HMP Shunt- hexose monophosphate shunt or pentose phosphate pathway
Glycogen Metabolism
3: Know what the pentose phosphate pathway does. Purpose- give 5-carbon sugars for DNA and RNA synthesis. Need NADPH for biosynthesis. In glycolytic pathway, you get NADH for catbolism. NADPH is needed to hydrogenate double bonds- does not come from glycolysis. NADPH is needed for fatty acid biosynthesis.
For NADPH, recycle 5-C to get 6-C sugar. Intermediates formed are summarized on the slide.
Know it is HMP or PP pathway.
6: 6-phosphogluconate dehydrogenase produces NADPH and CO2, converting glucose to D-ribulose 5 phosphate in fed state. (Cells want to grow and divide when well fed)
7: difference isomerase and epimerase: Isomerase changes aldehyde to ketone (or vice versa- reaction is reversible). Epimerase moves OH to other side of same carbon. Moved from one plane to another.
8: 5+5 (ribose-5-phosphate+xylulose-5-phosphate)=7+3(sedoheptulose-7-phosphate+glygeraldehyde-3-phosphate)
11: fructose 6 phosphate can be isomerized to glucose 6- phosphate. Know this figure. When glucose enters, has 3 possible pathways (glycolysis, pentose phosphate pathway, or glycogen synthesis). The arrows between fructose-6-phosphate and fructose 1,6 bisphosphate should not be doubled.
12: another depiction.
13: If you want ribose and not reducing equivalents, you can go directly from glyceraldehyde 3 phosphate.
14: Red blood cells have no nucleus and no mitochondria. they use the pentose phosphate pathway for NADPH. Red cells need NADPH to prevent oxidative damage to red blood cells. This deficiency reduces the half life of red blood cells. Causes hemolytic anemia.
16: processing of fructose. Muscle and fat have hexokinase to phosphorylate fructose, but it is difficult for fructose to get into them. Fructose messes up metabolism- has to be converted in liver.
17: Milk sugar is made of glucose and galactose. Galactose 6-p – get UDP galactose. UDP galactose is conv to UDP glucose. Get Glucose 1-phosphate.
18: Galactosemia is rare enzymatic defect. To treat the deficiency, reduce exposure to lactose until teenage years when alternate pathway becomes available.
19: Summary for galactose, fructose, and mannose. UDP glucose is used to make glycogen.
20: We store glucose in liver and muscle as glycogen.
22: Chain elongation requires UDP-glucose.
25: once you get 10 glucose linked, you need a branch point.
27: breakdown.
31: In test tube, you can make glycogen with glycogen phosphorylase with large amounts of glucose-1-phosphate(G-1-P), glucose, and limit dextrin. Not in vivo- not high enough concentration G-1P. In vitro they forced the enzyme to run backward.
33: We have receptors (in liver and muscle) in the cell membrane. Epinephrine added to membranes made cAMP that was heat stable. Added to liver extract, got second messenger to induce glycogen breakdown.
34: Phosphorylation activates phosphorylase kinase and inactivates glycogen synthase.
Review:
from 1-26 on exam review sheet.
17. basic pathway.
18. Why is virtually all fructose utilization in liver in humans? Liver ‘s function is to interconvert metabolic intermediates.
20. How do you make glycogen from glucose? Rapid Hydrolysis of pyrophosphate makes it reversible. Why need both enzymes? Linear polymer can get twisted and tangled. Branching works better. Less osmotic effect.
21. what does glycogenolysis require? debranching enzyme and phosphorylase, regulated by phosphorylase kinase.
Phosphorylate serine and/or threonine residues. Catecholamines regulate cAMP to cause covalent modification.
23-26: forget. His mistake. From other lectures. Not on test.
He has 3 questions, one for each lecture.
If you do not know A in a question on the test, do B and C for 6 pts instead of 10.
Thursday, November 30, 2006
Marion 4
Receptors in the immune system have incredible structural diversity along with constant structure that allows for consistent function. Took until early to mid 1970s to prove genetic structure- experiment by Tonegawa. Did Southern hybridization. Used sequence-specific endonucleases (restriction enzymes cleave at sitesof 3-6 nucleotides in length) . He used electrophoresis to separate the fragments on basis of size. This produced a smear of DNA on gel. Transfer to nitrocellulose and incubate with radiolabelled DNA fragment. Where fragment is homologous to DNA on nitrocellulose, get a band on radiograph. See slide 1 in PP Figs. Tonegawa took DNA from germ line (mouse liver DNA) and made 2 gels with 2 probes: one V region probe for a particular myeloma protein (plasma cytomas secrete myeloma proteins). Detected a band of a particular size. Same for a constant region probe- looked as though V and C region genes were separate. Took Myeloma DNA for protein and did same thing. Now with V and C region probes, identified same piece of DNA. Proved one protein originated from 2 pieces of DNA. Probe came from digesting cDNA from mRNA for the protein derived from the known protein sequence.
In order to generate the gene that encoded the antibody, something had to happen to bring pieces of DNA together. Somatic recombination.
What does light/heavy chain locus look like? See slide 2.
In humans, there are a series of 40 individual kappa genes separated by hundreds of kb. Jkappa there are 5, then space, then Ck constant region. Front- there is a leader sequence (L) at 5’ for each V region gene. For lambda, there are 30 variable region genes.
