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.
Kriwacki
Kriwacki Cell Cycle
Proteins that Regulate Cell Cycle
Read the Kirschner paper about fundamental role of protein degradation in regulation of the cell cycle. Concepts remain central to how aspects of cell cycle are regulated.
Slides are digitally intensive.
2
Discoveries were made in yeast due to ease of manipulation.
Cdk inhibition keeps kinases in check.
3
Cell cycle in S. Cerevisiae- from text assignment (Lodish 874-890). START is where decisions are made and boundaries crossed to replicate DNA and undergo budding process.
4
Screened for genes to complement temperature sensitive mutants. CDC 28 was one of 1st CDK s discovered.
Graphic shows cells harboring a ts mutation. These cells grow at 25 degrees C, but raise the temperature to 37 degrees and they exhibit defects with no budding, even with lots of nutrients. Library of genes in yeast was screened for effects of plasmids in helping yeast to bud.CDC28 was kinase.
5
Mechanisms common to all higher organisms. Cancer-cells lose ability to control proliferation.
6
Reed late 1980s discovered several cyclin regulatory partners. Similar experiment to earlier ones- factors regulate entry into S phase specifically. Mutation affected interaction of the kinase with its cyclin regulatory partner. Investigators did not know what the regulatory machinery consisted of.
7
CDK has regulatory factor bound in order for colonies to form in wild type. In ts cells, the mutant CDC28 protein could only bind to regulatory partner at low temperature. Used library in plasmids to express gene of plasmid at high levels. Able to shift equilibrium to bound and active state.
7
CLN3
Approach a little different- screened for plasmid to rescue defect in budding, but used inducible promoter in presence of low glucose. Expressed in absence of glucose. Used FACS analysis of yeast cells to determine whether expressing the gene rescued defect, The cells are labelled with fluorescence tag proportional to the amount of DNA in cell.G1=1 genome. G2= 2 copies. Cell number is on the on y axis. Normal disribution (distribution under normal conditions with normal cells) on left wih 2 peaks. With cyclin vector- not in presence of glucose, increase in peak for actively dividing cells. With glucose-2 traces similar. In C, Yeast defective in G1S phase cyclins with high glucose get stuck in G1. Without- could proceed to G2 with exogenous cyclin, albeit with lower level of replication due to using sick cells.
8
Role protein degradation in cell cycle regulation
Inhibitor of CDK is regulated by phosphorylation, which signals protein to be ubiquitinated, causing degradation and relieving inhibition of CDK complex. Mechanism illustrates the way the activity of one set CDK complexes is required to activate another set CDK complexes. One aspect of sequence of events- protein degradation is not easily reversible. Nomenclature is horrible. Clb 5 and 6 are S phase regulation. Inhibitor is Sic1. Cln 1 and 2 phosphorylate Sic1.
For exam, understand concepts, but refer to cyclins and CDKs by general type.
9
Diagram puts together results over years. Illustrates way activity of CDK complexes contributes to series of events taking cells into mitosis.
Yeast- only one CDK.Higher organisms- more kinases, more target proteins.
Cln3-CDK phosphorylates a transcription factor that upregulates cln-1 and cln-2 transcription, to pair with CDK to phosphorylate different transcription factors, etc resulting in sequential activation of transcription. Can also trigger degradation to eliminate activity after time needed or relieve repression. CDK has different partners.
10
Meshing of gears allowing cells to progress through cell cycle unidirectionally. Once cell enters mitosis, no longer needs active signal. Degrades regulatory subunit, cyclin. Whether to ubiquitinate cyclin depends on G1 CDK complex. Read this in text several times and read review.
11-12
Kirschner’s slides
This is more detail on what we have already covered.
13
APC pathway: cells need to have properly set up metaphase plate with appropriate pairing, etc, then go into anaphase. APC targets cyclin B to relieve inhibtion of exit from mitosis. Inactivated CDKs cause cell to sit in G1 until another cell division.
14
overview
15
Regulation by post-translational modification can be reversed, but regulation by proteolysis cannot.
READ the PAPER for the EXAM!!
Know differences between cyclin types, but not picky names.
16
Firing of replication complexes from origins of replication:
In order for replication to occur, must assemble regulatory factors. S phase CDK and DDK phosphorylate proteins involved in pre-replication complex. Phosphorylation signals replication to occur, disassembly of the pre-replication complex, and other factors to come in. RPA binds ssDNA exposed by helicases. Replication complete- origins get loaded w original complex, but in absence of CDK will not become active.
17
Mammalian cyclins and CDKs- concepts same, player different.
We have variety of cyclins and CDKs. CDK activates E2F, which phosphorylates retinoblastoma protein, frees E2F, causes transcription cyclins E and A. Follow cyclins through chart.
18
last page handout.
19
Cell Cycle 4:11, 1491-1494, Nov 2005
CDK1 and 2 may play redundant roles in early stages cell division. This reviews several papers looking for role for CDK1.
20
Which kinase dominant in normal cycles? Unknown. Mice wdeveloped normally, so the CDKs 1 and 2 seemed to act together in transitions.
21
Alignment of human CDK1 , cdk2, cdk4. Enzymes have distinct roles at different stages. CDK1 is essential for entry into mitosis. Enzymes are really similar. Thr 160 is regulatory Thr phosphorylated for CDKs to be active. Region near it is similar in all these kinases.
PSTAIRE Helix largely conserved.
22
Differences in activity come from differences in cyclin regulatory partners. A, B1,D1,E1 alignment. D1 and E are shorter. Much more variability in these sequences. Differences in structure make differences in function.
23
Functions:
Genes Dev. 2004: 18: 2699-711.
P107 and 130 Rb phosphorylation, liberates transcription factor to transcribe cyclin E, A, regulatory proteins, resulting in further expression of RB, E2F1, and enzymes involved in nucleic acid synthesis, DNA replication machinery.
Limited targets of kinase liberate a transcription factor controlling a variety of other factors.
E and A, phosphorylate RB at different sites to modify function of protein. P27 is inhibitory.
24
cdk activity regulates details in specific examples of cyclins and kinases- Will give copies of slides Monday.
CDK has to be paired with cyclin to have potential to be active.
CAK phosphorylates cyclin/cdk. P21 inhibits activity. Data shows when inhibitor is bound, some complexes are active, so the story is more complicated than we know right now.
25
Structural view of CDK2 with key features illustrated.
Kinases have N terminal beta sheet and helical domain at bottom. Active site is interface of these domains in middle in pocket. Regulatory element is PSTAIRE helix- cyclin binds on right and docks on face CDK2 to shove PSTAIRE in and reform active site. Thr phosphorylation is in loop that connects with helical lobe.
26
When cyclin A binds, shoves PSTAIRE in. Phospho transfer from ATP occurs.Cyclins interact with substrates.
31These slides may be misaligned.
Phosphorylation introduces negative charge, so basic residues in area are affected. They form pocket and rearrange complex due to phosphorylation. Opens area for substrate binding.
Tues- role of inhibitors and recent findings in field. Read text and paper. Ask questions Tues AM.
Kriwacki Cyclins II
Quick run-through of lecture 1:
At end of today will give review session. Nutshell: fundamental concepts of how cell cycle is regulated by CDKs and cyclins. Understand how key experiments were done in yeast and what significance of results were. 1989 cyclin paper- read and be able to describe from experimental and conceptual and significance perspectives. New paper in nature about the identity of 2 proteins phosphorylated by S phase CDK complex in yeast key in regulating DNA replication. Took 10 yrs to find kinase targets. We do not understand all of what the complexes are targeting. Look in January issue of Nature.
Specifically read and understand paper handed out today. Also look at protein degradation as a regulating mechanism. No-going-back type of regulation. Interplay between CDK complex. Understand how protein degradation is used to regulate cell cycle. Text and article by Kirchner hit key concepts. APC is an ubiquitination machine. Be able to describe key elements of a CDK that pertain to cyclin binding and conformational change in activation. Review in1999 JMB pavelitch is about structural results.
Main point of structural details- understand those.
Inhibitors of CDKs:
To activate, bind to cyclin and phosphorylate kinase. Levels of cyclins vary cyclically for control. If this were the only level of regulation, the cell could not prevent multiple rounds of division. Family of proteins called p16 act by binding to CDK 4 and 6 to affect entry into cell cycle at earliest stage. Cyclin D-CDK-4 is phosphorylated and phosphorylates Rb/E2F. Inhibiting CDK 4- prevents Rb phosphorylation and entry into S phase. P15,16,18,19 are CKIs.
p19 binds opposite side from PSTAIRE. It wedges in and locks N terminal lobe into open conformation to prevent binding of cyclin. P19 has 3 repeats of ankyrin motif, which is made up of 2 alpha helices and a beta hairpin.They stack together to form broad surface to form interface with CDK 6. Ankyrin repeat proteins are found in proteins involved in protein-protein interactions.
Lots done on biology of these.
P21 and p27 studied extensively since discovered in mid 90s. p27 inhibits most CDK/Cyclin complexes. IN cells, it promotes activity of CDK4 cyclin D. How? We do not know.
Activity of p27 is regulated by degradation through ubiquitination which is regulated by post-translational modification, specifically phosphorylation of p27.
When cells exit quiescence, they inherit high level of p27. Begins G1, but blocks progress. Needs to be reduced for crossing G1/S boundary. Ubiquitin ligase SCF/ Skp2 increase in mid G1 phase. This ubiquitin ligase targets p27 to reduce its levels so later complexes can be activated.
P27 levels reduced in most human cancers. Loss p27 deregulates G1/S checkpoint.
P27 bound to CDK2/CyclinA structure- binds like a molecular staple, one part to each subunit. C terminal regulatory domain does not interact w cyclin or kinase. Accessible for modification by enzymes. Activity of enzyme is required for phosphorylation p27, but p27 inhibits complex.
Structure function relationship p 11
Conserved domains have conserved structure function relationship. Linker helix domain is also conserved.
12
Domain 1, domain LH, domain 2 important.
Single turn at end is important to mechanism. 310 helix contains Tyr residue. Structure is highly dynamic. Flexibility plays a role in its function. 310 helix of p27 inserts into ATP binding procket preventing binding of ATP and substrate. Surface recognition by domain 1 is conserved in other cyclins in cell cycle. Helix has sequence MRAIL in cyclin A,B,D,E. Conserved binding site mediates specificity for p27.
What is conserved about linker is length.
Domain 2 wraps around side of CDK2 and positions 310 helix in active site for inhibition. P27 needs to interact with specific sites in order to inhibit.
Phosphorylation alters function of protein from inhibitor to activator and captive substrate of CDK2.
Domain 1 binds first, then forms basis of specificity.
What about the puzzle? How is p27 eliminated?
Tyrosine kinases can phosphorylate Tyr in 310 helix, causing local change to reactivate kinase to phosphorylate Thr residue and progress into cell cycle. Results tie Tyr kinase signalling into cell cycle.
Y88 sits in ATP binding pocket. Tail has to come close to active site once on a while for T187 to get phosphorylated.
