclic AMP pathway of signal transduction, including adenylyl cyclase and PKA
4. To understand the role of second messengers derived from phosphoinositide.
The details of signaling pathway involving PLC, PKC and calcium are required.
5. To understand the structure and activation of receptor tyrosine kinases (RTK) and activation of Ras by RTKs.
Know the mechanism involved in receptor activation and Ras activation
6. To understand the mechanism of activation of MAP kinase pathway.
Mechanism involved in activation of MAP kinase is important
7. To understand some of the features of integration and control of signals in tissues
Cytoskeleton
1. Structure of actin cytoskeleton
Structure of G-actin monomer and F-actin polymer
Polarity of actin filaments
Arrangement of actin filaments into bundles and networks
Cross linking proteins
2. Dynamics of actin polymerization
Different phases of polymerization
Critical concentration
Polarity of polymerization
Effects of toxins on actin dynamics
3. Actin binding proteins (when a name of the protein is given know what it does to microfilaments and how)
Actin polymerizing proteins
Actin severing proteins
Actin capping proteins
Nucleating proteins
4. Myosin-powered cellular movements
Different types of myosins and their structure and function
Movement of myosin heads along filaments
5. Role of actin and myosin in muscle contraction
Contractile apparatus - thick and thin filaments
6. Role of actin and myosin in cell migration
Four different steps in cell migration
Role of actin dynamics in different steps of cell migration
7. Microtubules
Know the similarities and differences between the structure and function of microfilaments and microtubules.
8. The structure of microtubules, which includes:
structure of tubulin subunits
organization of subunits into tubules
polarity of microtubules
microtubule organizing center
9. The dynamics of microtubules assembly and disassembly:
temperature dependence
kinetics of assembly
polarity of assembly
disruption of microtubules by drugs
dynamic instability of microtubules (know how to describe it, but you do not have to know the mechanism of it)
10. Role of microtubules and motor proteins in transport of vesicles:
vesicle transport in axons; role of microtubules, motor proteins and polarity of vesicle movement
microtubule motor proteins – kinesins - structure and function
11. The role of microtubules in the structure and function of mitotic apparatus:
different types of microtubules in mitotic apparatus and their functions
motor proteins in mitotic apparatus
kinetochore and attachment of chromosomes during prophase
stabilization of chromosomes at the equatorial plate of cell during metaphase
microtubules and motor proteins in separation of chromosomes during anaphase
Pfeffer
Pfeffer- questions straightforward. No bizarre answers. Read text and notes. Control of cell cycle, mitosis and meiosis. One question per lecture.
Lecture Objectives:
70. Overview of Cell Cycle
To understand:
General Cell Cycle control
Phases of cell cycle
Experimental systems used to study cell cycle
Regeneration potential of different cells
Role of checkpoints
How to determine length of cell cycle and its phases
Mitosis versus meiosis
71. Cell cycle control mechanisms
To understand:
Control of cell cycle in Xenopus laevis
Maturation promoting factor (MPF)
Role of cyclins in cell cycle
Control of cell cycle in yeast
Temperature sensitive mutants
Role of protein phosphorylation
Role of protein degradation
Mitotic index-what % of cells in mitosis. Labelling index- how many of mitotic cells are labelled.
Park
2 questions- one on nuclear receptors- structure function relationships.
Signalling- most emphasis on insulin and cAMP system. Focus on that. Remember CBP-brings in HAT activity./p300, steroid receptor coactivators, TRAP/DRIP/ARC complex, SRC-1
Learning objectives:
Thyroid and Glucocorticoid Receptors
To examine the regulation of gene expression by thyroid hormone
and glucocorticoids
common elements of receptor structure will be examined
intracellular localization will be examined
DNA binding properties of the NRs will be discussed
coactivator recruitment will be reviewed
classes of coactivators will be listed
actions of coactivators will be discussed
Common themes will be that the ligand will activate the receptor and increase gene expression
a nuclear localization signal (NLS) may be exposed
ligand binding will induce receptor activation
activated receptors will increase gene expression
activated receptors will recruit coactivators
To examine signaling pathways from the extracellular to the nucleus
Systems to be presented include:
cAMP and CREB activation (chapter 11/Fig 13-32)
Insulin and growth factors (chapter 14)
Cytokines and Jak/Stat (Chapter 14)
TNFalpha and NF-Kß (Chapter 14)
TGFß and Smads (chapter 13)
Steroid hormones and nuclear receptors
Common themes will be that an extracellular signal or ligand will activate a nuclear protein and increase gene expression
a nuclear localization signal (NLS) may be exposed
covalent modification of transcription factor may occur
ligand binding may induce factor activation
Kriwacki
Read and understand the Kirschner and Reed papers he handed out in class.
Understand Mechanisnms of cell cycle control including the cyclins, cyclin-dependent kinases, inhibitors.
Know how protein degradation pathways regulate the cell cycle.
Thursday, January 25, 2007
Ray II
15
If you have seen review articles, he picked simplified versions of slides to give general overview of apoptosis. If you want to read in detail, the information is for people who want to go into area. Otherwise you do not have to go into detail. Generating interest.