Slide 3: In germ line DNA, genes that can encode antibodies are scattered. To generate a functional gene for light and heavy chains, they have to be brought together. For light chains, 1 somatic recombination brings 1V with 1J. Choice seems to be random. Mechanism controlling recombination is controlled by 2 proteins: Recombination Activating Gene 1 and 2 (RAG 1 and 2) are the 2 proteins.
Slide 4: For every V, at 3’end, there is a 7-mer separated by 12 or 23 base pairs. For Vkappa, this is 12. Then there is a nonamer. This is RSS- recombination signal sequence. RSS is recognized by RAG1 and 2. Recognition obeys rule called. 12/23 rule. Somatic recombination puts together only one 12 with one 23 (representing one turn and 2 turns of DNA helix). Rule ensures correct function of somatic recombination machinery to put V with J correctly.
Slide 11: (This is from the nri1152.pdf article) Rag 1 and 2 generate a double-stranded break and make a DNA hairpin. Generates palindrome. Resolves hairpin by cutting randomly . Exonuclease at 3’ end of J region chews away nucleotides ramdomly. Then V and J ligated together. Ku70/80 DNA ligase 4 and DNA protein kinase are all essential for this. DNA protein kinase was discovered in SCID mouse (Mel Bosma). Slide 13: RAG mediated recombination- RAG binds to RSS, makes a double-stranded break, generates hairpin, RAg protein resolves hairpin by DSB in DNA on 5’end of J in palindromic region, Vin 3’ region, ligates, makes VJ. Because process is random, and nucleotides at ends are removed randomly, most likely to generate out of frame, useless coding sequence.
Slide 12: What happens to DNA in between? Thrown away. RAGs join RSSs to create a signal joint. A piece of circular DNA forms and is lost.
Heavy chain:
Slide 14:3 gene segments involved in formation of heavy chain variable region gene. Rag complex pulls out D and J first to make DJ joint. P nucleotides (palindromic) Exonuclease removal of nucleotides is random again, but another enzyme is involved called terminal deoxynucleotidyl transferase (TdT). Adds nucleotides without a template to 3’ end of a piece of DNA. Functions during heavy chain only. TdT transfers random number of nucleotides. Prefers G> C>T>A. Region between D and J often rich in G and C. Rag genes then add V.
Light: one recombination event. V-J.
Heavy: D-J, then V-DJ.
In humans, if you look at RSS, spacing seen as on slide 3. V does not go to J because of spacers in heavy chain. 23 only joins with 12.
Why not VD? DNA must be made accessible. Early B cell development- cryptic promoter upstream of D and J turns on before V promoter.
Consequence (other than generating coding sequence for light and heavy chain Ig):
FR (framework region)low variability. CDR(complementarity determining region)- high variability.
Once generated, transcribed. Primary transcript(see drawing on paper).
Heavy chain more complex. Tm is transmembrane exon.
Slide 9 and 16: What determines whether you get mu (membrane or secreted) or delta (membrane) is RNA splicing. Splicing determines which poly A site is incorporated into mRNA.Same cell can produce IgM and IgD with same variable region. This is unique to lymphocytes.
Slide 7:There are multiple classes: G(1-4), A(1,2) M, E,D. We know about M and D, but what about the others?
B cell becomes activated, it will die, become an antibody secreting plasma cell, or become a memory cell (long-lived).
Memory antibody response switches from IgM to IgG. Ab affinity goes up all the time in a process called affinity maturation. B cell activated undergoes differentiation. Can switch isotype it makes, and antibody may become better at binding. How would you keep specificity, but inprove affinity? 2 processes.
1. isotype switching
2. somatic mutation and clonal selection. Slide 8: Every round division, 1 mutation randomly inserted into variable region. Most likely to generate nonsense or missense. But occasionally mutation inserts and is functional. If it is likely to work better- selected for cloning. CDRs tend to accumulate mutations thorugh clonal selection. Improved cells are more likely to be selected. Mutations in FR interfere with beta sheets.
Slide 17: isotype switching:
In germline, mu and delta close together, then gamma3, gamma1, gamma2b, gamma2a, epsilon, alpha. In front of each is a switch signal sequence. S is repetitive sequence. What determines which is switched? Transcription. In B cell switching, signals from T cells and cytokines determine recognition of switch sequence.
Slide 15: T cell receptor:
alpha like light chain-VJ. Beta like heavy chain-VDJ. Has TdT added N sequence. These are alpha beta cells.
There are others- gammadelta T cells are more like innate immunity.Gamma like light chain with its own locus with V and J genes. Delta sits in middle of alpha locus – lots of Ds and Js with its own constant region. Alpha rearrangement eliminates D. Delta uses same V regions as alpha, but develops along a different pathway.
Diversity is generated by V gene pairs, random joining, N-P sequences. For T cells you get 1014-18 potential different cells. Amazing.
He will modify and reprint notes- added some powerpoint figures to blackboard as well. These are referenced in my notes above. Images are from Janeway text. Note to All: the fifth edition of the Janeway, et al.Immunobiology text is in the Books section of Pubmed if you can’t get to the library. He uses the 6th ed, but a lot of the information is the same.
In order to generate the gene that encoded the antibody, something had to happen to bring pieces of DNA together. Somatic recombination.
What does light/heavy chain locus look like? See slide 2.
In humans, there are a series of 40 individual kappa genes separated by hundreds of kb. Jkappa there are 5, then space, then Ck constant region. Front- there is a leader sequence (L) at 5’ for each V region gene. For lambda, there are 30 variable region genes.