NMR- 2D spectra for free p27 amide nitrogens and protons in peptide. Shifts cluster in the free state because the parts of the molecule average to have similar chemical environments due to the molecule’s flexibility.Right- single conformation with effects of neighbors spread out. Other proteins are not labelled, and are invisible. Most resonances can be labelled.Yellow domain 1, green domain 2, red 310 helix region. Right spectrum is non phosphorylated and reflects inhibitory conformation. Resonance changes are fingerprint of structure when bound to complex. Magnitude of changes shown in bar graph on p. 16
Same experiment with phosphorylated p27 on Y88 prepared in vitro- looked at same pair of spectra and plotted histogram- Pattern of chemical shift changes is same, except for Y88 in 310 helix region. No change free to bound when phosphorylated. Phosphorylation of Tyr causes portion inhibitor to be ejected from active site of complex. How can residue be phosphorylated by Tyr kinase when it is bound in ATP binding pocket? It can’t. Must occasionally swing out and get phosphorylated, then it cant go back in. It is naturally flexible.
Phosphorylation reactivates CDK2. Hypothesized that phosphorylation of the Tyr would promote phosphorylation on Thr. Demonstrated using kinase assay. Phosphorylation of the Thr depends on concentration of complex added to reaction with P23 labelled ATP.
Even if most molecules are bound, can still have small amount free kinase to cause reaction even without phosphorylation Tyr. Graphs illustrate levels phosphorylation. Bimolecular reaction. Tyr phosphorylation ejects helix, tail encounters active site CDK2, Thr phosphorylation (intracomplex reaction with inherent rate depending on how often tail encounters active site)- creates linear concentration dependence.
BcrAbl is Tyr kinase active in some leukemias, constitutively active. Promotes chronic myelogenous leukemia and acute lymphoblastic leukemia. This kinase act causes premature eliminates p27. Phosphorylation at early stage targets p27 for degradation. Lin kinase also does this.
Gleevec acts to target Bcr-Abl to make degradation p27 more normal. The Tyr phosphorylated structure may be a novel drug target.
Be able to answer how this mechanism ties post-translational modification and degradation together. This mechanism ties Tyr kinase signalling into cell cycle control.
Known that short segments of the c-terminal tail are recognized only when Thr is phosphorylated. Parts of CDK2/cyclinA also recognized. There are also several Ks in tail that are probably sites of ubiquitination. What residues ubiquitinated and how it happens mechanistically are not shown yet. Lots of regulation probably goes on that we don’t know about yet.
Review:
Details of CDK2 and features preserved in other CDKs.
Ubiquitination machinery slides page 21- not on exam, but covered elsewhere. SCF complex regulates E and D and p27. APC regulates later stages as covered in Kirschner paper.
Exam format- simple questions to describe some of topics pointed out as he went through today. May give words to include in essay on topic. READ ASSIGNED PAPERS!!
Proteins that Regulate Cell Cycle
Read the Kirschner paper about fundamental role of protein degradation in regulation of the cell cycle. Concepts remain central to how aspects of cell cycle are regulated.
Slides are digitally intensive.
2
Discoveries were made in yeast due to ease of manipulation.
Cdk inhibition keeps kinases in check.
3
Cell cycle in S. Cerevisiae- from text assignment (Lodish 874-890). START is where decisions are made and boundaries crossed to replicate DNA and undergo budding process.
4
Screened for genes to complement temperature sensitive mutants. CDC 28 was one of 1st CDK s discovered.
Graphic shows cells harboring a ts mutation. These cells grow at 25 degrees C, but raise the temperature to 37 degrees and they exhibit defects with no budding, even with lots of nutrients. Library of genes in yeast was screened for effects of plasmids in helping yeast to bud.CDC28 was kinase.
5
Mechanisms common to all higher organisms. Cancer-cells lose ability to control proliferation.
6
Reed late 1980s discovered several cyclin regulatory partners. Similar experiment to earlier ones- factors regulate entry into S phase specifically. Mutation affected interaction of the kinase with its cyclin regulatory partner. Investigators did not know what the regulatory machinery consisted of.
7
CDK has regulatory factor bound in order for colonies to form in wild type. In ts cells, the mutant CDC28 protein could only bind to regulatory partner at low temperature. Used library in plasmids to express gene of plasmid at high levels. Able to shift equilibrium to bound and active state.
7
CLN3
Approach a little different- screened for plasmid to rescue defect in budding, but used inducible promoter in presence of low glucose. Expressed in absence of glucose. Used FACS analysis of yeast cells to determine whether expressing the gene rescued defect, The cells are labelled with fluorescence tag proportional to the amount of DNA in cell.G1=1 genome. G2= 2 copies. Cell number is on the on y axis. Normal disribution (distribution under normal conditions with normal cells) on left wih 2 peaks. With cyclin vector- not in presence of glucose, increase in peak for actively dividing cells. With glucose-2 traces similar. In C, Yeast defective in G1S phase cyclins with high glucose get stuck in G1. Without- could proceed to G2 with exogenous cyclin, albeit with lower level of replication due to using sick cells.
8
Role protein degradation in cell cycle regulation
Inhibitor of CDK is regulated by phosphorylation, which signals protein to be ubiquitinated, causing degradation and relieving inhibition of CDK complex. Mechanism illustrates the way the activity of one set CDK complexes is required to activate another set CDK complexes. One aspect of sequence of events- protein degradation is not easily reversible. Nomenclature is horrible. Clb 5 and 6 are S phase regulation. Inhibitor is Sic1. Cln 1 and 2 phosphorylate Sic1.
For exam, understand concepts, but refer to cyclins and CDKs by general type.
9
Diagram puts together results over years. Illustrates way activity of CDK complexes contributes to series of events taking cells into mitosis.
Yeast- only one CDK.Higher organisms- more kinases, more target proteins.
Cln3-CDK phosphorylates a transcription factor that upregulates cln-1 and cln-2 transcription, to pair with CDK to phosphorylate different transcription factors, etc resulting in sequential activation of transcription. Can also trigger degradation to eliminate activity after time needed or relieve repression. CDK has different partners.
10
Meshing of gears allowing cells to progress through cell cycle unidirectionally. Once cell enters mitosis, no longer needs active signal. Degrades regulatory subunit, cyclin. Whether to ubiquitinate cyclin depends on G1 CDK complex. Read this in text several times and read review.
11-12
Kirschner’s slides
This is more detail on what we have already covered.
13
APC pathway: cells need to have properly set up metaphase plate with appropriate pairing, etc, then go into anaphase. APC targets cyclin B to relieve inhibtion of exit from mitosis. Inactivated CDKs cause cell to sit in G1 until another cell division.
14
overview
15
Regulation by post-translational modification can be reversed, but regulation by proteolysis cannot.
READ the PAPER for the EXAM!!
Know differences between cyclin types, but not picky names.
16
Firing of replication complexes from origins of replication:
In order for replication to occur, must assemble regulatory factors. S phase CDK and DDK phosphorylate proteins involved in pre-replication complex. Phosphorylation signals replication to occur, disassembly of the pre-replication complex, and other factors to come in. RPA binds ssDNA exposed by helicases. Replication complete- origins get loaded w original complex, but in absence of CDK will not become active.
17
Mammalian cyclins and CDKs- concepts same, player different.
We have variety of cyclins and CDKs. CDK activates E2F, which phosphorylates retinoblastoma protein, frees E2F, causes transcription cyclins E and A. Follow cyclins through chart.
18
last page handout.
19
Cell Cycle 4:11, 1491-1494, Nov 2005
CDK1 and 2 may play redundant roles in early stages cell division. This reviews several papers looking for role for CDK1.
20
Which kinase dominant in normal cycles? Unknown. Mice wdeveloped normally, so the CDKs 1 and 2 seemed to act together in transitions.
21
Alignment of human CDK1 , cdk2, cdk4. Enzymes have distinct roles at different stages. CDK1 is essential for entry into mitosis. Enzymes are really similar. Thr 160 is regulatory Thr phosphorylated for CDKs to be active. Region near it is similar in all these kinases.
PSTAIRE Helix largely conserved.
22
Differences in activity come from differences in cyclin regulatory partners. A, B1,D1,E1 alignment. D1 and E are shorter. Much more variability in these sequences. Differences in structure make differences in function.
23
Functions:
Genes Dev. 2004: 18: 2699-711.
P107 and 130 Rb phosphorylation, liberates transcription factor to transcribe cyclin E, A, regulatory proteins, resulting in further expression of RB, E2F1, and enzymes involved in nucleic acid synthesis, DNA replication machinery.
Limited targets of kinase liberate a transcription factor controlling a variety of other factors.
E and A, phosphorylate RB at different sites to modify function of protein. P27 is inhibitory.
24
cdk activity regulates details in specific examples of cyclins and kinases- Will give copies of slides Monday.
CDK has to be paired with cyclin to have potential to be active.
CAK phosphorylates cyclin/cdk. P21 inhibits activity. Data shows when inhibitor is bound, some complexes are active, so the story is more complicated than we know right now.
25
Structural view of CDK2 with key features illustrated.
Kinases have N terminal beta sheet and helical domain at bottom. Active site is interface of these domains in middle in pocket. Regulatory element is PSTAIRE helix- cyclin binds on right and docks on face CDK2 to shove PSTAIRE in and reform active site. Thr phosphorylation is in loop that connects with helical lobe.
26
When cyclin A binds, shoves PSTAIRE in. Phospho transfer from ATP occurs.Cyclins interact with substrates.
31These slides may be misaligned.
Phosphorylation introduces negative charge, so basic residues in area are affected. They form pocket and rearrange complex due to phosphorylation. Opens area for substrate binding.
Tues- role of inhibitors and recent findings in field. Read text and paper. Ask questions Tues AM.
Kriwacki Cyclins II
Quick run-through of lecture 1:
At end of today will give review session. Nutshell: fundamental concepts of how cell cycle is regulated by CDKs and cyclins. Understand how key experiments were done in yeast and what significance of results were. 1989 cyclin paper- read and be able to describe from experimental and conceptual and significance perspectives. New paper in nature about the identity of 2 proteins phosphorylated by S phase CDK complex in yeast key in regulating DNA replication. Took 10 yrs to find kinase targets. We do not understand all of what the complexes are targeting. Look in January issue of Nature.
Specifically read and understand paper handed out today. Also look at protein degradation as a regulating mechanism. No-going-back type of regulation. Interplay between CDK complex. Understand how protein degradation is used to regulate cell cycle. Text and article by Kirchner hit key concepts. APC is an ubiquitination machine. Be able to describe key elements of a CDK that pertain to cyclin binding and conformational change in activation. Review in1999 JMB pavelitch is about structural results.
Main point of structural details- understand those.
Inhibitors of CDKs:
To activate, bind to cyclin and phosphorylate kinase. Levels of cyclins vary cyclically for control. If this were the only level of regulation, the cell could not prevent multiple rounds of division. Family of proteins called p16 act by binding to CDK 4 and 6 to affect entry into cell cycle at earliest stage. Cyclin D-CDK-4 is phosphorylated and phosphorylates Rb/E2F. Inhibiting CDK 4- prevents Rb phosphorylation and entry into S phase. P15,16,18,19 are CKIs.
p19 binds opposite side from PSTAIRE. It wedges in and locks N terminal lobe into open conformation to prevent binding of cyclin. P19 has 3 repeats of ankyrin motif, which is made up of 2 alpha helices and a beta hairpin.They stack together to form broad surface to form interface with CDK 6. Ankyrin repeat proteins are found in proteins involved in protein-protein interactions.