16
Most caspases are initiators, executors, or inflammatory caspases. Inflammation not involved in apoptosis. Caspase 1 is related to ICE. Difference initiator and executor : Inititators are autoactivated after receiving signal. Autoactivation requires close proximity of more than one molecule. Clustering required. Receptor mediated pathways allow initiator caspases to form a complex, acting as modulators. 8 and 9 are major players in almost all cell types. 3,6,7 execute. No turning back. If 3,6,7 are inhibited, prevent cell death. If massive insult to the cell occurs with executor caspase inhibition, cells undergo necrosis.Mild condition- during a short window of tiime, you can prevent caspase 3 activation if you remove the insult.
17
Caspase structure and regulation:30 kD protein. Have prodomain, large, small domain. Removal prodomain results in release of large and small fragments. Complex forms active caspase. Must be processing at 2 sites for activation. Need amplifying signal to complete.
Caspases differ according to structure of domains. Variations affect binding. Any caspase with CARD domain does not act alone.Must recruit proteins to get activated.
18
List of caspases having CARD required for protein-protein interactions. Sequence is well-conserved.
19
DFMO inhibits enzyme to synthesize polyamines and inhibits apoptosis.
Add put, restore function.
With TNF alpha, which increases apoptosis in dose-dependent manner, active form appears of caspase3. Add compound back- get active Caspase 3. Western blot, but you could use ELISA to see it as well.
20
Caspase regulation: Cytochrome C released from mitochondrion as result of insult. It is released into cytoplasm. Binds APAF 1 and forms complex to recruit caspase 9. More recruited, more formed. Caspase 3 and all executor caspases need other caspase upstream in hierarchy.
21
Process of apoptosis is regulated like rheostat. Upregulation and downregulation.
Inhibitors can bind APAF complex or bind caspases directly.
22
Role of mitochondrion. Signal from outside can lead to release cytochrome C from periplasmic space. Extensive damage from insult- cytochrome C. formation of apopotosome complex requires ATP to enhance formation. Can be inhibited by IAP- inhibitor of apoptosis protein. AIF directly causes DNA fragmentation. Caspase independent.
23
Mitochondrion membrane permeability transition pore complex (PTP) regulated by several proteins. Bcl2 1st identified from B cell lymphoma cancers. Bcl-2 was anti-apoptoic. Accumulation of cells by increasing number or preventing decreasing number can cause cancer. There are different forms in different cell types. But function is same.Bcl-2 can form homodimer. Can form heterodimer. Bcl-2-Bax is balanced. Bax-Bax allows formation PTP pore. In cell where Bcl-2 expression is high, it inhibits Bax
24
Bcl-2 can regulate severity of any of these proteins.
25
Mcl is Bcl-2. All antiapoptosis proteins have all 4 homology domains. Pro-apoptosis lack one or more domains.
26
Transmembrane protein is required to insert protein in membrane. It has a membrane localization signal.
27
How are these proteins regulated?
Bid cleaved to t-Bid which can enter mitochondrial membrane. Bad is present in cytosol sequestered by other proteins like 14-3-3. When 14-3-3 is phosphorylated, releases Bad and Bad can translocate. Also phosphorylated for inactivation .. Regulated by signalling mechanism.Proteins subjected to additional level regulation. Amplification of signal from mitochondrion increases activation of caspase 3,6,7.
Process is complex.
28
Tip of villus, cells slough off and apoptosis. Nobody knows which comes first.
29
In animal experiment, Irradiated animal had increased caspase 3. Inhibition does not completely block caspase. Can reduce severity of response. Radiation increases Bax. DFMO inhibits ornithine carboxylase. Activates anti-apoptotic singnalling pathways.
30
Survival induction
Erk can phosphorylate Bad.Akt can also phosphorylate Bad. Under normal circumstances growth factors cause prolifreraton through upregulation of expression of genes required for cell proliferation. Induce stress- activates Jnk kinase. Jun kinase is apoptotic or anti-apoptotic. Must determine which way it goes.With TNF-alpha- gets phosphorylated. Compound to inhibit Kinase, Establish JNK can be blocked by inhibition or stimulated by TNF alpha.
31
Saw caspase 9 activation. Sig indicating mit damage. Assessed mit damage by mitosensor dye. Monomer fl green, multimer red. In mit fl red b/c it aggregates . Green indicates apoptosis.JNk inh decreased mit damage. JNk is pro-apoptosis in the cells illustrated.
32
more evidence of above. Caspase 3 same pattern b/c is downsream of caspase 9.
33 comprehensive slide.
Okadaic acid is inhibiting protein phosphatase. PP2a inhibited. Prevents dephosphorylation. Accumulation of phosphorylated proteins results. Fostreicin does same thing. Inhibiting phosphatases increases phosphorylated protein, or survival proteins.Ser-Thr phosphatases amplifies signalling in apoptosis process
PP2a regulates Jnk and a wide variety of other proteins.
34
What pathway leads to protection of cells- found increased ERK activity. ERK involved in proliferation. These cells were serum starved, so they would not transfer into proliferation. Increased Bad dephosphorylation increases ERK. Phosphroylation of BAD increases apoptosis resistance.
35
Bcl-2 practical example with rat tumors. P53 knocked out- got some tumor resistance to cell death, so p53 was necessary for apoptosis. All animals treated with cyclophosphamide. With p53 mutation, chemoresistance resulted.
High level Bcl-2, chemoresistance and animals did not respond. P53 mutant had reduced response to chemotherapy.
36
Bcl-2 is not part of apoptosis machine but part of mitochondrial homeostasis and membrane maintenance.