Slide 3: In germ line DNA, genes that can encode antibodies are scattered. To generate a functional gene for light and heavy chains, they have to be brought together. For light chains, 1 somatic recombination brings 1V with 1J. Choice seems to be random. Mechanism controlling recombination is controlled by 2 proteins: Recombination Activating Gene 1 and 2 (RAG 1 and 2) are the 2 proteins.
Slide 4: For every V, at 3’end, there is a 7-mer separated by 12 or 23 base pairs. For Vkappa, this is 12. Then there is a nonamer. This is RSS- recombination signal sequence. RSS is recognized by RAG1 and 2. Recognition obeys rule called. 12/23 rule. Somatic recombination puts together only one 12 with one 23 (representing one turn and 2 turns of DNA helix). Rule ensures correct function of somatic recombination machinery to put V with J correctly.
Slide 11: (This is from the nri1152.pdf article) Rag 1 and 2 generate a double-stranded break and make a DNA hairpin. Generates palindrome. Resolves hairpin by cutting randomly . Exonuclease at 3’ end of J region chews away nucleotides ramdomly. Then V and J ligated together. Ku70/80 DNA ligase 4 and DNA protein kinase are all essential for this. DNA protein kinase was discovered in SCID mouse (Mel Bosma). Slide 13: RAG mediated recombination- RAG binds to RSS, makes a double-stranded break, generates hairpin, RAg protein resolves hairpin by DSB in DNA on 5’end of J in palindromic region, Vin 3’ region, ligates, makes VJ. Because process is random, and nucleotides at ends are removed randomly, most likely to generate out of frame, useless coding sequence.
Slide 12: What happens to DNA in between? Thrown away. RAGs join RSSs to create a signal joint. A piece of circular DNA forms and is lost.
Heavy chain:
Slide 14:3 gene segments involved in formation of heavy chain variable region gene. Rag complex pulls out D and J first to make DJ joint. P nucleotides (palindromic) Exonuclease removal of nucleotides is random again, but another enzyme is involved called terminal deoxynucleotidyl transferase (TdT). Adds nucleotides without a template to 3’ end of a piece of DNA. Functions during heavy chain only. TdT transfers random number of nucleotides. Prefers G> C>T>A. Region between D and J often rich in G and C. Rag genes then add V.
Light: one recombination event. V-J.
Heavy: D-J, then V-DJ.
In humans, if you look at RSS, spacing seen as on slide 3. V does not go to J because of spacers in heavy chain. 23 only joins with 12.
Why not VD? DNA must be made accessible. Early B cell development- cryptic promoter upstream of D and J turns on before V promoter.
Consequence (other than generating coding sequence for light and heavy chain Ig):
FR (framework region)low variability. CDR(complementarity determining region)- high variability.
Once generated, transcribed. Primary transcript(see drawing on paper).
Heavy chain more complex. Tm is transmembrane exon.
Slide 9 and 16: What determines whether you get mu (membrane or secreted) or delta (membrane) is RNA splicing. Splicing determines which poly A site is incorporated into mRNA.Same cell can produce IgM and IgD with same variable region. This is unique to lymphocytes.
Slide 7:There are multiple classes: G(1-4), A(1,2) M, E,D. We know about M and D, but what about the others?
B cell becomes activated, it will die, become an antibody secreting plasma cell, or become a memory cell (long-lived).
Memory antibody response switches from IgM to IgG. Ab affinity goes up all the time in a process called affinity maturation. B cell activated undergoes differentiation. Can switch isotype it makes, and antibody may become better at binding. How would you keep specificity, but inprove affinity? 2 processes.
1. isotype switching
2. somatic mutation and clonal selection. Slide 8: Every round division, 1 mutation randomly inserted into variable region. Most likely to generate nonsense or missense. But occasionally mutation inserts and is functional. If it is likely to work better- selected for cloning. CDRs tend to accumulate mutations thorugh clonal selection. Improved cells are more likely to be selected. Mutations in FR interfere with beta sheets.
Slide 17: isotype switching:
In germline, mu and delta close together, then gamma3, gamma1, gamma2b, gamma2a, epsilon, alpha. In front of each is a switch signal sequence. S is repetitive sequence. What determines which is switched? Transcription. In B cell switching, signals from T cells and cytokines determine recognition of switch sequence.
Slide 15: T cell receptor:
alpha like light chain-VJ. Beta like heavy chain-VDJ. Has TdT added N sequence. These are alpha beta cells.
There are others- gammadelta T cells are more like innate immunity.Gamma like light chain with its own locus with V and J genes. Delta sits in middle of alpha locus – lots of Ds and Js with its own constant region. Alpha rearrangement eliminates D. Delta uses same V regions as alpha, but develops along a different pathway.
Diversity is generated by V gene pairs, random joining, N-P sequences. For T cells you get 1014-18 potential different cells. Amazing.
He will modify and reprint notes- added some powerpoint figures to blackboard as well. These are referenced in my notes above. Images are from Janeway text. Note to All: the fifth edition of the Janeway, et al.Immunobiology text is in the Books section of Pubmed if you can’t get to the library. He uses the 6th ed, but a lot of the information is the same.
Marion Immunology 3
Comments:
Recommended reading is in text Immunobiology in library on reserve. AM lecture- a lot of factual information was presented. We are not expected to remember all the facts, but come away with realization that there are specific receptors to recognize different types of molecules on pathogen surfaces or RNA and DNA. For signalling, remember that the end result is activation of NFkB and MAP(?) transcription factors.Also there is common adaptor factor MyD88. Study the concepts, not minute details.