Lots done on biology of these.
P21 and p27 studied extensively since discovered in mid 90s. p27 inhibits most CDK/Cyclin complexes. IN cells, it promotes activity of CDK4 cyclin D. How? We do not know.
Activity of p27 is regulated by degradation through ubiquitination which is regulated by post-translational modification, specifically phosphorylation of p27.
When cells exit quiescence, they inherit high level of p27. Begins G1, but blocks progress. Needs to be reduced for crossing G1/S boundary. Ubiquitin ligase SCF/ Skp2 increase in mid G1 phase. This ubiquitin ligase targets p27 to reduce its levels so later complexes can be activated.
P27 levels reduced in most human cancers. Loss p27 deregulates G1/S checkpoint.
P27 bound to CDK2/CyclinA structure- binds like a molecular staple, one part to each subunit. C terminal regulatory domain does not interact w cyclin or kinase. Accessible for modification by enzymes. Activity of enzyme is required for phosphorylation p27, but p27 inhibits complex.
Structure function relationship p 11
Conserved domains have conserved structure function relationship. Linker helix domain is also conserved.
12
Domain 1, domain LH, domain 2 important.
Single turn at end is important to mechanism. 310 helix contains Tyr residue. Structure is highly dynamic. Flexibility plays a role in its function. 310 helix of p27 inserts into ATP binding procket preventing binding of ATP and substrate. Surface recognition by domain 1 is conserved in other cyclins in cell cycle. Helix has sequence MRAIL in cyclin A,B,D,E. Conserved binding site mediates specificity for p27.
What is conserved about linker is length.
Domain 2 wraps around side of CDK2 and positions 310 helix in active site for inhibition. P27 needs to interact with specific sites in order to inhibit.
Phosphorylation alters function of protein from inhibitor to activator and captive substrate of CDK2.
Domain 1 binds first, then forms basis of specificity.
What about the puzzle? How is p27 eliminated?
Tyrosine kinases can phosphorylate Tyr in 310 helix, causing local change to reactivate kinase to phosphorylate Thr residue and progress into cell cycle. Results tie Tyr kinase signalling into cell cycle.
Y88 sits in ATP binding pocket. Tail has to come close to active site once on a while for T187 to get phosphorylated.
NMR- 2D spectra for free p27 amide nitrogens and protons in peptide. Shifts cluster in the free state because the parts of the molecule average to have similar chemical environments due to the molecule’s flexibility.Right- single conformation with effects of neighbors spread out. Other proteins are not labelled, and are invisible. Most resonances can be labelled.Yellow domain 1, green domain 2, red 310 helix region. Right spectrum is non phosphorylated and reflects inhibitory conformation. Resonance changes are fingerprint of structure when bound to complex. Magnitude of changes shown in bar graph on p. 16
Same experiment with phosphorylated p27 on Y88 prepared in vitro- looked at same pair of spectra and plotted histogram- Pattern of chemical shift changes is same, except for Y88 in 310 helix region. No change free to bound when phosphorylated. Phosphorylation of Tyr causes portion inhibitor to be ejected from active site of complex. How can residue be phosphorylated by Tyr kinase when it is bound in ATP binding pocket? It can’t. Must occasionally swing out and get phosphorylated, then it cant go back in. It is naturally flexible.
Phosphorylation reactivates CDK2. Hypothesized that phosphorylation of the Tyr would promote phosphorylation on Thr. Demonstrated using kinase assay. Phosphorylation of the Thr depends on concentration of complex added to reaction with P23 labelled ATP.
Even if most molecules are bound, can still have small amount free kinase to cause reaction even without phosphorylation Tyr. Graphs illustrate levels phosphorylation. Bimolecular reaction. Tyr phosphorylation ejects helix, tail encounters active site CDK2, Thr phosphorylation (intracomplex reaction with inherent rate depending on how often tail encounters active site)- creates linear concentration dependence.
BcrAbl is Tyr kinase active in some leukemias, constitutively active. Promotes chronic myelogenous leukemia and acute lymphoblastic leukemia. This kinase act causes premature eliminates p27. Phosphorylation at early stage targets p27 for degradation. Lin kinase also does this.
Gleevec acts to target Bcr-Abl to make degradation p27 more normal. The Tyr phosphorylated structure may be a novel drug target.
Be able to answer how this mechanism ties post-translational modification and degradation together. This mechanism ties Tyr kinase signalling into cell cycle control.
Known that short segments of the c-terminal tail are recognized only when Thr is phosphorylated. Parts of CDK2/cyclinA also recognized. There are also several Ks in tail that are probably sites of ubiquitination. What residues ubiquitinated and how it happens mechanistically are not shown yet. Lots of regulation probably goes on that we don’t know about yet.
Review:
Details of CDK2 and features preserved in other CDKs.
Ubiquitination machinery slides page 21- not on exam, but covered elsewhere. SCF complex regulates E and D and p27. APC regulates later stages as covered in Kirschner paper.
Exam format- simple questions to describe some of topics pointed out as he went through today. May give words to include in essay on topic. READ ASSIGNED PAPERS!!
Scott Cancer Lectures
His notes are on Blackboard. They were good for the first one. Below are my notes for 2 and 3. He stressed studying his notes.
Cancer Notes 2
Terminology and definitions
People mix up words in practice often. It gets confusing. You have to understand a bit of histology. Skin is example.Skin normally has 10-15 layers above basal layer. As skin cells grow they differentiate and eventually slough off. 85% of human cancers derive from epithelial cells. With respect to cancer,this fact is important to understand. Any surface in contact with lumen or outside environment on outside of basement membrane is epithelium. Lungs, GI, salivary glands, liver, pancreas, part of thyroid may be epithelial. Kidney, uterus, included. Tissues that line these surface are epithelial. Cancers of epithelial tissues are called carcinomas. All others are sarcomas (brain, bone, blood vessels, cartilage, blood).
What can happen to an epithelium to lead to cancer?
Hyperplasia- defined in handout. Grows too fast. Does not mean it is cancer. Increased cell proliferation for increased number of cells. Can be normal. Physiological response that is reversible normally. If it goes on long enough tissues can proliferate even without stim. Give rise to cancer. Normal human body takes 10-20 years to get cancer.
Hyperplastic skin is thicker with ridges of basement membrane.
Hypertrophy
More size due to increase size of cells. Bigger cells. Example: lifting weights. Hypertrophy has nothing to do with cancer.
4 key terms to KNOW proliferation, differentiation, apoptosis, senescence.Senescence is process of normal cells losing ability to grow (losing proliferative potential) as you age. Hayflick discovered this concept as a tissue culture phenomenon. Cells divide 30-40 times, stop in G1. Quit growing. Telomerases came out of this. You lose chromosome ends due to loss of telomerase activity. Senescence is not related to differentiation as in neurons. They are terminally differentiated. Not related to telomerases. Senescence did not originally apply to real humans. Progeria and Werner’s syndrome (aging syndromes) led to applying senescence to people.
Metaplasia is primarily a differentiation term. It is an adaptation to stimuli in which one cell type substitutes for another to increase resistance to environmental stress. Lining of bronchs can change from ciliated epithelium to squamous cells due to smoking and/or pollution. Squamous tougher than ciliated. Changes differentiation.
More mixing up of proliferation, differentiation, apoptosis, senescence- more progress towrd cancer.
Dysplasia- screws up proliferation, differentiation, maybe apoptosis and senescence. Alterations in multiple genes, but not malignant. Is progressing towrd cancer. “Carcinoma in situ”= very close to cancer. Almost there. Severe dysplasia- cells dividing all the way to top instead of seeing clear layers differentiated cells.
Invasion is required for cancer. Must become invasive- some cells penetrate basement membrane. Cell crosses membrane and grows where it is not supposed to be-cancer.
A clone of cells in a severe dysplasia can learn to invade.
Tumor- swelling or mass-inflammation, cyst, clot, not necessarily cancer. Could be, or not. Tumor does not equal cancer.
Neoplasia- new growth. Can be quite benign.
Benign tumor- Growth localized.
Metastasis- spread to distant site.
Cancer- malignant, invasive.
Examples of terms for benign tumors- Can have benign or malignant tumors of all tissues. Non-epithelial tissues- oma means benign. Lipoma- benign fat tumor. See his notes for other names.
Epithelial benign growths- papillomas, adenomas, etc. OMA means benign.
Sarcoma is cancer of non-epithelial tissues. Carcinoma means malignant tumor. Adenocarcinoma,
Exception to terminology- melanoma is often term used for malignant pigmented skin cell tumor. Some cancers are named after a famous doctor. Hodgkin’s- malignant tumor of the lymph cells.
Cancer is disease of clonal evolution. Cell clone evolution. What does that mean?See chart at top of page. Field concept of carcinogenesis says that if you expose agroup of cells in a location to cancer causing agents, cells will undergo mutations randomly.
Aneuploidy- abnormal chromosome number. Too many or few. 92 in humans is tetraploidy. Aneuploidy is inappropriate sorting. 95% of cancers are aneuploid. Many premalignant lesions are also aneuploid. Chromosomal imbalance- no mutation required.
Probability is that cells underdosed with chromosomes will die. Overdosed may tend to live. Survival in aneuploidy above 46. Early on, some cells become aneuploid. These events may not happen in the order on chart, so don’t take it as a sequence. But these 5 things happen in course of early cancer. Anything that messes with mitotic spindle (drugs, estrogen, etc) can cause aneuploidy.
Lesion in proliferation gene- mutate oncogene controlling cell cycle and G1 checkpoint does not work. Could screw up any point of cell cycle. Must hit all 5 categories to get cancer.
Mutate differentiation genes- Cell differentiates, malignancy chance slim. Not proliferating if further along in differentiation.
Mess up telomerase- cells no longer senesce. Mess up apoptosis- becomes invasive and malignant.
This is field concept. Clones have different hits in different functions. One may become cancer. Keep zapping, may get mutations in others to make cancer. One clone becomes malignant first. If you sequence each as they become invasive, each clone is different with different mutational profiles. Why is every cancer different? There are lots of genes in the above pathways.
Clonal selection based on field concept- selection for an advantageous trait. Survival advantage. If B is mutated in a way that does not confer advantage, it may die. Each of the categories in the chart fits this theory. Philosophy is idea that “field” is exposed to carcinogens, and clonal evolution and selection goes on. Over years, right hits occur.
Next step is ability to be invasive (selected for ), then metastasis (other set of genes).
Tomorrow- mechanisms of invasion and metastasis.
Cancer stem cell concept suggests that normal stem cells differentiate into cancer. Basal cells with proliferative potential may beome cancer, but skin stem cells sit at certain spots. They are prime targets of carcinogens. Every cancer starts from one cell, but that is a misnomer. The end cells after clonal selection are quite different from the original stem cell. Within the evolving lesion are multiple cells, but one makes it. Cancer is monoclonal disease, but premalignant tumor goes through many selection processes. Variations of this model will probably be correct in the end.