37
p53 was characterized a long time ago as tumor suppressor protein. S to G1-p53 goes up. It is a transcription Factor that can also bind to apoptosis proteins.Induced by list on left. Can lead to apoptosis or cell cycle arrest depending on context. Transcibes p21, which negatively regulates CDK activity. Prevents phosphorylation CDK2 to block cell cycle progression.
38
IAPs
39
Bir1,2,3 bacculovirus repeats. Conserved domains bind caspases to prevent access of substrate. Activity is blocked by protein. Upstream process is fine, but do not see effect of caspase 3.
These are from different organismss or different compartments. All do same job.
41
All growth factors activate pathways to phosphorylate Bad. Bad has 7-Ser phosphorylation site to be phosphor¥lated by different kinases.
Ser, Thr, Tyr can be switches to adapt a protein for a different job.
42
AKT activates nfkappaB. When there is a growth factor signal, converges to nucleus to increase transcription to put cells in proliferative or survival mode.
43
Everything is linked in chain.
RAC1 is part of GTPase and NADPH complex in mitochondrion for respiration. Activates or inactivates causing changes in redox state of th mitochondrion, which is also involved in apoptosis.
NFkB negatively regulates apoptosis by leading to increased transcription. IAP proteins to block process. Does not totally block- changes the balance toward survival.
44
Jnk is rate modulator, not direct effector. Depends on conditions in model.
45
Death receptor showing another pathway.
Read the articles.
NFkB inc transcription Mcl1. Has NLS sequence. STAT3 also translocates to nucleus, binds promoter region, activates transcription. Other cytokines activate transcription other genes.
If you have seen review articles, he picked simplified versions of slides to give general overview of apoptosis. If you want to read in detail, the information is for people who want to go into area. Otherwise you do not have to go into detail. Generating interest.
16
Most caspases are initiators, executors, or inflammatory caspases. Inflammation not involved in apoptosis. Caspase 1 is related to ICE. Difference initiator and executor : Inititators are autoactivated after receiving signal. Autoactivation requires close proximity of more than one molecule. Clustering required. Receptor mediated pathways allow initiator caspases to form a complex, acting as modulators. 8 and 9 are major players in almost all cell types. 3,6,7 execute. No turning back. If 3,6,7 are inhibited, prevent cell death. If massive insult to the cell occurs with executor caspase inhibition, cells undergo necrosis.Mild condition- during a short window of tiime, you can prevent caspase 3 activation if you remove the insult.
17
Caspase structure and regulation:30 kD protein. Have prodomain, large, small domain. Removal prodomain results in release of large and small fragments. Complex forms active caspase. Must be processing at 2 sites for activation. Need amplifying signal to complete.
Caspases differ according to structure of domains. Variations affect binding. Any caspase with CARD domain does not act alone.Must recruit proteins to get activated.
18
List of caspases having CARD required for protein-protein interactions. Sequence is well-conserved.
19
DFMO inhibits enzyme to synthesize polyamines and inhibits apoptosis.
Add put, restore function.
With TNF alpha, which increases apoptosis in dose-dependent manner, active form appears of caspase3. Add compound back- get active Caspase 3. Western blot, but you could use ELISA to see it as well.
20
Caspase regulation: Cytochrome C released from mitochondrion as result of insult. It is released into cytoplasm. Binds APAF 1 and forms complex to recruit caspase 9. More recruited, more formed. Caspase 3 and all executor caspases need other caspase upstream in hierarchy.
21
Process of apoptosis is regulated like rheostat. Upregulation and downregulation.
Inhibitors can bind APAF complex or bind caspases directly.
22
Role of mitochondrion. Signal from outside can lead to release cytochrome C from periplasmic space. Extensive damage from insult- cytochrome C. formation of apopotosome complex requires ATP to enhance formation. Can be inhibited by IAP- inhibitor of apoptosis protein. AIF directly causes DNA fragmentation. Caspase independent.
23
Mitochondrion membrane permeability transition pore complex (PTP) regulated by several proteins. Bcl2 1st identified from B cell lymphoma cancers. Bcl-2 was anti-apoptoic. Accumulation of cells by increasing number or preventing decreasing number can cause cancer. There are different forms in different cell types. But function is same.Bcl-2 can form homodimer. Can form heterodimer. Bcl-2-Bax is balanced. Bax-Bax allows formation PTP pore. In cell where Bcl-2 expression is high, it inhibits Bax
24
Bcl-2 can regulate severity of any of these proteins.
25
Mcl is Bcl-2. All antiapoptosis proteins have all 4 homology domains. Pro-apoptosis lack one or more domains.
26
Transmembrane protein is required to insert protein in membrane. It has a membrane localization signal.
27
How are these proteins regulated?
Bid cleaved to t-Bid which can enter mitochondrial membrane. Bad is present in cytosol sequestered by other proteins like 14-3-3. When 14-3-3 is phosphorylated, releases Bad and Bad can translocate. Also phosphorylated for inactivation .. Regulated by signalling mechanism.Proteins subjected to additional level regulation. Amplification of signal from mitochondrion increases activation of caspase 3,6,7.
Process is complex.
28
Tip of villus, cells slough off and apoptosis. Nobody knows which comes first.
29
In animal experiment, Irradiated animal had increased caspase 3. Inhibition does not completely block caspase. Can reduce severity of response. Radiation increases Bax. DFMO inhibits ornithine carboxylase. Activates anti-apoptotic singnalling pathways.