This lecture: Adaptive immune receptors and their structure and specificities. Tomorrow: genetic mechanisms.
Structure of monomeric immunoglobulin IgG: dimer of a heterodimer. Heavy chains vary (50-70 kD), light chains 25kD. Heterodimers are covalently bound by disulfide bond. One or 2-3 disulfide bonds in region called the hinge region hold it together.
We can divide the molecule into 2 distinct regions:
C or constant region provides for biological function (for cell surface and protein-protein interactions).
V or variable region provides for antigen binding.
Mid 1950s, after discovery DNA, Burnet, based on work of Medawar, proposed clonal selection hypothesis.It says that the potential for immune responses exists and develops in the absence of knowledge of what the antigen would be. Diversity is built into the system generating the receptors. Antibodies are formed prior to exposure to antigen. People who opposed this theory were instructionists. They proposed that cells took in antigen and wrapped protein around it as template and spit it out. That was shot down well by work from Dreyer and Bennett in 1965. They proposed from protein sequencing of IgGs that about 1/3 of heavy chain and 1/3 of light chain had variable sequence from one chain to another. They examined myeloma proteins. A given tumor cell produced only one type. Other part was constant. Allelic differences within the constant region were inherited as polymorphisms. Hypothesized that for Igs, 2 genes encoded one polypeptide. They were right. Alternative theory was mutational theory. It said that there was one variable region gene that was mutated by B cells. That isn’t how B cells develop the receptor they start with.
Basic building block of antibody molecule is immunoglobulin domain. Light chains have 2 domains, 1 V and 1 C. Heavy chains, some have 4 (1 V, 3 C), some 5(1 V, 4C). Of all proteins encoded by human genome, the Ig domain is most represented. Why? Stability. Among protein structures, this is most stable. Why? See slide 1. 2 parallel beta sheets disulfide linked. Formed of antiparallel strands connected by alpha helical loops. These are very stable.Structures of Ig domains in proteins differ, but the function depends on the overall structural stability.
Variability is in alpha helical loops.
The constant region of IgM and IgG is formed from 3-4 constant region Ig domains.
IgGs-3 CH1, CH2, CH3. Primary structure is different, but secondary structure is the same.
variable domain- sequence varies, but amount of variability differs from one part to another.
Slide 2:Wu and Kabat were early investigators of Ig structure. Took all known Ig sequences, aligned to study, looked for relationships one to another. Compared. Constant regions were the same, but variable regions had periodicity. Some parts have much less variability than others. “Framework” regions represent B strands. Structural integrity is maintained by relatively constant sequence.
Variable regions can be divided into 10-12 families in humans. Red on graph- complementarity determining regions- CDRs. Also called hypervariable regions. These are parts of molecule contacting the epitope.
Epitope- the molecular determinant of antigen (Ag)to which antibody(Ab) binds.
antigen- something that an antibody or T cell receptor (TCR) can bind.
immunogen- antigen that can induce an immune response. All antigens are not immunogens. There are some things an antibody can bind that are not immunogenic.
Igs have 5 classes, depending on the C region structure.
IgM
IgG
IgA
IgE
IgD
All found in circulation except for D. D is never secreted. Found on D cells.
Classes also referred to as isotypes. Isotype explains antigenicity of constant region. Can immunize mouse with human IgG. Isotypes are mu, gamma, alpha, epsilon, delta. Refers to heavy chain for each class. Two isotypes light chain: kappa and lambda.
allotypes: 2 of each for heavy and light chains.
One other type of antigenic determinant: idiotype refers to the structure created by heavy and light chain variable regions. There are idiotypic differences from one molecule to another.
In addition to 5 classes, IgG has subclasses 1,2,3,4. IgA has 1 and 2.
Mouse IgG subclasses IgG 1, 2a, 2b, 3. identified on the basis of structural variation in variable regions. Different subclasses dominate different immune responses depending on the antigen stimulating the response.
IgM and IgA are secreted in polymeric form (IgA dimeric, IgM pentamer). Plasma cell secretes J polypeptide, which disulfide binds 2 IgM monomers to polymerize. Creates 5 IgM monomers bound in constant region. For IgA, J forms dimer in mucosal secretions only.
Beginning in 1940s and 50s first studies of Ig structure were performed by proteolysis by papain and pepsin. ONLY relevant to IgG. Papain cleaves molecule into 3 frags: Fab- antigen binding. Fc-crystallizable. Pepsin forms F(ab’)2 and peptides.
F(ab’)2 could agglutinate or aggregate. Could act like intact antibody. Monomeric IgG had to be a dimer of a heterodimer.
How antibodies bind to antigens:
binding is noncovalent, kinetic interaction. KA can be measured by equilibrium dialysis. KA is dependent on association and dissociation. Referred to as affinity of binding an antibody. Measures interaction of a single Fab and one Ag. Affinity of an antibody molecule is always higher because they may be dimers to pentamers. Avidity is relative affinity that is consequence of its valence (number of monomers together) and its true affinity. When you measure interactions, single interaction is independent to a point, depending on how many determinants are on the molecule. Multiple epitopes increase the apparent affinity. When one of the interactors is immobilized to a solid phase, interaction is changed to first order reaction and changes relative affinity by as much as four logs.