Scott III: Metastasis and Invasion
Multistep Clonal Selection Concept of Carcinogenesis
1. enabling event-genomic instability-aneuploidy
2. preneoplasia:Selection of Cell clones with a proliferation and survival advantage
a.proliferation control autonomy
b. resistance to terminal differentiation
c. resistance to senescence
d. resistance to apoptosis
3. malignant conversion: Development of invasive cancer cell clones
a. disruption of cell-cell interactions
b. production of metalloproteinases and related enzymes that disrupt basement membranes
c. increased cell motility
d. increased angiogenesis
4. Cancer dissemination
a. ability to invade vascular or lymphatic channels
b. potential survival in atypical microenvironments
c. resistance to immune or traumatic activity
d. ability to establish distant cancer foci
e. potential to proliferate and survive at metastatic sites
Advantage of this concept is its explanation of cancer in real world. Takes 20 yrs to go from normal to cancer. Stages along the way happen, and the selection takes time. This theory has a lot of data behind it and makes sense in real people.
Yesterday was premalignant steps. No certain order, but the evidence indicates that aneuploidy occurs early. Human genome is resistant to mutation and had DNA repair mechanisms. If there have to be 140 mutations in 93 genes to make cancer in cell with all those repair mechanisms- probability small unless control is released somehow. Hopkins group looked at first malignant lesion they could study. Smallest polyps called cancer, earliest common characteristic was aneuploidy and genetic instability, plus some of the characteristics in the above list.
1 and 2 above can be characteristics of carcinomas. List above does not apply to leukemias. Leukemias are only 5-7% of cancers.
Dysplastic lesions contain between 4 and 8 different subclones mixed with normal cells. That does not mean there are not more- we have to be able to detect them.
For carconoma to become malignant, must become invasive and metastatic.
What happens during invasion?
Clone loses ability to interact with neighbors- intercellular interaction lost. Cell accentuates interaction with fibronectins and other molecules in basement membrane. Then develops mutations in genes to digest basement membrane, then cell migrates across and starts to invade. This is textbook general process. Problem: What is selective advantage of invading? E-cadherin mutated so cell adhesion is messed up. 20-30 genes have to be mutated for it to get throgh membrane. New discoveries shed light on it. Cells under basement membrane are in stroma. Some of these make factors that tell skin cells to stay up where they belong. The basement membrane is made by eipthelial cells and stromal cells in cooperation, but it is not some kind of lead barrier. IN normal conditions epithelial cells stay where they belong due to signals. This is not all worked out yet.Process becomes more complex. What if a wound creates a hole? Basement membrane is not a magic barrier. Lots of companies are developing drugs and treatments to help wound healing.
Application to cancer-A clone invades, basement membrane looks defective. Actually some of the stromal cells may be mutated and not working right. Cancer may be more than defects in just the cancer cells- may be defects in other cells as well. Clonal selection may not just be for clone, but how clone responds to signal as well. There may be a master switch enabling cells to “hear” signals from other cells- regulation of communication may be key to invasion.
Real lesion- melanoma are embryological descendants of the same ancestors as brain cells. Some call them melanosarcomas. Malignant melanomas are classic invaders. They invade fast. Further it invades, more likely it is to metastasize or spread.
Metastasis
Cancer cells are not all metastatic. All carcinomas are invasive, not metastatic. Not all invasive cells are malignant- immune cells can invade other tissues normally. Lymphocytes, granulocytes, placenta are normally invasive, not cancerous.
All carcinomas are invasive-true.
Cell must be invasive to be malignant, but not necessarily metastatic.
Metastasis means cells can spread to other regions without being in contact with primary tumor. Spreads to distant site, losing contact with primary tumor.
Example of normal metastasis- trophoblasts- some pregnant women placental trophoblasts can be in lung from uterus. Don’t cause cancer, go away after pregnancy. All definitions are not big generalizations. There are exceptions to rules.
Modes metastasis:
1. lymphatic-common
2. Hemotogenous- these 2 are most common. When cancer cells spread hemotogenously- spread most commonly through veins. Arteries have thick muscular walls and veins do not. Mechanically easier to get through vein. Artery will hit capillary bed and tumor could get stuck. No gates like that for veins. Top 2 here most common.
Veins go to lung next. 1st capillary bed is in lung in path from hand. Cells can get stuck in lung. Lymphatic spread- hits lymph nodes. Breast cancer – look at lymph nodes to see spread. Goes to axilla.Any breast cancer in lymph nodes- get chemotherapy.
3. Interstitial complicated
4. gravity through”body spaces” Brain through CNS to spine. Liver to perineum to pelvis.
5. Iatrogenic-induced by doctor. Doctor gets cells on glove when removing cancer and spreads them to other sites.
Cancer can be primary(skin), secondary(lung), tertiary (brain)
Key to metastasis- data shows that if you have tumor size of golf ball, it sheds 1 million cells per day into blood. They mostly die. What happens? Clonal selection. Invasive cells invade vein, cancer cells shed to lung or brain- must survive multistep process to travel, stick, and grow in new environment. Metastatic cells are subclone with different mutation profile.
Primary cancer- grows, becomes vascularized, invades, get in vein in little clumps (intravasate),detach, embolize through veins, arrest at new site, extravasation to invade basement membrane of capillary, grow in new site. One primary tumor can yield a variety of subclones with different metastatic potential.
Take high one. Could reproduce low middle and high after passage through 20 cultures. Genomic instability yields a lot of mutation. Normal cells are genetically stable.
Cancers of different types prefer different places to go. Renal to thyroid or bone. Prostate carcinoma likes arm bone. Breast cancer- lymph nodes, adrenals, bone.
When you treat a cancer, you do clonal selection for cells that are resistant to therapeutic agents. To avoid resistance, people are treated with cocktails these days.
Cancer Notes 2
Terminology and definitions
People mix up words in practice often. It gets confusing. You have to understand a bit of histology. Skin is example.Skin normally has 10-15 layers above basal layer. As skin cells grow they differentiate and eventually slough off. 85% of human cancers derive from epithelial cells. With respect to cancer,this fact is important to understand. Any surface in contact with lumen or outside environment on outside of basement membrane is epithelium. Lungs, GI, salivary glands, liver, pancreas, part of thyroid may be epithelial. Kidney, uterus, included. Tissues that line these surface are epithelial. Cancers of epithelial tissues are called carcinomas. All others are sarcomas (brain, bone, blood vessels, cartilage, blood).
What can happen to an epithelium to lead to cancer?
Hyperplasia- defined in handout. Grows too fast. Does not mean it is cancer. Increased cell proliferation for increased number of cells. Can be normal. Physiological response that is reversible normally. If it goes on long enough tissues can proliferate even without stim. Give rise to cancer. Normal human body takes 10-20 years to get cancer.
Hyperplastic skin is thicker with ridges of basement membrane.
Hypertrophy
More size due to increase size of cells. Bigger cells. Example: lifting weights. Hypertrophy has nothing to do with cancer.
4 key terms to KNOW proliferation, differentiation, apoptosis, senescence.Senescence is process of normal cells losing ability to grow (losing proliferative potential) as you age. Hayflick discovered this concept as a tissue culture phenomenon. Cells divide 30-40 times, stop in G1. Quit growing. Telomerases came out of this. You lose chromosome ends due to loss of telomerase activity. Senescence is not related to differentiation as in neurons. They are terminally differentiated. Not related to telomerases. Senescence did not originally apply to real humans. Progeria and Werner’s syndrome (aging syndromes) led to applying senescence to people.
Metaplasia is primarily a differentiation term. It is an adaptation to stimuli in which one cell type substitutes for another to increase resistance to environmental stress. Lining of bronchs can change from ciliated epithelium to squamous cells due to smoking and/or pollution. Squamous tougher than ciliated. Changes differentiation.
More mixing up of proliferation, differentiation, apoptosis, senescence- more progress towrd cancer.
Dysplasia- screws up proliferation, differentiation, maybe apoptosis and senescence. Alterations in multiple genes, but not malignant. Is progressing towrd cancer. “Carcinoma in situ”= very close to cancer. Almost there. Severe dysplasia- cells dividing all the way to top instead of seeing clear layers differentiated cells.
Invasion is required for cancer. Must become invasive- some cells penetrate basement membrane. Cell crosses membrane and grows where it is not supposed to be-cancer.
A clone of cells in a severe dysplasia can learn to invade.
Tumor- swelling or mass-inflammation, cyst, clot, not necessarily cancer. Could be, or not. Tumor does not equal cancer.
Neoplasia- new growth. Can be quite benign.
Benign tumor- Growth localized.
Metastasis- spread to distant site.
Cancer- malignant, invasive.
Examples of terms for benign tumors- Can have benign or malignant tumors of all tissues. Non-epithelial tissues- oma means benign. Lipoma- benign fat tumor. See his notes for other names.
Epithelial benign growths- papillomas, adenomas, etc. OMA means benign.
Sarcoma is cancer of non-epithelial tissues. Carcinoma means malignant tumor. Adenocarcinoma,
Exception to terminology- melanoma is often term used for malignant pigmented skin cell tumor. Some cancers are named after a famous doctor. Hodgkin’s- malignant tumor of the lymph cells.
Cancer is disease of clonal evolution. Cell clone evolution. What does that mean?See chart at top of page. Field concept of carcinogenesis says that if you expose agroup of cells in a location to cancer causing agents, cells will undergo mutations randomly.
Aneuploidy- abnormal chromosome number. Too many or few. 92 in humans is tetraploidy. Aneuploidy is inappropriate sorting. 95% of cancers are aneuploid. Many premalignant lesions are also aneuploid. Chromosomal imbalance- no mutation required.
Probability is that cells underdosed with chromosomes will die. Overdosed may tend to live. Survival in aneuploidy above 46. Early on, some cells become aneuploid. These events may not happen in the order on chart, so don’t take it as a sequence. But these 5 things happen in course of early cancer. Anything that messes with mitotic spindle (drugs, estrogen, etc) can cause aneuploidy.
Lesion in proliferation gene- mutate oncogene controlling cell cycle and G1 checkpoint does not work. Could screw up any point of cell cycle. Must hit all 5 categories to get cancer.
Mutate differentiation genes- Cell differentiates, malignancy chance slim. Not proliferating if further along in differentiation.
Mess up telomerase- cells no longer senesce. Mess up apoptosis- becomes invasive and malignant.
This is field concept. Clones have different hits in different functions. One may become cancer. Keep zapping, may get mutations in others to make cancer. One clone becomes malignant first. If you sequence each as they become invasive, each clone is different with different mutational profiles. Why is every cancer different? There are lots of genes in the above pathways.
Clonal selection based on field concept- selection for an advantageous trait. Survival advantage. If B is mutated in a way that does not confer advantage, it may die. Each of the categories in the chart fits this theory. Philosophy is idea that “field” is exposed to carcinogens, and clonal evolution and selection goes on. Over years, right hits occur.