30
Survival induction
Erk can phosphorylate Bad.Akt can also phosphorylate Bad. Under normal circumstances growth factors cause prolifreraton through upregulation of expression of genes required for cell proliferation. Induce stress- activates Jnk kinase. Jun kinase is apoptotic or anti-apoptotic. Must determine which way it goes.With TNF-alpha- gets phosphorylated. Compound to inhibit Kinase, Establish JNK can be blocked by inhibition or stimulated by TNF alpha.
31
Saw caspase 9 activation. Sig indicating mit damage. Assessed mit damage by mitosensor dye. Monomer fl green, multimer red. In mit fl red b/c it aggregates . Green indicates apoptosis.JNk inh decreased mit damage. JNk is pro-apoptosis in the cells illustrated.
32
more evidence of above. Caspase 3 same pattern b/c is downsream of caspase 9.
33 comprehensive slide.
Okadaic acid is inhibiting protein phosphatase. PP2a inhibited. Prevents dephosphorylation. Accumulation of phosphorylated proteins results. Fostreicin does same thing. Inhibiting phosphatases increases phosphorylated protein, or survival proteins.Ser-Thr phosphatases amplifies signalling in apoptosis process
PP2a regulates Jnk and a wide variety of other proteins.
34
What pathway leads to protection of cells- found increased ERK activity. ERK involved in proliferation. These cells were serum starved, so they would not transfer into proliferation. Increased Bad dephosphorylation increases ERK. Phosphroylation of BAD increases apoptosis resistance.
35
Bcl-2 practical example with rat tumors. P53 knocked out- got some tumor resistance to cell death, so p53 was necessary for apoptosis. All animals treated with cyclophosphamide. With p53 mutation, chemoresistance resulted.
High level Bcl-2, chemoresistance and animals did not respond. P53 mutant had reduced response to chemotherapy.
36
Bcl-2 is not part of apoptosis machine but part of mitochondrial homeostasis and membrane maintenance.
37
p53 was characterized a long time ago as tumor suppressor protein. S to G1-p53 goes up. It is a transcription Factor that can also bind to apoptosis proteins.Induced by list on left. Can lead to apoptosis or cell cycle arrest depending on context. Transcibes p21, which negatively regulates CDK activity. Prevents phosphorylation CDK2 to block cell cycle progression.
38
IAPs
39
Bir1,2,3 bacculovirus repeats. Conserved domains bind caspases to prevent access of substrate. Activity is blocked by protein. Upstream process is fine, but do not see effect of caspase 3.
These are from different organismss or different compartments. All do same job.
41
All growth factors activate pathways to phosphorylate Bad. Bad has 7-Ser phosphorylation site to be phosphor¥lated by different kinases.
Ser, Thr, Tyr can be switches to adapt a protein for a different job.
42
AKT activates nfkappaB. When there is a growth factor signal, converges to nucleus to increase transcription to put cells in proliferative or survival mode.
43
Everything is linked in chain.
RAC1 is part of GTPase and NADPH complex in mitochondrion for respiration. Activates or inactivates causing changes in redox state of th mitochondrion, which is also involved in apoptosis.
NFkB negatively regulates apoptosis by leading to increased transcription. IAP proteins to block process. Does not totally block- changes the balance toward survival.
44
Jnk is rate modulator, not direct effector. Depends on conditions in model.
45
Death receptor showing another pathway.
Read the articles.
NFkB inc transcription Mcl1. Has NLS sequence. STAT3 also translocates to nucleus, binds promoter region, activates transcription. Other cytokines activate transcription other genes.
Shanklin Notes
Shanklin Notes
Tomorrow-stay home and study. Net week-think.
Many aspects of disease are straightforward. Neoplasms, inflammation, etc. The fifth column is development. How does the organism get to a mature state?
Gametes merge to form a blastocyst. Programming for development is in DNA.
Development is change from gametic genetic structure to a recognizable meso and gross structure which can contribute to furtherance of the species. Virus only does it with proper host.
What about bacteria?
Bacteria need a host to thrive.
Function of organisms is a function of the environment in which you find them.
Conserved genetic structures have common features. Adults are generally in stasis in terms of cessation of development. Not all tissues are at end of their developmental possibilities. Development is not always a straight line reaction. Sometimes there are retrogressions. Bile duct comes off liver. In healthy adult person it is a cylinder. In developing embryo it is a solid core of cells which have to change by apoptosis in center to restructure. Some cells differentiate into lining biliary epithelium.
Early aorta has series of arches in neck .Some retrogress by apoptosis or become incorporated. in other structures like left brachial artery.
Vertebrate body has rough bilateral symmetry. Adult kidneys are the third set to be formed. Babies with malformed kidneys are arrested at second stage.
Some fish have no glomeruli.
Kidneys have endocrine function-Vitamin D, erythropoetin, renin. We think of them as excretory, but has function above that.
Ex: lung has to do with estrogen metabolism in female.
Some deviations in development are significant- malformations or anomalies, or deformity in clinical situations. Malformation is alteration in structure.
During 3 stages of kidney formation, body is getting longer. Kidneys start near urethra. Body grows toward head from stimulus from neural tube.Kidneys move to wind up in mid body. Energy is used to reposition kidney. 1st gives way to mesonephros. 1st pronephros first, then meso (involved in movement), then metanephros. If signal does not go through, embryo gets stuck with mesonephros. Cannot adjust, so become cystic (cystic dysplasia). iThis can happen only on one side. Inducer after a period of time is within the organ itself.
