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.
Wednesday, January 31, 2007
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.
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.
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