Next step is ability to be invasive (selected for ), then metastasis (other set of genes).
Tomorrow- mechanisms of invasion and metastasis.
Cancer stem cell concept suggests that normal stem cells differentiate into cancer. Basal cells with proliferative potential may beome cancer, but skin stem cells sit at certain spots. They are prime targets of carcinogens. Every cancer starts from one cell, but that is a misnomer. The end cells after clonal selection are quite different from the original stem cell. Within the evolving lesion are multiple cells, but one makes it. Cancer is monoclonal disease, but premalignant tumor goes through many selection processes. Variations of this model will probably be correct in the end.
Scott III: Metastasis and Invasion
Multistep Clonal Selection Concept of Carcinogenesis
1. enabling event-genomic instability-aneuploidy
2. preneoplasia:Selection of Cell clones with a proliferation and survival advantage
a.proliferation control autonomy
b. resistance to terminal differentiation
c. resistance to senescence
d. resistance to apoptosis
3. malignant conversion: Development of invasive cancer cell clones
a. disruption of cell-cell interactions
b. production of metalloproteinases and related enzymes that disrupt basement membranes
c. increased cell motility
d. increased angiogenesis
4. Cancer dissemination
a. ability to invade vascular or lymphatic channels
b. potential survival in atypical microenvironments
c. resistance to immune or traumatic activity
d. ability to establish distant cancer foci
e. potential to proliferate and survive at metastatic sites
Advantage of this concept is its explanation of cancer in real world. Takes 20 yrs to go from normal to cancer. Stages along the way happen, and the selection takes time. This theory has a lot of data behind it and makes sense in real people.
Yesterday was premalignant steps. No certain order, but the evidence indicates that aneuploidy occurs early. Human genome is resistant to mutation and had DNA repair mechanisms. If there have to be 140 mutations in 93 genes to make cancer in cell with all those repair mechanisms- probability small unless control is released somehow. Hopkins group looked at first malignant lesion they could study. Smallest polyps called cancer, earliest common characteristic was aneuploidy and genetic instability, plus some of the characteristics in the above list.
1 and 2 above can be characteristics of carcinomas. List above does not apply to leukemias. Leukemias are only 5-7% of cancers.
Dysplastic lesions contain between 4 and 8 different subclones mixed with normal cells. That does not mean there are not more- we have to be able to detect them.
For carconoma to become malignant, must become invasive and metastatic.
What happens during invasion?
Clone loses ability to interact with neighbors- intercellular interaction lost. Cell accentuates interaction with fibronectins and other molecules in basement membrane. Then develops mutations in genes to digest basement membrane, then cell migrates across and starts to invade. This is textbook general process. Problem: What is selective advantage of invading? E-cadherin mutated so cell adhesion is messed up. 20-30 genes have to be mutated for it to get throgh membrane. New discoveries shed light on it. Cells under basement membrane are in stroma. Some of these make factors that tell skin cells to stay up where they belong. The basement membrane is made by eipthelial cells and stromal cells in cooperation, but it is not some kind of lead barrier. IN normal conditions epithelial cells stay where they belong due to signals. This is not all worked out yet.Process becomes more complex. What if a wound creates a hole? Basement membrane is not a magic barrier. Lots of companies are developing drugs and treatments to help wound healing.
Application to cancer-A clone invades, basement membrane looks defective. Actually some of the stromal cells may be mutated and not working right. Cancer may be more than defects in just the cancer cells- may be defects in other cells as well. Clonal selection may not just be for clone, but how clone responds to signal as well. There may be a master switch enabling cells to “hear” signals from other cells- regulation of communication may be key to invasion.
Real lesion- melanoma are embryological descendants of the same ancestors as brain cells. Some call them melanosarcomas. Malignant melanomas are classic invaders. They invade fast. Further it invades, more likely it is to metastasize or spread.
Metastasis
Cancer cells are not all metastatic. All carcinomas are invasive, not metastatic. Not all invasive cells are malignant- immune cells can invade other tissues normally. Lymphocytes, granulocytes, placenta are normally invasive, not cancerous.
All carcinomas are invasive-true.
Cell must be invasive to be malignant, but not necessarily metastatic.
Metastasis means cells can spread to other regions without being in contact with primary tumor. Spreads to distant site, losing contact with primary tumor.
Example of normal metastasis- trophoblasts- some pregnant women placental trophoblasts can be in lung from uterus. Don’t cause cancer, go away after pregnancy. All definitions are not big generalizations. There are exceptions to rules.
Modes metastasis:
1. lymphatic-common
2. Hemotogenous- these 2 are most common. When cancer cells spread hemotogenously- spread most commonly through veins. Arteries have thick muscular walls and veins do not. Mechanically easier to get through vein. Artery will hit capillary bed and tumor could get stuck. No gates like that for veins. Top 2 here most common.
Veins go to lung next. 1st capillary bed is in lung in path from hand. Cells can get stuck in lung. Lymphatic spread- hits lymph nodes. Breast cancer – look at lymph nodes to see spread. Goes to axilla.Any breast cancer in lymph nodes- get chemotherapy.
3. Interstitial complicated
4. gravity through”body spaces” Brain through CNS to spine. Liver to perineum to pelvis.
5. Iatrogenic-induced by doctor. Doctor gets cells on glove when removing cancer and spreads them to other sites.
Cancer can be primary(skin), secondary(lung), tertiary (brain)
Key to metastasis- data shows that if you have tumor size of golf ball, it sheds 1 million cells per day into blood. They mostly die. What happens? Clonal selection. Invasive cells invade vein, cancer cells shed to lung or brain- must survive multistep process to travel, stick, and grow in new environment. Metastatic cells are subclone with different mutation profile.
Primary cancer- grows, becomes vascularized, invades, get in vein in little clumps (intravasate),detach, embolize through veins, arrest at new site, extravasation to invade basement membrane of capillary, grow in new site. One primary tumor can yield a variety of subclones with different metastatic potential.
Take high one. Could reproduce low middle and high after passage through 20 cultures. Genomic instability yields a lot of mutation. Normal cells are genetically stable.
Cancers of different types prefer different places to go. Renal to thyroid or bone. Prostate carcinoma likes arm bone. Breast cancer- lymph nodes, adrenals, bone.
When you treat a cancer, you do clonal selection for cells that are resistant to therapeutic agents. To avoid resistance, people are treated with cocktails these days.
Saturday, January 20, 2007
Notes past few days
Drs Dohan and Scott provided their own notes in class and say anything they ask will come from there. Here are notes for Kriwacki, though he did not provide all his slides and they are not posted yet. The articles cited in the notes below from Cell Cycle and Genes and Development are freely available, and they pretty well cover the slides we did not get. He handed out the Science article in class and STRONGLY URGED us to read and understand it and the Lodish assigned reading for the exam.
Kriwacki Cell Cycle
Proteins that Regulate Cell Cycle
Read the Kirschner paper about fundamental role of protein degradation in regulation of the cell cycle. Concepts remain central to how aspects of cell cycle are regulated.
Slides are digitally intensive.
2
Discoveries were made in yeast due to ease of manipulation.
Cdk inhibition keeps kinases in check.
3
Cell cycle in S. Cerevisiae- from text assignment (Lodish 874-890). START is where decisions are made and boundaries crossed to replicate DNA and undergo budding process.
4
Screened for genes to complement temperature sensitive mutants. CDC 28 was one of 1st CDK s discovered.
Graphic shows cells harboring a ts mutation. These cells grow at 25 degrees C, but raise the temperature to 37 degrees and they exhibit defects with no budding, even with lots of nutrients. Library of genes in yeast was screened for effects of plasmids in helping yeast to bud.CDC28 was kinase.
5
Mechanisms common to all higher organisms. Cancer-cells lose ability to control proliferation.
6
Reed late 1980s discovered several cyclin regulatory partners. Similar experiment to earlier ones- factors regulate entry into S phase specifically. Mutation affected interaction of the kinase with its cyclin regulatory partner. Investigators did not know what the regulatory machinery consisted of.
7
CDK has regulatory factor bound in order for colonies to form in wild type. In ts cells, the mutant CDC28 protein could only bind to regulatory partner at low temperature. Used library in plasmids to express gene of plasmid at high levels. Able to shift equilibrium to bound and active state.
7
CLN3
Approach a little different- screened for plasmid to rescue defect in budding, but used inducible promoter in presence of low glucose. Expressed in absence of glucose. Used FACS analysis of yeast cells to determine whether expressing the gene rescued defect, The cells are labelled with fluorescence tag proportional to the amount of DNA in cell.G1=1 genome. G2= 2 copies. Cell number is on the on y axis. Normal disribution (distribution under normal conditions with normal cells) on left wih 2 peaks. With cyclin vector- not in presence of glucose, increase in peak for actively dividing cells. With glucose-2 traces similar. In C, Yeast defective in G1S phase cyclins with high glucose get stuck in G1. Without- could proceed to G2 with exogenous cyclin, albeit with lower level of replication due to using sick cells.
8
Role protein degradation in cell cycle regulation
Inhibitor of CDK is regulated by phosphorylation, which signals protein to be ubiquitinated, causing degradation and relieving inhibition of CDK complex. Mechanism illustrates the way the activity of one set CDK complexes is required to activate another set CDK complexes. One aspect of sequence of events- protein degradation is not easily reversible. Nomenclature is horrible. Clb 5 and 6 are S phase regulation. Inhibitor is Sic1. Cln 1 and 2 phosphorylate Sic1.
For exam, understand concepts, but refer to cyclins and CDKs by general type.
9
Diagram puts together results over years. Illustrates way activity of CDK complexes contributes to series of events taking cells into mitosis.
Yeast- only one CDK.Higher organisms- more kinases, more target proteins.
Cln3-CDK phosphorylates a transcription factor that upregulates cln-1 and cln-2 transcription, to pair with CDK to phosphorylate different transcription factors, etc resulting in sequential activation of transcription. Can also trigger degradation to eliminate activity after time needed or relieve repression. CDK has different partners.
10
Meshing of gears allowing cells to progress through cell cycle unidirectionally. Once cell enters mitosis, no longer needs active signal. Degrades regulatory subunit, cyclin. Whether to ubiquitinate cyclin depends on G1 CDK complex. Read this in text several times and read review.
11-12
Kirschner’s slides
This is more detail on what we have already covered.
13
APC pathway: cells need to have properly set up metaphase plate with appropriate pairing, etc, then go into anaphase. APC targets cyclin B to relieve inhibtion of exit from mitosis. Inactivated CDKs cause cell to sit in G1 until another cell division.
14
overview
15
Regulation by post-translational modification can be reversed, but regulation by proteolysis cannot.
READ the PAPER for the EXAM!!
Know differences between cyclin types, but not picky names.
16
Firing of replication complexes from origins of replication:
In order for replication to occur, must assemble regulatory factors. S phase CDK and DDK phosphorylate proteins involved in pre-replication complex. Phosphorylation signals replication to occur, disassembly of the pre-replication complex, and other factors to come in. RPA binds ssDNA exposed by helicases. Replication complete- origins get loaded w original complex, but in absence of CDK will not become active.