Each kidney has one renal artery most of the time. Accessory renal artery can exist because supernumary vessel can be connected near upper pole. Ureter grows and gets longer. If you have a lower pole accessory it contacts ureter.Physical pressure could obstruct ureter. Backup causes renal pelvis to dilate.Residual pressure shuts down opening and backs up into kidney proper-interstital response- secretion- high blood pressure. Failure at 10-11weeks of development causes high blood pressure later, cured if renal artery is cut. If cyst is left from deviation, can alter flow and efficiency.
Renal interstitial cell= Renal arteries break up into lobars or interlobar into arcuate arteries into interlobular .Control depends on nerve sructure. We have between 3 nd 4 times the kidney we need.
Structure is highly integrated functionally. What does renal tubule do?
Developing kidney passes urine into amniotic fluid. The fluid turns over . Turnover rate of sodium is 30 min. Tracer substance can be used to determine this. (deuterium oxide in solution) Loss followed over time to get exchange rate.
Low dose Na24 and see what is picked up . mg/s exchange.
When organs start to function but are not mature,function augments their maturation. Know when functions begin.
Original heart is a tube with a receiving chamber and a pumping chamber. It has serial components. How does it become parallel?
What part of early embryo produces lungs? Part of foregut forms trachea and bronchi which secrete inducers into parenchyma to create lung.
When we look at the original truncus, it has cushions on either side. They grow to meet in middle. A helical form diverts one functional flow away from the other. Endocardial cushion is a complex process. Structures get bigger and orient to split.One to protolung and one to periphery. Malformations arise which are important. Endocardial cushion defect-Root of aorta meets membranous portion of interatrial spetum. This membrane is gone in the defect. Biventricular heart do not survive. Mild is correctible with surgery.
One of remnants of arch is ductus arteriosus- shunts blood from right ventricle back to aorta. regresses after birth. Once oxygen tension rises with breathing it constricts, then undergoes fibrous replacement by apoptotic involution. Not proper- blood retrogrades into lung and harms vessels- pulmonary hypertension. What if media is incorporated into aorta- causes coarctation. The localization of specialized tissue is as important as its function. Common bile duct passes through pancreas before emptying into duodenum. What if duct is misplaced or absent? Buffering is necessary for operation of intestinal enzymes.
Pyloric stenosis, annular pancreas- cause problems.
Lesion- tumor can be one, inflammatory response can be, bullet hole can be. What about a biochemical lesion? Absence of enzyme, disordered electrolyte compartment, rise in serum potassium following muscle injury? Often before you see change in cells you see changes in function.Lesion is abnormality usually defined by location and sequence of events leading to disease. For Tuesday- think about:
Down’s synrome has genetic element. Think about evidences for epigenetic contribution. What evidence would it take to consider challenging the axiom that it is a genetic disorder?
Couldn’t make heads or tails of the lecture today? You are not alone. Try this.
http://embryology.med.unsw.edu.au/embryo.htm
Also an idea for Monday:
Original Research Communications
Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome1,2,3
S Jill James, Marta Pogribna, Igor P Pogribny, Stepan Melnyk, R Jean Hine, James B Gibson, Ping Yi, Dixie L Tafoya, David H Swenson, Vincent L Wilson and David W Gaylor
1 From the Food and Drug Administration–National Center for Toxicological Research, the Division of Biochemical Toxicology, Jefferson, AR; the University of Arkansas for Medical Sciences, the Department of Biochemistry and Molecular Biology and the Department of Dietetics and Nutrition, Little Rock; the Arkansas Children's Hospital, the Division of Pediatric Genetics, Little Rock; Trisomy-21 Research, Inc, San Jose, CA; the Saginaw Valley State University, the Department of Chemistry, University Center, MI; and the Institute for Environmental Studies and Institute for Mutagenesis, Louisiana State University, Baton Rouge.
Background: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age.
Objective: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677CT) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers.
Design: The frequency of the MTHFR 677CT mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status.
Results: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677CT mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03).
Conclusion: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.
American Journal of Clinical Nutrition, Vol. 70, No. 4, 495-501, October 1999
© 1999 American Society for Clinical Nutrition
Tomorrow-stay home and study. Net week-think.
Many aspects of disease are straightforward. Neoplasms, inflammation, etc. The fifth column is development. How does the organism get to a mature state?
Gametes merge to form a blastocyst. Programming for development is in DNA.
Development is change from gametic genetic structure to a recognizable meso and gross structure which can contribute to furtherance of the species. Virus only does it with proper host.
What about bacteria?
Bacteria need a host to thrive.
Function of organisms is a function of the environment in which you find them.
Conserved genetic structures have common features. Adults are generally in stasis in terms of cessation of development. Not all tissues are at end of their developmental possibilities. Development is not always a straight line reaction. Sometimes there are retrogressions. Bile duct comes off liver. In healthy adult person it is a cylinder. In developing embryo it is a solid core of cells which have to change by apoptosis in center to restructure. Some cells differentiate into lining biliary epithelium.
Early aorta has series of arches in neck .Some retrogress by apoptosis or become incorporated. in other structures like left brachial artery.
Vertebrate body has rough bilateral symmetry. Adult kidneys are the third set to be formed. Babies with malformed kidneys are arrested at second stage.
Some fish have no glomeruli.
Kidneys have endocrine function-Vitamin D, erythropoetin, renin. We think of them as excretory, but has function above that.
Ex: lung has to do with estrogen metabolism in female.
Some deviations in development are significant- malformations or anomalies, or deformity in clinical situations. Malformation is alteration in structure.