T cell receptors:
Essentially antibody Fab. Found membrane bound, and only on T cells. 2 polypeptides make the receptors:
alpha and beta.Alpha is like the Ig light chain. B is like the heavy chain structurally. Had V and C regions. Variability in V region allows recognition of antigens.
Antibody and B cell receptor can bind native antigen. That means antibody can bind protein or carbohydrate which does not have to be changed for it to bind.
T cell receptors don’t do that. they are selected for their ability to bind peptide in MHC molecule.
MHC-Major histocompatibility complex. these molecules are detected as transplantation antigens. MHC encoded molecules generate the strongest transplantation responses.
2 types MHC:
Class I
Class II
Both bind peptides derived from proteins proteolytically digested in antigen presenting cells (APCs). APCs are cells that proteolytically process proteins into peptides bound by MHC. All nucleated cells make class I MHC. Only a subset (Professional APCs) express class II (macrophages, dendritic cells, B cells). Allows peptides from antigens inside and outside the cell to be expressed inside one of these to be sampled. TcR is selected to bind only to peptides in Class I or II MHC. T cell does not do a thing to virus or bacterium- recognizes infected or tumor cells.
Depending on type of T cell, immune system divides and conquers. CD4 T cells class II, CD8 in class I. Selection for specificity happens in thymus.
Slide 6: Pockets, grooves, or planar surfaces can be formed by heavy and light chains. interactions and affinities are electrostatic, hydrophobic,e tc.
MHC restriction- Peter dougherty discovered this concept: T cells only bind to antigens bound to MHC class one or II. If MHC wrong- no binding. Wrong peptide- no binding.
12: T cell receptors interacting with peptide and Class II MHC.
13: Ig is co-expressed with Ig alpha and beta, which engage sark like kinases. receptor functions to bind.
T cell receptor coexpressed with CD3. Zeta is signal for T cell receptor. ITAM phosphorylated, attract kinases to initiate signalling.
Recommended reading is in text Immunobiology in library on reserve. AM lecture- a lot of factual information was presented. We are not expected to remember all the facts, but come away with realization that there are specific receptors to recognize different types of molecules on pathogen surfaces or RNA and DNA. For signalling, remember that the end result is activation of NFkB and MAP(?) transcription factors.Also there is common adaptor factor MyD88. Study the concepts, not minute details.
This lecture: Adaptive immune receptors and their structure and specificities. Tomorrow: genetic mechanisms.
Structure of monomeric immunoglobulin IgG: dimer of a heterodimer. Heavy chains vary (50-70 kD), light chains 25kD. Heterodimers are covalently bound by disulfide bond. One or 2-3 disulfide bonds in region called the hinge region hold it together.
We can divide the molecule into 2 distinct regions:
C or constant region provides for biological function (for cell surface and protein-protein interactions).
V or variable region provides for antigen binding.
Mid 1950s, after discovery DNA, Burnet, based on work of Medawar, proposed clonal selection hypothesis.It says that the potential for immune responses exists and develops in the absence of knowledge of what the antigen would be. Diversity is built into the system generating the receptors. Antibodies are formed prior to exposure to antigen. People who opposed this theory were instructionists. They proposed that cells took in antigen and wrapped protein around it as template and spit it out. That was shot down well by work from Dreyer and Bennett in 1965. They proposed from protein sequencing of IgGs that about 1/3 of heavy chain and 1/3 of light chain had variable sequence from one chain to another. They examined myeloma proteins. A given tumor cell produced only one type. Other part was constant. Allelic differences within the constant region were inherited as polymorphisms. Hypothesized that for Igs, 2 genes encoded one polypeptide. They were right. Alternative theory was mutational theory. It said that there was one variable region gene that was mutated by B cells. That isn’t how B cells develop the receptor they start with.
Basic building block of antibody molecule is immunoglobulin domain. Light chains have 2 domains, 1 V and 1 C. Heavy chains, some have 4 (1 V, 3 C), some 5(1 V, 4C). Of all proteins encoded by human genome, the Ig domain is most represented. Why? Stability. Among protein structures, this is most stable. Why? See slide 1. 2 parallel beta sheets disulfide linked. Formed of antiparallel strands connected by alpha helical loops. These are very stable.Structures of Ig domains in proteins differ, but the function depends on the overall structural stability.
Variability is in alpha helical loops.
The constant region of IgM and IgG is formed from 3-4 constant region Ig domains.
IgGs-3 CH1, CH2, CH3. Primary structure is different, but secondary structure is the same.
variable domain- sequence varies, but amount of variability differs from one part to another.
Slide 2:Wu and Kabat were early investigators of Ig structure. Took all known Ig sequences, aligned to study, looked for relationships one to another. Compared. Constant regions were the same, but variable regions had periodicity. Some parts have much less variability than others. “Framework” regions represent B strands. Structural integrity is maintained by relatively constant sequence.
Variable regions can be divided into 10-12 families in humans. Red on graph- complementarity determining regions- CDRs. Also called hypervariable regions. These are parts of molecule contacting the epitope.
Epitope- the molecular determinant of antigen (Ag)to which antibody(Ab) binds.
antigen- something that an antibody or T cell receptor (TCR) can bind.
immunogen- antigen that can induce an immune response. All antigens are not immunogens. There are some things an antibody can bind that are not immunogenic.
Igs have 5 classes, depending on the C region structure.
IgM
IgG
IgA
IgE
IgD
All found in circulation except for D. D is never secreted. Found on D cells.