17
Mammalian cyclins and CDKs- concepts same, player different.
We have variety of cyclins and CDKs. CDK activates E2F, which phosphorylates retinoblastoma protein, frees E2F, causes transcription cyclins E and A. Follow cyclins through chart.
18
last page handout.
19
Cell Cycle 4:11, 1491-1494, Nov 2005
CDK1 and 2 may play redundant roles in early stages cell division. This reviews several papers looking for role for CDK1.
20
Which kinase dominant in normal cycles? Unknown. Mice wdeveloped normally, so the CDKs 1 and 2 seemed to act together in transitions.
21
Alignment of human CDK1 , cdk2, cdk4. Enzymes have distinct roles at different stages. CDK1 is essential for entry into mitosis. Enzymes are really similar. Thr 160 is regulatory Thr phosphorylated for CDKs to be active. Region near it is similar in all these kinases.
PSTAIRE Helix largely conserved.
22
Differences in activity come from differences in cyclin regulatory partners. A, B1,D1,E1 alignment. D1 and E are shorter. Much more variability in these sequences. Differences in structure make differences in function.
23
Functions:
Genes Dev. 2004: 18: 2699-711.
P107 and 130 Rb phosphorylation, liberates transcription factor to transcribe cyclin E, A, regulatory proteins, resulting in further expression of RB, E2F1, and enzymes involved in nucleic acid synthesis, DNA replication machinery.
Limited targets of kinase liberate a transcription factor controlling a variety of other factors.
E and A, phosphorylate RB at different sites to modify function of protein. P27 is inhibitory.
24
cdk activity regulates details in specific examples of cyclins and kinases- Will give copies of slides Monday.
CDK has to be paired with cyclin to have potential to be active.
CAK phosphorylates cyclin/cdk. P21 inhibits activity. Data shows when inhibitor is bound, some complexes are active, so the story is more complicated than we know right now.
25
Structural view of CDK2 with key features illustrated.
Kinases have N terminal beta sheet and helical domain at bottom. Active site is interface of these domains in middle in pocket. Regulatory element is PSTAIRE helix- cyclin binds on right and docks on face CDK2 to shove PSTAIRE in and reform active site. Thr phosphorylation is in loop that connects with helical lobe.
26
When cyclin A binds, shoves PSTAIRE in. Phospho transfer from ATP occurs.Cyclins interact with substrates.
31These slides may be misaligned.
Phosphorylation introduces negative charge, so basic residues in area are affected. They form pocket and rearrange complex due to phosphorylation. Opens area for substrate binding.
Tues- role of inhibitors and recent findings in field. Read text and paper. Ask questions Tues AM.
Kriwacki Cell Cycle
Proteins that Regulate Cell Cycle
Read the Kirschner paper about fundamental role of protein degradation in regulation of the cell cycle. Concepts remain central to how aspects of cell cycle are regulated.
Slides are digitally intensive.
2
Discoveries were made in yeast due to ease of manipulation.
Cdk inhibition keeps kinases in check.
3
Cell cycle in S. Cerevisiae- from text assignment (Lodish 874-890). START is where decisions are made and boundaries crossed to replicate DNA and undergo budding process.
4
Screened for genes to complement temperature sensitive mutants. CDC 28 was one of 1st CDK s discovered.
Graphic shows cells harboring a ts mutation. These cells grow at 25 degrees C, but raise the temperature to 37 degrees and they exhibit defects with no budding, even with lots of nutrients. Library of genes in yeast was screened for effects of plasmids in helping yeast to bud.CDC28 was kinase.
5
Mechanisms common to all higher organisms. Cancer-cells lose ability to control proliferation.
6
Reed late 1980s discovered several cyclin regulatory partners. Similar experiment to earlier ones- factors regulate entry into S phase specifically. Mutation affected interaction of the kinase with its cyclin regulatory partner. Investigators did not know what the regulatory machinery consisted of.
7
CDK has regulatory factor bound in order for colonies to form in wild type. In ts cells, the mutant CDC28 protein could only bind to regulatory partner at low temperature. Used library in plasmids to express gene of plasmid at high levels. Able to shift equilibrium to bound and active state.
7
CLN3
Approach a little different- screened for plasmid to rescue defect in budding, but used inducible promoter in presence of low glucose. Expressed in absence of glucose. Used FACS analysis of yeast cells to determine whether expressing the gene rescued defect, The cells are labelled with fluorescence tag proportional to the amount of DNA in cell.G1=1 genome. G2= 2 copies. Cell number is on the on y axis. Normal disribution (distribution under normal conditions with normal cells) on left wih 2 peaks. With cyclin vector- not in presence of glucose, increase in peak for actively dividing cells. With glucose-2 traces similar. In C, Yeast defective in G1S phase cyclins with high glucose get stuck in G1. Without- could proceed to G2 with exogenous cyclin, albeit with lower level of replication due to using sick cells.
8
Role protein degradation in cell cycle regulation
Inhibitor of CDK is regulated by phosphorylation, which signals protein to be ubiquitinated, causing degradation and relieving inhibition of CDK complex. Mechanism illustrates the way the activity of one set CDK complexes is required to activate another set CDK complexes. One aspect of sequence of events- protein degradation is not easily reversible. Nomenclature is horrible. Clb 5 and 6 are S phase regulation. Inhibitor is Sic1. Cln 1 and 2 phosphorylate Sic1.
For exam, understand concepts, but refer to cyclins and CDKs by general type.
9
Diagram puts together results over years. Illustrates way activity of CDK complexes contributes to series of events taking cells into mitosis.
Yeast- only one CDK.Higher organisms- more kinases, more target proteins.
Cln3-CDK phosphorylates a transcription factor that upregulates cln-1 and cln-2 transcription, to pair with CDK to phosphorylate different transcription factors, etc resulting in sequential activation of transcription. Can also trigger degradation to eliminate activity after time needed or relieve repression. CDK has different partners.
10
Meshing of gears allowing cells to progress through cell cycle unidirectionally. Once cell enters mitosis, no longer needs active signal. Degrades regulatory subunit, cyclin. Whether to ubiquitinate cyclin depends on G1 CDK complex. Read this in text several times and read review.
11-12
Kirschner’s slides
This is more detail on what we have already covered.
13
APC pathway: cells need to have properly set up metaphase plate with appropriate pairing, etc, then go into anaphase. APC targets cyclin B to relieve inhibtion of exit from mitosis. Inactivated CDKs cause cell to sit in G1 until another cell division.
14
overview
15
Regulation by post-translational modification can be reversed, but regulation by proteolysis cannot.
READ the PAPER for the EXAM!!
Know differences between cyclin types, but not picky names.
16
Firing of replication complexes from origins of replication:
In order for replication to occur, must assemble regulatory factors. S phase CDK and DDK phosphorylate proteins involved in pre-replication complex. Phosphorylation signals replication to occur, disassembly of the pre-replication complex, and other factors to come in. RPA binds ssDNA exposed by helicases. Replication complete- origins get loaded w original complex, but in absence of CDK will not become active.
17
Mammalian cyclins and CDKs- concepts same, player different.
We have variety of cyclins and CDKs. CDK activates E2F, which phosphorylates retinoblastoma protein, frees E2F, causes transcription cyclins E and A. Follow cyclins through chart.
18
last page handout.
19
Cell Cycle 4:11, 1491-1494, Nov 2005
CDK1 and 2 may play redundant roles in early stages cell division. This reviews several papers looking for role for CDK1.
20
Which kinase dominant in normal cycles? Unknown. Mice wdeveloped normally, so the CDKs 1 and 2 seemed to act together in transitions.
21
Alignment of human CDK1 , cdk2, cdk4. Enzymes have distinct roles at different stages. CDK1 is essential for entry into mitosis. Enzymes are really similar. Thr 160 is regulatory Thr phosphorylated for CDKs to be active. Region near it is similar in all these kinases.
PSTAIRE Helix largely conserved.
22
Differences in activity come from differences in cyclin regulatory partners. A, B1,D1,E1 alignment. D1 and E are shorter. Much more variability in these sequences. Differences in structure make differences in function.
23
Functions:
Genes Dev. 2004: 18: 2699-711.
P107 and 130 Rb phosphorylation, liberates transcription factor to transcribe cyclin E, A, regulatory proteins, resulting in further expression of RB, E2F1, and enzymes involved in nucleic acid synthesis, DNA replication machinery.
Limited targets of kinase liberate a transcription factor controlling a variety of other factors.
E and A, phosphorylate RB at different sites to modify function of protein. P27 is inhibitory.
24
cdk activity regulates details in specific examples of cyclins and kinases- Will give copies of slides Monday.
CDK has to be paired with cyclin to have potential to be active.
CAK phosphorylates cyclin/cdk. P21 inhibits activity. Data shows when inhibitor is bound, some complexes are active, so the story is more complicated than we know right now.
25
Structural view of CDK2 with key features illustrated.
Kinases have N terminal beta sheet and helical domain at bottom. Active site is interface of these domains in middle in pocket. Regulatory element is PSTAIRE helix- cyclin binds on right and docks on face CDK2 to shove PSTAIRE in and reform active site. Thr phosphorylation is in loop that connects with helical lobe.
26
When cyclin A binds, shoves PSTAIRE in. Phospho transfer from ATP occurs.Cyclins interact with substrates.
31These slides may be misaligned.
Phosphorylation introduces negative charge, so basic residues in area are affected. They form pocket and rearrange complex due to phosphorylation. Opens area for substrate binding.
Tues- role of inhibitors and recent findings in field. Read text and paper. Ask questions Tues AM.
Wednesday, January 17, 2007
Coagulation Notes
Gregory Tetrault
These notes correlate to the slides in the powerpoint lecture. Handout in class follows slides as well.
Handouts, etc. on Blackboard.
Not too many notes to take.
2
Clinical pathology covers all testing and blood banking.
Hemoostasis-regulation of blood volume inside you. Hemodynamics is about blood flow.
Sepsis- one of main casuses of death is inappropriate coagulation in all capillaries of body triggered by bacterial toxin.
3
Objectives
(topics in handout file)
Many homeostatic systems have multiple checks and balances.
4
Abbreviated schematic of major parts of coagulation cascade. Intrinsic pathway statrts at endothelial surface. Thrombin reacts there. Factor 11 converts to little a activated factor. Little a form is smaller because a part is cleaved off. Often you get 2-3 cleavage products, one of which is active product.
5
Clotting factors cleave proteins. A cascade of enzymatic reactions occurs. Each curved arrow is an enzymatic reaction. At end protein fibrinogen cleaved to fibrin, crosslinked, stable clot forms. Not crosslink, clot will not hold and washes away.
6
important facts- Many steps need free ionized Ca. Without Ca, clotting does not occur. Why doesn’t donated blood clot? Bag contains citrate to chelate Ca. EDTA or citrate can be used when drawing blood. Binds Ca.
Intrinsic pathway needs HMWK and prekallikrein(not a proteolytic enzyme).
Platelets are part of regular clotting. They can form plugs to block leakage and can release phospholipids. If they do not work, cannot prevent any kind of bleeding.
Some active factors trigger formation of anticoagulants as part of feedback regulation.