During 3 stages of kidney formation, body is getting longer. Kidneys start near urethra. Body grows toward head from stimulus from neural tube.Kidneys move to wind up in mid body. Energy is used to reposition kidney. 1st gives way to mesonephros. 1st pronephros first, then meso (involved in movement), then metanephros. If signal does not go through, embryo gets stuck with mesonephros. Cannot adjust, so become cystic (cystic dysplasia). iThis can happen only on one side. Inducer after a period of time is within the organ itself.
Each kidney has one renal artery most of the time. Accessory renal artery can exist because supernumary vessel can be connected near upper pole. Ureter grows and gets longer. If you have a lower pole accessory it contacts ureter.Physical pressure could obstruct ureter. Backup causes renal pelvis to dilate.Residual pressure shuts down opening and backs up into kidney proper-interstital response- secretion- high blood pressure. Failure at 10-11weeks of development causes high blood pressure later, cured if renal artery is cut. If cyst is left from deviation, can alter flow and efficiency.
Renal interstitial cell= Renal arteries break up into lobars or interlobar into arcuate arteries into interlobular .Control depends on nerve sructure. We have between 3 nd 4 times the kidney we need.
Structure is highly integrated functionally. What does renal tubule do?
Developing kidney passes urine into amniotic fluid. The fluid turns over . Turnover rate of sodium is 30 min. Tracer substance can be used to determine this. (deuterium oxide in solution) Loss followed over time to get exchange rate.
Low dose Na24 and see what is picked up . mg/s exchange.
When organs start to function but are not mature,function augments their maturation. Know when functions begin.
Original heart is a tube with a receiving chamber and a pumping chamber. It has serial components. How does it become parallel?
What part of early embryo produces lungs? Part of foregut forms trachea and bronchi which secrete inducers into parenchyma to create lung.
When we look at the original truncus, it has cushions on either side. They grow to meet in middle. A helical form diverts one functional flow away from the other. Endocardial cushion is a complex process. Structures get bigger and orient to split.One to protolung and one to periphery. Malformations arise which are important. Endocardial cushion defect-Root of aorta meets membranous portion of interatrial spetum. This membrane is gone in the defect. Biventricular heart do not survive. Mild is correctible with surgery.
One of remnants of arch is ductus arteriosus- shunts blood from right ventricle back to aorta. regresses after birth. Once oxygen tension rises with breathing it constricts, then undergoes fibrous replacement by apoptotic involution. Not proper- blood retrogrades into lung and harms vessels- pulmonary hypertension. What if media is incorporated into aorta- causes coarctation. The localization of specialized tissue is as important as its function. Common bile duct passes through pancreas before emptying into duodenum. What if duct is misplaced or absent? Buffering is necessary for operation of intestinal enzymes.
Pyloric stenosis, annular pancreas- cause problems.
Lesion- tumor can be one, inflammatory response can be, bullet hole can be. What about a biochemical lesion? Absence of enzyme, disordered electrolyte compartment, rise in serum potassium following muscle injury? Often before you see change in cells you see changes in function.Lesion is abnormality usually defined by location and sequence of events leading to disease. For Tuesday- think about:
Down’s synrome has genetic element. Think about evidences for epigenetic contribution. What evidence would it take to consider challenging the axiom that it is a genetic disorder?
Couldn’t make heads or tails of the lecture today? You are not alone. Try this.
http://embryology.med.unsw.edu.au/embryo.htm
Also an idea for Monday:
Original Research Communications
Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome1,2,3
S Jill James, Marta Pogribna, Igor P Pogribny, Stepan Melnyk, R Jean Hine, James B Gibson, Ping Yi, Dixie L Tafoya, David H Swenson, Vincent L Wilson and David W Gaylor
1 From the Food and Drug Administration–National Center for Toxicological Research, the Division of Biochemical Toxicology, Jefferson, AR; the University of Arkansas for Medical Sciences, the Department of Biochemistry and Molecular Biology and the Department of Dietetics and Nutrition, Little Rock; the Arkansas Children's Hospital, the Division of Pediatric Genetics, Little Rock; Trisomy-21 Research, Inc, San Jose, CA; the Saginaw Valley State University, the Department of Chemistry, University Center, MI; and the Institute for Environmental Studies and Institute for Mutagenesis, Louisiana State University, Baton Rouge.
Background: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age.
Objective: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677CT) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers.
Design: The frequency of the MTHFR 677CT mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status.
Results: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677CT mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03).
Conclusion: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.
American Journal of Clinical Nutrition, Vol. 70, No. 4, 495-501, October 1999
© 1999 American Society for Clinical Nutrition
Wednesday, January 24, 2007
Ramesh Ray 1
Ramesh Ray
Overview Apoptosis
Contact him for more info.His office at 521 Nash next to Dr. Rao.
Main aim of science is to increase life expectancy and quality of life.
During embryonic development life and death coexist. Cell death forms interdigital spaces of fingers.
Last decade apoptosis was discovered.
2
Word “apoptosis” is “falling off” -natural process by which normal cell dies.Also called programmed cell death.
Adult human body- every day 10 billion cells made, so same number cells continuously eliminated.
Before 1972- most cell death seemed to be by necrosis. Cells die randomly, rupture, and contents of cell relesed. Apoptosis is a lot cleaner.