Classes also referred to as isotypes. Isotype explains antigenicity of constant region. Can immunize mouse with human IgG. Isotypes are mu, gamma, alpha, epsilon, delta. Refers to heavy chain for each class. Two isotypes light chain: kappa and lambda.
allotypes: 2 of each for heavy and light chains.
One other type of antigenic determinant: idiotype refers to the structure created by heavy and light chain variable regions. There are idiotypic differences from one molecule to another.
In addition to 5 classes, IgG has subclasses 1,2,3,4. IgA has 1 and 2.
Mouse IgG subclasses IgG 1, 2a, 2b, 3. identified on the basis of structural variation in variable regions. Different subclasses dominate different immune responses depending on the antigen stimulating the response.
IgM and IgA are secreted in polymeric form (IgA dimeric, IgM pentamer). Plasma cell secretes J polypeptide, which disulfide binds 2 IgM monomers to polymerize. Creates 5 IgM monomers bound in constant region. For IgA, J forms dimer in mucosal secretions only.
Beginning in 1940s and 50s first studies of Ig structure were performed by proteolysis by papain and pepsin. ONLY relevant to IgG. Papain cleaves molecule into 3 frags: Fab- antigen binding. Fc-crystallizable. Pepsin forms F(ab’)2 and peptides.
F(ab’)2 could agglutinate or aggregate. Could act like intact antibody. Monomeric IgG had to be a dimer of a heterodimer.
How antibodies bind to antigens:
binding is noncovalent, kinetic interaction. KA can be measured by equilibrium dialysis. KA is dependent on association and dissociation. Referred to as affinity of binding an antibody. Measures interaction of a single Fab and one Ag. Affinity of an antibody molecule is always higher because they may be dimers to pentamers. Avidity is relative affinity that is consequence of its valence (number of monomers together) and its true affinity. When you measure interactions, single interaction is independent to a point, depending on how many determinants are on the molecule. Multiple epitopes increase the apparent affinity. When one of the interactors is immobilized to a solid phase, interaction is changed to first order reaction and changes relative affinity by as much as four logs.
T cell receptors:
Essentially antibody Fab. Found membrane bound, and only on T cells. 2 polypeptides make the receptors:
alpha and beta.Alpha is like the Ig light chain. B is like the heavy chain structurally. Had V and C regions. Variability in V region allows recognition of antigens.
Antibody and B cell receptor can bind native antigen. That means antibody can bind protein or carbohydrate which does not have to be changed for it to bind.
T cell receptors don’t do that. they are selected for their ability to bind peptide in MHC molecule.
MHC-Major histocompatibility complex. these molecules are detected as transplantation antigens. MHC encoded molecules generate the strongest transplantation responses.
2 types MHC:
Class I
Class II
Both bind peptides derived from proteins proteolytically digested in antigen presenting cells (APCs). APCs are cells that proteolytically process proteins into peptides bound by MHC. All nucleated cells make class I MHC. Only a subset (Professional APCs) express class II (macrophages, dendritic cells, B cells). Allows peptides from antigens inside and outside the cell to be expressed inside one of these to be sampled. TcR is selected to bind only to peptides in Class I or II MHC. T cell does not do a thing to virus or bacterium- recognizes infected or tumor cells.
Depending on type of T cell, immune system divides and conquers. CD4 T cells class II, CD8 in class I. Selection for specificity happens in thymus.
Slide 6: Pockets, grooves, or planar surfaces can be formed by heavy and light chains. interactions and affinities are electrostatic, hydrophobic,e tc.
MHC restriction- Peter dougherty discovered this concept: T cells only bind to antigens bound to MHC class one or II. If MHC wrong- no binding. Wrong peptide- no binding.
12: T cell receptors interacting with peptide and Class II MHC.
13: Ig is co-expressed with Ig alpha and beta, which engage sark like kinases. receptor functions to bind.
T cell receptor coexpressed with CD3. Zeta is signal for T cell receptor. ITAM phosphorylated, attract kinases to initiate signalling.
English Immunology Notes
He will focus on innate immunity and Toll-like receptors.
2 Innate immunity turns on and adapts adaptive immunity. Triggered by recognition of conserved molecular patterns.
4 LPS (lipopolysaccharide) exists only in bacteria.
7 Innate immune system should be perfect at recognition self vs nonself. SLE- lupus may have something wrong in toll-like receptor 9 which distinguishes human DNA from bacterial DNA.
10 Innate immunity- he focuses on signalling pathway. Opsonin is a word from the Greek language meaning “to prepare something to eat”- coat bacteria for easier ingestion.
15 Janeway- father of innate immunity.
16 PRRs recognize PAMPs. LPS is lipopolysacharride.
18 PRRs can be classified by location or by function.
19: complement coats microorganisms for phagocytosis. Pentraxin- C-reactive protein is a marker of acute infection. Pentraxin 3 may have role in pathogenesis of sepsis.
20: Dectin 1 senses fungi.
21: interferon induced proteins are antiviral.
nod-like receptors sense bits of broken down cell walls.
22: NOD 2 was discovered to be mutated in subset of patients with Crohn’s disease- may disrupt ability of normal flora to protect from infection.
26: Aspergillus is an opportunistic pathogen. toll negative ones died- normal ones were fine.
27: Toll receptors work completely differently in drosophila and man.
28: endotoxin is a misnomer- it is outside cell. It is lipopolysaccharide. ON surface.
Point mutation in TLR4 in mice makes them unable to respons to LPS.