8
Every clot triggering event is damage to epithelial layer of blood vessel. Exposure of collagen activates factor XII, Tissue factor can leak from damaged tissue like skin. Factor 11a circulates. Kallikrein stays at injury site in complex.
9
HMWK acts as anchor. XIa cleaves IX to IXa in a Ca dependent way.
10
Factors numbered in order of discovery, not function.
11
Always small amount of thrombin in plasma.
13
Damage to subendothelial cell membranes- tissue factor without collagen and HMWK activates extrinsic pathway. Can trigger boh pathways activating at same time.
15
Common pathway
Clotting factors trigger more and more activation as you go along.
16
Thrombin action.
XIIIa catalyses crosslinking monomers.
When thrombin cleaves receptor on platelet, causes aggregation. Degranulation provide phospholipids.
17
Photomicrograph shows early microscopic clot with platelets in fibrin mesh.
18
Natural anticoagulantss prevent runaway clotting- do not work perfectly.
19
Once damage is patched, need clot to dissolve. Otherwise would have permanent clots and reduced function.
Can run out of fibrinogen. Uncontrolled clotting can result in uncontrolled bleeding from other sites.
20
Heparin used to prevent unwanted clotting, but almost never used outside hospital. Must be injected or given intravenously.Potentiates Antithrombin III. Easy to overdose. Must be monitored carefully. There is a reversing drug- polycation binds to heparin to deactivate.
21
Inhibits here.
22
heparin cofactor II is native inhibitor of clotting. Inhibits thrombin only. Binds thrombin and physically blocks active site. Heparan sulfate and dermatan sulfate are GAGs or acid mucopolysaccharides on cell membranes. Since GAGs are on cell membranes, they inhibit thrombin near intact cells so clot does not cover them. Clot limited to site to damage.
23
3rd inhibitory system proteins labelled with letters. Endothelial cell Thrombin receptor activates protein C. Protein S attaches and complex inactivates factrs V and VIII (common pathway)
24
4th systems
tissue factor pathway inhibitor
protein circulates in blood. Inhibits extrinsic pathway, not intrinsic pathway.
Coagulation reversal
25
Acute scenario- all you need is clotting and inhibition. Gives balance so clots form when needed and don’t when not needed.
Plasmin breaks down fibirn network of clot.
tPA cleaves plasminogen
26
FDPs protein fragments released into blood. Inhibit more clot formation. D dimers have structure that can be analysed. Concentration tells rough size of how much clot degraded.
26-28
Clotting factor facts in table. Will encounter literature that uses these terms.
29+
biochemistry
30
FactorXII
Low-medium molecular weight protein. Sequence homology with plasminogen activator and fibronectin. Clotting activator has homology with clot dissolver component. Fibronectin in pregnancy is involved with integration of placenta with uterine wall. Marker of premature labor. False labor, baby is not in jeopardy. Differentiate true from false by monitoring level of fibronectin. Premature labor needs to be monitored and drugs used.
Has EGF-like activity. Not normal function. EGF helps skin and mucus membrane to grow.
31
Fragments do not separate after cleavage, but conformational change reveals active site. Heavy chain may be degraded further.
32
Factor XI
Least common form of hemophilia is due to this. Most mutations are harmless. Forms 2 products. Smaller is active as before. Deactivates by alpha-1-antitrypsin. Enzyme produced by liver. Activated trypsin is supposed to only be in intestine. If it gets into blood, really dangerous. General protease. Specific enzyme cleaves trypsin.Also deactivated Factor XI.
There can be deficiencies of antitrypsin- get liver cirrhosis and pulmonary fibrosis. Causes liver transplant. New liver has right genes. Transplant is curative if do it before lungs get too bad.
33
Factor 9 –hemoB Gene on X chromosome. Woman carrier, male affected.
Requires vitamin K- many clotting factors require vitamin K for polycarboxylation to occur for factor formation in liver. Vitamin K deficiency causes coagulation problems. Coumadin is rat poison. Warfarin inhibits vitamin K.
Stays together after cleavage. Heavy chain has EGF like region, unknown why.
Heavy chain is where polycarboxylation happens.
34
Large protein. Most common form hemophilia. Multiple chains. Gene with 26 exons on X chromosome. Takes 2 cleavages to get activated factor.
35
Factor X 11-12 Ca binding sites. Affected by Ca deficiency. Alters conformation and confers enzymatic activity.
36
Factor V
Vitamin K dependent
Factor V can deactivate itself if high concentration Va exists. Ca dependent. Ca bridges hold parts together. Leiden mutation- molecule works fine, but does not get degraded by protein C. Causes excess clotting. Risky for pregnant women- pelvic veins can clot off.
37
requires vitamin K. Ca dependent area. Needs Ca and binding to phospholipids.
Heavy part is active form because it is a small protein.
38-39
Factor I
Fibrinogen. Blue are alpha chains. Mirror image proteins.Green is beta. Cleavage is at small black arrows in center. End can bind to gamma chain on different fibrin strand.
40
Staggered fibrils form. Forming link requires factor XIIIa. Need cleavage, then catalysis and assistance for steric lining up.
XIII transglutaminase.
42
Crosslink forms Gln-lys bridge to stabilize loose interactions. Harder to fall apart.
43-44
Conclusion
Reversal pathway has no redundancy.
These notes correlate to the slides in the powerpoint lecture. Handout in class follows slides as well.
Handouts, etc. on Blackboard.
Not too many notes to take.
2
Clinical pathology covers all testing and blood banking.
Hemoostasis-regulation of blood volume inside you. Hemodynamics is about blood flow.
Sepsis- one of main casuses of death is inappropriate coagulation in all capillaries of body triggered by bacterial toxin.
3
Objectives
(topics in handout file)
Many homeostatic systems have multiple checks and balances.
4
Abbreviated schematic of major parts of coagulation cascade. Intrinsic pathway statrts at endothelial surface. Thrombin reacts there. Factor 11 converts to little a activated factor. Little a form is smaller because a part is cleaved off. Often you get 2-3 cleavage products, one of which is active product.
5
Clotting factors cleave proteins. A cascade of enzymatic reactions occurs. Each curved arrow is an enzymatic reaction. At end protein fibrinogen cleaved to fibrin, crosslinked, stable clot forms. Not crosslink, clot will not hold and washes away.
6
important facts- Many steps need free ionized Ca. Without Ca, clotting does not occur. Why doesn’t donated blood clot? Bag contains citrate to chelate Ca. EDTA or citrate can be used when drawing blood. Binds Ca.
Intrinsic pathway needs HMWK and prekallikrein(not a proteolytic enzyme).
Platelets are part of regular clotting. They can form plugs to block leakage and can release phospholipids. If they do not work, cannot prevent any kind of bleeding.
Some active factors trigger formation of anticoagulants as part of feedback regulation.
8
Every clot triggering event is damage to epithelial layer of blood vessel. Exposure of collagen activates factor XII, Tissue factor can leak from damaged tissue like skin. Factor 11a circulates. Kallikrein stays at injury site in complex.
9
HMWK acts as anchor. XIa cleaves IX to IXa in a Ca dependent way.
10
Factors numbered in order of discovery, not function.
11
Always small amount of thrombin in plasma.
13
Damage to subendothelial cell membranes- tissue factor without collagen and HMWK activates extrinsic pathway. Can trigger boh pathways activating at same time.
15
Common pathway
Clotting factors trigger more and more activation as you go along.
16
Thrombin action.
XIIIa catalyses crosslinking monomers.
When thrombin cleaves receptor on platelet, causes aggregation. Degranulation provide phospholipids.
17
Photomicrograph shows early microscopic clot with platelets in fibrin mesh.
18
Natural anticoagulantss prevent runaway clotting- do not work perfectly.
19
Once damage is patched, need clot to dissolve. Otherwise would have permanent clots and reduced function.
Can run out of fibrinogen. Uncontrolled clotting can result in uncontrolled bleeding from other sites.
20
Heparin used to prevent unwanted clotting, but almost never used outside hospital. Must be injected or given intravenously.Potentiates Antithrombin III. Easy to overdose. Must be monitored carefully. There is a reversing drug- polycation binds to heparin to deactivate.
21
Inhibits here.
22
heparin cofactor II is native inhibitor of clotting. Inhibits thrombin only. Binds thrombin and physically blocks active site. Heparan sulfate and dermatan sulfate are GAGs or acid mucopolysaccharides on cell membranes. Since GAGs are on cell membranes, they inhibit thrombin near intact cells so clot does not cover them. Clot limited to site to damage.
23
3rd inhibitory system proteins labelled with letters. Endothelial cell Thrombin receptor activates protein C. Protein S attaches and complex inactivates factrs V and VIII (common pathway)
24
4th systems
tissue factor pathway inhibitor
protein circulates in blood. Inhibits extrinsic pathway, not intrinsic pathway.
Coagulation reversal
25
Acute scenario- all you need is clotting and inhibition. Gives balance so clots form when needed and don’t when not needed.
Plasmin breaks down fibirn network of clot.
tPA cleaves plasminogen
26
FDPs protein fragments released into blood. Inhibit more clot formation. D dimers have structure that can be analysed. Concentration tells rough size of how much clot degraded.
26-28
Clotting factor facts in table. Will encounter literature that uses these terms.
29+
biochemistry
30
FactorXII
Low-medium molecular weight protein. Sequence homology with plasminogen activator and fibronectin. Clotting activator has homology with clot dissolver component. Fibronectin in pregnancy is involved with integration of placenta with uterine wall. Marker of premature labor. False labor, baby is not in jeopardy. Differentiate true from false by monitoring level of fibronectin. Premature labor needs to be monitored and drugs used.
Has EGF-like activity. Not normal function. EGF helps skin and mucus membrane to grow.
31
Fragments do not separate after cleavage, but conformational change reveals active site. Heavy chain may be degraded further.
32
Factor XI
Least common form of hemophilia is due to this. Most mutations are harmless. Forms 2 products. Smaller is active as before. Deactivates by alpha-1-antitrypsin. Enzyme produced by liver. Activated trypsin is supposed to only be in intestine. If it gets into blood, really dangerous. General protease. Specific enzyme cleaves trypsin.Also deactivated Factor XI.
There can be deficiencies of antitrypsin- get liver cirrhosis and pulmonary fibrosis. Causes liver transplant. New liver has right genes. Transplant is curative if do it before lungs get too bad.
33
Factor 9 –hemoB Gene on X chromosome. Woman carrier, male affected.
Requires vitamin K- many clotting factors require vitamin K for polycarboxylation to occur for factor formation in liver. Vitamin K deficiency causes coagulation problems. Coumadin is rat poison. Warfarin inhibits vitamin K.
Stays together after cleavage. Heavy chain has EGF like region, unknown why.
Heavy chain is where polycarboxylation happens.
34
Large protein. Most common form hemophilia. Multiple chains. Gene with 26 exons on X chromosome. Takes 2 cleavages to get activated factor.
35
Factor X 11-12 Ca binding sites. Affected by Ca deficiency. Alters conformation and confers enzymatic activity.