3
2 processes contrasted. How to differentiate? Know these hallmarks. Apoptosis- all intracellular organelles intact. Cell forms blebs with small vesicles. Those vesicles are engulfed by neighboring or phagocytic cells. Necrosis is opposite. Cell ruptures and contents released.
All necrotic processes lead to inflammatory response. Apoptosis does not cause inflammatory response.
4
See Lodish for diagram. SEM shows blebbing.
5
TNF alpha
How to quantify cells dying of apoptosis? When you induce apoptosis in cell culture- See DNA fragments in gel. Nucleosomes- eukaryotic DNA wrapped around histones.Each element of Dna and histone is nucleosome. Specific endonucleases are activated to cut open area. Each histone has about 150 kbp. See multiples of 150 on gel. Ladder shows up because endonucleases are activated sequentially. ELISA done in top graph. Antibody against histone used. Can use quantitatively.TNF alpha induces apoptosis in many systems.Cyclohexamide is an antibiotic that is inhibiting protein synthesis by inhibiting ribosomes. Combination causes apoptosis. In presence of TNF alpha- cells synthesizes survivor proteins. The antibiotic keeps these proteins from being synthesized and promotes apoptosis in a dose-dependent way.
6
Development of the immune system is dependent on proper apoptosis. Not-RA, diabetes, other autimmune disease.
Adulthood- fixed number neurons maintained by regulation of apoptosis.
7
To understand, must have a model to study. C. Elegans had fixed number cells. Fixed number undergoes apoptosis at different stages of development. Bcl-2 prevents apoptosis in adult life.
8
Knocking out certain developmental genes in c. elegans is not fatal.GFP tags can be used to follow proteins in a spatiotemporal way in these animals.
9
We should be able to see relationships between model and human. The information from elegans translates in a conserved way so far.
CED means Cell death or c.elegans death genes. APAF- apoptosis activating factor. Caspases kill cell. There is also caspase independent-death. It is a Failsafe mechanism with more than one alternative pathway.
10
If we know components, we can put together picture of how cellular machinery works
Adapters are same component in 2 different systems. Higher level organization, more complexity. Vertebrates have more components.
11
2 mechanisms cell death. Threat can be from outside or inside. One is extrinsic other intrinsic. Threat outside- extrinsic pathway. Threat sensed by death receptors on membrane. Signal transmitted inside for response. Fas and other receptors recognize ligand. Receptors have multimeric components. Binding opens intracellular domains and induces binding of interior proteins. Apoptosis proteins not active until start signal given. Regulator binds receptor, adaptors bind,DD is death domain. Cell tries to correct damage or meet threat first. Halts cell cycle (cell cycle arrest) first. Then brings processes to basal level and assesses damage. Then tries to repair damage. If damage too much, apoptosis commitment starts. DISC involves lots of proteins.(5 or 6). TNF signal forms TNFADD. Death complex forms and activates Caspase 8, which activates caspase 3. Caspase 3 is commited step. Granzymes activate caspase 3 or cause caspase independentendent death.
12
Intrinsic pathway
Internal threat includes damage to DNA. 1500 nucleotides added per second in replication. Most mistakes are repaired, but presence of certain chemicals or lack of cytokines in medium can cause damage or cell death. Caspase 1 originally known as interleukin converting enzyme (ICE).
Mitochondrion is important for respiration and for sensing internal and external trouble in cell. Low ATP in cell gives signal for apoptosis.
Deplete ATP-induce apoptosis.BCl-2 is a negative regulator of apoptosis, promoting cell survival. It regulates cytochrome-c. Cytochrome C is between inner and outer membrane. Released- out of place. Threat signal .Cytochrome c binds APAF-1 to form apoptosome. The apoptosome is made of a number of APAFs and recruits caspase 9 molecules. Other proteins also leak out from periplasmic space to bind inhibitors of apoptosis molecules to encourage apoptosis.
13
Genotoxic damage can be caused by drugs.
Cytokine deprivation- withdraw IL-3. Initiates cell death signal.Checkpoint- decision is made.BCl-2 is anti-apoptosis. BAX is pro-apoptosis. Mitochondrion is on left. Once capsases activated, going toward commitment. Today about 250 known proteins are known targets for caspase cleavage.
14
Pathway based on information from past 10 years. Slide illustrates classic TNF alpha induced apoptosis. TNF binds trimeric receptor, goes inside to TRADD or FADD. Those proteins bind inside to give signal that something is bound outside. Procaspase 8 binds to the bound FADD. Procaspase is like a zymogen or proenzyme. Activated by cleavage. Active fragment is caspase 8. When multiple molecules bind, one activates other and signal is amplified. Procaspase 3 forms caspases 3,6,7, which are executional caspases. Earlier ones are initiational caspases.
When caspase 8 activates, cleaves Bid (BCl family protein). Increases permeability of mitochondrion. Bad is pro-apoptosis protein. Pores form in the mitochondrial membrane to allow leakage of cytochrome C.Cytochrome-C with APAF activates caspase 9, caspase 3, apoptosis.
15
Will stop here. Caspases cleave Cys after Asp in specific sequences. Cell chops up big proteins into small fragments into vesicles for neighboring cells to clear.
Overview Apoptosis
Contact him for more info.His office at 521 Nash next to Dr. Rao.
Main aim of science is to increase life expectancy and quality of life.
During embryonic development life and death coexist. Cell death forms interdigital spaces of fingers.
Last decade apoptosis was discovered.
2
Word “apoptosis” is “falling off” -natural process by which normal cell dies.Also called programmed cell death.