29: LRR- leucine rich region. TIR (Toll/Interleukin1 Receptor) homologous domain is signalling domain.
30: TLR 11 may prevent UTIs.
31: Two groups TLRs. The ones on surface react with complement of microorganisms on surface or released into cytoplasm. Other group recognizes components in endosomes. Bacterial DNA in cell binds to TLR9. MyD88 is signalling molecule for downstream function. MyD88 is required for TLR 9 and 5. TLR4 needs at least 3 other signalling molecules. Downstream are IRAKs.
32: TLRs in Drosophila are entirely indirect.
37: Krieg worked with antisense DNAs. Cytokines were activated in his controls. CpG DNA was recognized by innate system as foreign. In humans- CpG seen less often.
40 Chloroquine blocks endosomal acidification. Inhibitory oligo also blocks (iCpG). iCpG is the only blocker for a TLR.
48: DC are dendritic cells.
53: Exaggerated inflammatory response kills in septic shock.
“cytokine storm” gets cells to site of infection.
59: Most immune defects are in adaptive immunity.
In systemic lupus, patient may make immune response to his or her own DNA complexed to histones. Leading theory of autoimmunity is that it results from aberrant immune response to an infection.
2 Innate immunity turns on and adapts adaptive immunity. Triggered by recognition of conserved molecular patterns.
4 LPS (lipopolysaccharide) exists only in bacteria.
7 Innate immune system should be perfect at recognition self vs nonself. SLE- lupus may have something wrong in toll-like receptor 9 which distinguishes human DNA from bacterial DNA.
10 Innate immunity- he focuses on signalling pathway. Opsonin is a word from the Greek language meaning “to prepare something to eat”- coat bacteria for easier ingestion.
15 Janeway- father of innate immunity.
16 PRRs recognize PAMPs. LPS is lipopolysacharride.
18 PRRs can be classified by location or by function.
19: complement coats microorganisms for phagocytosis. Pentraxin- C-reactive protein is a marker of acute infection. Pentraxin 3 may have role in pathogenesis of sepsis.
20: Dectin 1 senses fungi.
21: interferon induced proteins are antiviral.
nod-like receptors sense bits of broken down cell walls.
22: NOD 2 was discovered to be mutated in subset of patients with Crohn’s disease- may disrupt ability of normal flora to protect from infection.
26: Aspergillus is an opportunistic pathogen. toll negative ones died- normal ones were fine.
27: Toll receptors work completely differently in drosophila and man.
28: endotoxin is a misnomer- it is outside cell. It is lipopolysaccharide. ON surface.
Point mutation in TLR4 in mice makes them unable to respons to LPS.
29: LRR- leucine rich region. TIR (Toll/Interleukin1 Receptor) homologous domain is signalling domain.
30: TLR 11 may prevent UTIs.
31: Two groups TLRs. The ones on surface react with complement of microorganisms on surface or released into cytoplasm. Other group recognizes components in endosomes. Bacterial DNA in cell binds to TLR9. MyD88 is signalling molecule for downstream function. MyD88 is required for TLR 9 and 5. TLR4 needs at least 3 other signalling molecules. Downstream are IRAKs.
32: TLRs in Drosophila are entirely indirect.
37: Krieg worked with antisense DNAs. Cytokines were activated in his controls. CpG DNA was recognized by innate system as foreign. In humans- CpG seen less often.
40 Chloroquine blocks endosomal acidification. Inhibitory oligo also blocks (iCpG). iCpG is the only blocker for a TLR.
48: DC are dendritic cells.
53: Exaggerated inflammatory response kills in septic shock.
“cytokine storm” gets cells to site of infection.
59: Most immune defects are in adaptive immunity.
In systemic lupus, patient may make immune response to his or her own DNA complexed to histones. Leading theory of autoimmunity is that it results from aberrant immune response to an infection.
Fain 52
we will review a bit at the end.What he tests on will come from 1-26 and statements on first page under 51b.
Today: pathways of glycolysis. No inborn errors known in these enzymes- if they aremissing, you die in utero.
E. Coli and man- glycolysis is the same process.
Gluconeogenesis is not the foundation of life- mammals must make glucose in liver to supply brain.
3: summary of next few days.
Pyruvate to acetyl coA is the only one-way pathway on this chart. Ethanol comes in as acetyl coA, so if 2 people are stranded, one with alcohol and one with sugar, the person with sugar lasts longer.
Anaerobic oxidation yields lactate in man.
4: 2 pyruvates from every glucose. 2ATPs to prime. Yields 4 ATPs. net:2 ATPs. Glucose to G6P isomerized to fructose 6-phosphate. Fructose can’t come in here- has to be handled in liver. rxn 2 is reversible using a phosphatase. Reversible steps are common to glycolysis and gluconeogenesis.
5: Know structure of pyruvate, acetate, lactate.
9: Know irreversible steps and Rate Limiting Step in glycolysis. PFK-1 is committed step. It is also the rate- limitng step. Hexokinase is not because product can proceed through different pathways.PFK-1 is allosterically regulated.
11: Most enzymes are inhibited by their products. When you have lots of ATP in a cell, you don’t want to make more by breaking down glucose. PFK-1 is activated by AMP. Citrate is from TCA cycle, indicating Acetyl-coA is high. These are 3 irreversible enzymes of glycolysis. Fructose 2,6 bisphosphate is a regulator to prevent futile cycling. Activates glycolysis and blocks gluconeogenesis.Dont want breakdown and synthesis at same time.