36
Factor V
Vitamin K dependent
Factor V can deactivate itself if high concentration Va exists. Ca dependent. Ca bridges hold parts together. Leiden mutation- molecule works fine, but does not get degraded by protein C. Causes excess clotting. Risky for pregnant women- pelvic veins can clot off.
37
requires vitamin K. Ca dependent area. Needs Ca and binding to phospholipids.
Heavy part is active form because it is a small protein.
38-39
Factor I
Fibrinogen. Blue are alpha chains. Mirror image proteins.Green is beta. Cleavage is at small black arrows in center. End can bind to gamma chain on different fibrin strand.
40
Staggered fibrils form. Forming link requires factor XIIIa. Need cleavage, then catalysis and assistance for steric lining up.
XIII transglutaminase.
42
Crosslink forms Gln-lys bridge to stabilize loose interactions. Harder to fall apart.
43-44
Conclusion
Reversal pathway has no redundancy.
Pfeffer 1
lpfeffer@utmem.edu
Overview
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
Test questions directly off poweroints and objectives.
All powerpoints on web.
This list is on blackboard separately. Pfeffer directs new cancer building.
2
Eukaryotic cell cycle 4 phases: G1, G2, M,S.
Cell cycle controlled by discrete regulatory pathways. Orchestrated carefully with checkpoints where cell can sense if something is not right and arrest or die.
Central point of control is cyclically activated cyclin-dependent kinases.
Last 5 years 2 sets of Nobel prizes were awarded for discovery of cell cycle control and ubiquitination pathway.
3
Typical mitosis in mammalian cell takes 1 hour. Human cell cycle 24 hours. M phase is linear series of events to get cell out of mitosis. Deformation- checkpoint stops cells before telophase in somatic cells. Best understood part of cell cycle is mitosis. Cytokinesis is dividing cytoplasm. Cytokinesis is the only part of mitosis that is not exact. One cell may get a bit more or less cytoplasm than the other. Gap phases provide time for monitoring signals to prepare for next phase. G1- all preparation for S completed. G2- all proteins for M manufactured except for cyclin.
4
DNA is only synthesized during S phase.
5
pre 1950 could only see mitosis and interphase under microsope.
Swift found discrete part of cell cycle when DNA was replicated.
H and P named S phase for synthesis, and G1 and G2 for gaps in cycle.
Xenopus laevis is African clawed toad.
Kinds of things controlling cell cycle in organisms from surf clam to yeast to frog are same.
6
Xenopus oocyte is big.
Fibroblasts are used in culture because they grow well (lower picture). Piece of skin in plastic Petri dish wth serum and media- they grow easily.
7
Duration of cell cycle listed here.
Amount of DNA listed here too. 1n haploid, 2n diploid, 4n tetraploid.
8
Why frog eggs?
Enormous- can see with naked eye. Egg has million times amount cytoplasm as fully mature frog cell. Easy to inject something into it. Oocyte G1 and G2 done away with. Has eveything in cytoplasm ready to divide. MPF is maturation or mitosis promoting factor. It is a complex sufficient to drive cell from G2 into mitosis.
During fertilization- pseudohaploid polar body becomes halploid cell, which mixes with 1n from sperm to make diploid cell.
9
Mammalian cells a lot smaller. Grow well. MPF from frog can be used to drive fibroblasts into mitosis.
Mutant cell lines with defects have been used to study cell cycle control. G1/S and G2/M transition studied and well understood. Bromodeoxyuridine looks like thymidine.
Flow cytometer- get pattern on left.
10
3 kinds of cells in body listed here.
Permanent are not thought to regenerate.
Stable- hepatocytes. In rat liver you can cut out 2 out of 3 lobules. Regenerate in one week.
Labile cells continuously divide.
11
To enter M phase the cell must assemble a mitotic spindle. Get out- anaphase and cytokinesis. Checkpoints located G1/S, G2/M.
12
Checkpoint questions. Cell in low serum, goes into G0. Nutrients are not there, so cell slows down.
13
Cell mass increases to critical point, chromosomes segregate, cell division happens.
M phase cyclins just made during mitosis.
14
Classic experiment. Fused cells in different parts of cycle.
Something in S phase cell can drive DNA synthesis for G1 cell, but not G2. Cell must be competent for signal. There is a soluble factor to tell G1 to go into S. Something in M cell drives other cells into M phase.
16
G1 critical part cell cycle. If cell not ready, goes into sleep or G0 phase.
17
Studies in late 1950s and 60s by Hayflick. Lifespan of human fibroblasts is closely regulated. Newborn- 50 -60 doublings. Older 15-20. There is an inverse relationship between age and proliferation potential of fibroblasts.
Progeria- something limits lifespan of cells.
Telomeres shorten as you age. One way to immortalize cells- express telomerase gene.
Senescence is a specific cell state in which protein and DNA synthesis slow down and proliferation potential ends.
18
Cell labelling
Radioactively labelled thymidine
BrdU
23
Feed cells label, take cells out, figure out # labels
25
Percent labelled mitosis: Only S phase cells are labelled.
26-27
Graph generated in experiment.
28
Shows doubling time 20 hours, not 22.
29 Cancers can grow slow or fast, but they usually have similar doubling time to counterpart in normal cells. They just do not know when to stop.
30
Flow cytometry is easier.
Label with DNA dye.
31
Fluorescent detector
35
Different tumors have varying doubling times.
36
Mitotic cells change shape.
One way to get synchronized cells- 4% of cells at any one time are undergoing mitosis. Telandek (this is phonetic spelling)- grow fibroblasts on roller bottle with tubing that sprayed media- shake- all mitotic cells came off. 95% pure.
39
Start seeing mitotic spindle.
40
Metaphase plate with chromosomes lined up in middle.
45
Mitosis vs meiosis.
Somatic cells undergo mitosis, gametes meiosis.
Meiosis one produces pseudodiploid cell. Second meiotic division yields 1n amount of DNA.
48
platelet derived growth factor was one of first.
50-51
Hydroxyurea blocks cells going into DNA synthesis and causes cell death. All cells in S phase die. Remove and wash- cells march synchronouslty thru cell cycle.
Overview
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
Test questions directly off poweroints and objectives.
All powerpoints on web.
This list is on blackboard separately. Pfeffer directs new cancer building.
2
Eukaryotic cell cycle 4 phases: G1, G2, M,S.
Cell cycle controlled by discrete regulatory pathways. Orchestrated carefully with checkpoints where cell can sense if something is not right and arrest or die.
Central point of control is cyclically activated cyclin-dependent kinases.
Last 5 years 2 sets of Nobel prizes were awarded for discovery of cell cycle control and ubiquitination pathway.
3
Typical mitosis in mammalian cell takes 1 hour. Human cell cycle 24 hours. M phase is linear series of events to get cell out of mitosis. Deformation- checkpoint stops cells before telophase in somatic cells. Best understood part of cell cycle is mitosis. Cytokinesis is dividing cytoplasm. Cytokinesis is the only part of mitosis that is not exact. One cell may get a bit more or less cytoplasm than the other. Gap phases provide time for monitoring signals to prepare for next phase. G1- all preparation for S completed. G2- all proteins for M manufactured except for cyclin.
4
DNA is only synthesized during S phase.
5
pre 1950 could only see mitosis and interphase under microsope.
Swift found discrete part of cell cycle when DNA was replicated.
H and P named S phase for synthesis, and G1 and G2 for gaps in cycle.
Xenopus laevis is African clawed toad.
Kinds of things controlling cell cycle in organisms from surf clam to yeast to frog are same.
6
Xenopus oocyte is big.
Fibroblasts are used in culture because they grow well (lower picture). Piece of skin in plastic Petri dish wth serum and media- they grow easily.
7
Duration of cell cycle listed here.
Amount of DNA listed here too. 1n haploid, 2n diploid, 4n tetraploid.
8
Why frog eggs?
Enormous- can see with naked eye. Egg has million times amount cytoplasm as fully mature frog cell. Easy to inject something into it. Oocyte G1 and G2 done away with. Has eveything in cytoplasm ready to divide. MPF is maturation or mitosis promoting factor. It is a complex sufficient to drive cell from G2 into mitosis.
During fertilization- pseudohaploid polar body becomes halploid cell, which mixes with 1n from sperm to make diploid cell.
9
Mammalian cells a lot smaller. Grow well. MPF from frog can be used to drive fibroblasts into mitosis.
Mutant cell lines with defects have been used to study cell cycle control. G1/S and G2/M transition studied and well understood. Bromodeoxyuridine looks like thymidine.
Flow cytometer- get pattern on left.
10
3 kinds of cells in body listed here.
Permanent are not thought to regenerate.
Stable- hepatocytes. In rat liver you can cut out 2 out of 3 lobules. Regenerate in one week.
Labile cells continuously divide.
11
To enter M phase the cell must assemble a mitotic spindle. Get out- anaphase and cytokinesis. Checkpoints located G1/S, G2/M.
12
Checkpoint questions. Cell in low serum, goes into G0. Nutrients are not there, so cell slows down.
13
Cell mass increases to critical point, chromosomes segregate, cell division happens.
M phase cyclins just made during mitosis.
14
Classic experiment. Fused cells in different parts of cycle.
Something in S phase cell can drive DNA synthesis for G1 cell, but not G2. Cell must be competent for signal. There is a soluble factor to tell G1 to go into S. Something in M cell drives other cells into M phase.
16
G1 critical part cell cycle. If cell not ready, goes into sleep or G0 phase.
17
Studies in late 1950s and 60s by Hayflick. Lifespan of human fibroblasts is closely regulated. Newborn- 50 -60 doublings. Older 15-20. There is an inverse relationship between age and proliferation potential of fibroblasts.
Progeria- something limits lifespan of cells.
Telomeres shorten as you age. One way to immortalize cells- express telomerase gene.
Senescence is a specific cell state in which protein and DNA synthesis slow down and proliferation potential ends.
18
Cell labelling
Radioactively labelled thymidine
BrdU
23
Feed cells label, take cells out, figure out # labels
25
Percent labelled mitosis: Only S phase cells are labelled.
26-27
Graph generated in experiment.
28
Shows doubling time 20 hours, not 22.
29 Cancers can grow slow or fast, but they usually have similar doubling time to counterpart in normal cells. They just do not know when to stop.
30
Flow cytometry is easier.
Label with DNA dye.
31
Fluorescent detector
35
Different tumors have varying doubling times.
36
Mitotic cells change shape.
One way to get synchronized cells- 4% of cells at any one time are undergoing mitosis. Telandek (this is phonetic spelling)- grow fibroblasts on roller bottle with tubing that sprayed media- shake- all mitotic cells came off. 95% pure.
39
Start seeing mitotic spindle.
40
Metaphase plate with chromosomes lined up in middle.
45
Mitosis vs meiosis.
Somatic cells undergo mitosis, gametes meiosis.
Meiosis one produces pseudodiploid cell. Second meiotic division yields 1n amount of DNA.
48
platelet derived growth factor was one of first.
50-51
Hydroxyurea blocks cells going into DNA synthesis and causes cell death. All cells in S phase die. Remove and wash- cells march synchronouslty thru cell cycle.
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