Adult human body- every day 10 billion cells made, so same number cells continuously eliminated.
Before 1972- most cell death seemed to be by necrosis. Cells die randomly, rupture, and contents of cell relesed. Apoptosis is a lot cleaner.
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2 processes contrasted. How to differentiate? Know these hallmarks. Apoptosis- all intracellular organelles intact. Cell forms blebs with small vesicles. Those vesicles are engulfed by neighboring or phagocytic cells. Necrosis is opposite. Cell ruptures and contents released.
All necrotic processes lead to inflammatory response. Apoptosis does not cause inflammatory response.
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See Lodish for diagram. SEM shows blebbing.
5
TNF alpha
How to quantify cells dying of apoptosis? When you induce apoptosis in cell culture- See DNA fragments in gel. Nucleosomes- eukaryotic DNA wrapped around histones.Each element of Dna and histone is nucleosome. Specific endonucleases are activated to cut open area. Each histone has about 150 kbp. See multiples of 150 on gel. Ladder shows up because endonucleases are activated sequentially. ELISA done in top graph. Antibody against histone used. Can use quantitatively.TNF alpha induces apoptosis in many systems.Cyclohexamide is an antibiotic that is inhibiting protein synthesis by inhibiting ribosomes. Combination causes apoptosis. In presence of TNF alpha- cells synthesizes survivor proteins. The antibiotic keeps these proteins from being synthesized and promotes apoptosis in a dose-dependent way.
6
Development of the immune system is dependent on proper apoptosis. Not-RA, diabetes, other autimmune disease.
Adulthood- fixed number neurons maintained by regulation of apoptosis.
7
To understand, must have a model to study. C. Elegans had fixed number cells. Fixed number undergoes apoptosis at different stages of development. Bcl-2 prevents apoptosis in adult life.
8
Knocking out certain developmental genes in c. elegans is not fatal.GFP tags can be used to follow proteins in a spatiotemporal way in these animals.
9
We should be able to see relationships between model and human. The information from elegans translates in a conserved way so far.
CED means Cell death or c.elegans death genes. APAF- apoptosis activating factor. Caspases kill cell. There is also caspase independent-death. It is a Failsafe mechanism with more than one alternative pathway.
10
If we know components, we can put together picture of how cellular machinery works
Adapters are same component in 2 different systems. Higher level organization, more complexity. Vertebrates have more components.
11
2 mechanisms cell death. Threat can be from outside or inside. One is extrinsic other intrinsic. Threat outside- extrinsic pathway. Threat sensed by death receptors on membrane. Signal transmitted inside for response. Fas and other receptors recognize ligand. Receptors have multimeric components. Binding opens intracellular domains and induces binding of interior proteins. Apoptosis proteins not active until start signal given. Regulator binds receptor, adaptors bind,DD is death domain. Cell tries to correct damage or meet threat first. Halts cell cycle (cell cycle arrest) first. Then brings processes to basal level and assesses damage. Then tries to repair damage. If damage too much, apoptosis commitment starts. DISC involves lots of proteins.(5 or 6). TNF signal forms TNFADD. Death complex forms and activates Caspase 8, which activates caspase 3. Caspase 3 is commited step. Granzymes activate caspase 3 or cause caspase independentendent death.
12
Intrinsic pathway
Internal threat includes damage to DNA. 1500 nucleotides added per second in replication. Most mistakes are repaired, but presence of certain chemicals or lack of cytokines in medium can cause damage or cell death. Caspase 1 originally known as interleukin converting enzyme (ICE).
Mitochondrion is important for respiration and for sensing internal and external trouble in cell. Low ATP in cell gives signal for apoptosis.
Deplete ATP-induce apoptosis.BCl-2 is a negative regulator of apoptosis, promoting cell survival. It regulates cytochrome-c. Cytochrome C is between inner and outer membrane. Released- out of place. Threat signal .Cytochrome c binds APAF-1 to form apoptosome. The apoptosome is made of a number of APAFs and recruits caspase 9 molecules. Other proteins also leak out from periplasmic space to bind inhibitors of apoptosis molecules to encourage apoptosis.
13
Genotoxic damage can be caused by drugs.
Cytokine deprivation- withdraw IL-3. Initiates cell death signal.Checkpoint- decision is made.BCl-2 is anti-apoptosis. BAX is pro-apoptosis. Mitochondrion is on left. Once capsases activated, going toward commitment. Today about 250 known proteins are known targets for caspase cleavage.
14
Pathway based on information from past 10 years. Slide illustrates classic TNF alpha induced apoptosis. TNF binds trimeric receptor, goes inside to TRADD or FADD. Those proteins bind inside to give signal that something is bound outside. Procaspase 8 binds to the bound FADD. Procaspase is like a zymogen or proenzyme. Activated by cleavage. Active fragment is caspase 8. When multiple molecules bind, one activates other and signal is amplified. Procaspase 3 forms caspases 3,6,7, which are executional caspases. Earlier ones are initiational caspases.
When caspase 8 activates, cleaves Bid (BCl family protein). Increases permeability of mitochondrion. Bad is pro-apoptosis protein. Pores form in the mitochondrial membrane to allow leakage of cytochrome C.Cytochrome-C with APAF activates caspase 9, caspase 3, apoptosis.
15
Will stop here. Caspases cleave Cys after Asp in specific sequences. Cell chops up big proteins into small fragments into vesicles for neighboring cells to clear.
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