13: Aldolase splits fructose 1,6 bisphosphate.
15: no energy change in isomerization.
16: Glyceraldehyde-3-phosphate dehydrogenase used as a marker because it is in every cell. Converts aldehyde to hydroxyl group. 2Hs removed, one from water.
17: Change phosphorylation to phosphorolysis.
18: Sulfhydryl group is in active site of enzyme. NAD is essential coenzyme. Step 4: phosphorylytic attack. Ordinalily water would attack there. Active site is deep within enzyme and excludes water, but has binding site for phosphate.
25: Ferment- life without oxygen. Lactate dehydrogenase converts pyruvate to lactate. Dehydrogenates lactate to give you pyruvate.
27: Important step. Cannot go back. Get Ethanol and NAD+. Yeast does this. Good test question: If a certain child is sick and does not eat, gets hypoglycemic coma. Happy all the time- producing alcohol- problem- bleeding glucose needed for brain into alcohol. Yeast make ethanol. Can be further fermented to acetic acid.
This does not happen in people in real life- made up test question.
30: energetics.
32: Your body only gets 33% of work out to ATP production. Rest goes to generate heat.
33: Repeat slide. Know this.
Allosteric- any site except catalytic site to which something binds. Hexokinase has more than one site for binding of ATP.
35: Pyruvate comes from amino acids.
39: What biotin does.
40: Reaction involves GTP.
42: Mammals have glucose 6-phosphatase only in liver.
43: Cori cycle- lactate to liver to convert to pyruvate and glucose. Alanine cycle gets amine to liver in chronic starvation, breaking down muscle protein.
44: Cori cycle.
45: Alanine cycle. Nitrogen converted to urea in liver.
46: Substrate cycling is not the same as futile cycling.From pyruvate to glucose, need 4 ATP.
52: Why both functions on same enzyme? one function can be turned on, the other off.
Few minutes of review:
on 1-26 on handout. Most are statements of what he expects us to know.
4. What are 2 functions of pentose phosphate pathway that can be carried out by glycogenolysis.
What are 3 irreversible steps in glycolysis?
7. Which enzyme forms NADH in glycolysis? G3PdH.
8. Name 2 enzymes.
9. Where does lactate go in man vs. yeast? What happens to NADH?
11. No enzyme to bypass pyruvate decarboxylase.
12. Name steps.
13. What is role of biotin?
14 and 15- irreversible steps.
16. Why is lactate released from muscle during anaerobic exercise, alanine during starvation?
Turn back to 51b.
What form is energy captured in? ion gradients. What are they used to do? Pump substances against their gradients or make ATP.
Why can you convert creatine phosphate to ATP but not Glucose 1-p to ATP? phosphoryl potential.
Digestion in gut does what for metabolism?
What is difference between use of NADPH and NADH?
Where do the major reactions of metabolism listed occur in glycolysis? (not ligation)
How do we regulate metabolism?
1. amount substrate
2. allosteric modification or covalent modification by phosphorylation
3. synthesis and/or degradation to control the amount of enzyme
Be familiar with glycolysis and gluconeogenesis.
Today: pathways of glycolysis. No inborn errors known in these enzymes- if they aremissing, you die in utero.
E. Coli and man- glycolysis is the same process.
Gluconeogenesis is not the foundation of life- mammals must make glucose in liver to supply brain.
3: summary of next few days.
Pyruvate to acetyl coA is the only one-way pathway on this chart. Ethanol comes in as acetyl coA, so if 2 people are stranded, one with alcohol and one with sugar, the person with sugar lasts longer.
Anaerobic oxidation yields lactate in man.
4: 2 pyruvates from every glucose. 2ATPs to prime. Yields 4 ATPs. net:2 ATPs. Glucose to G6P isomerized to fructose 6-phosphate. Fructose can’t come in here- has to be handled in liver. rxn 2 is reversible using a phosphatase. Reversible steps are common to glycolysis and gluconeogenesis.
5: Know structure of pyruvate, acetate, lactate.
9: Know irreversible steps and Rate Limiting Step in glycolysis. PFK-1 is committed step. It is also the rate- limitng step. Hexokinase is not because product can proceed through different pathways.PFK-1 is allosterically regulated.
11: Most enzymes are inhibited by their products. When you have lots of ATP in a cell, you don’t want to make more by breaking down glucose. PFK-1 is activated by AMP. Citrate is from TCA cycle, indicating Acetyl-coA is high. These are 3 irreversible enzymes of glycolysis. Fructose 2,6 bisphosphate is a regulator to prevent futile cycling. Activates glycolysis and blocks gluconeogenesis.Dont want breakdown and synthesis at same time.
13: Aldolase splits fructose 1,6 bisphosphate.
15: no energy change in isomerization.
16: Glyceraldehyde-3-phosphate dehydrogenase used as a marker because it is in every cell. Converts aldehyde to hydroxyl group. 2Hs removed, one from water.
17: Change phosphorylation to phosphorolysis.
18: Sulfhydryl group is in active site of enzyme. NAD is essential coenzyme. Step 4: phosphorylytic attack. Ordinalily water would attack there. Active site is deep within enzyme and excludes water, but has binding site for phosphate.
25: Ferment- life without oxygen. Lactate dehydrogenase converts pyruvate to lactate. Dehydrogenates lactate to give you pyruvate.
27: Important step. Cannot go back. Get Ethanol and NAD+. Yeast does this. Good test question: If a certain child is sick and does not eat, gets hypoglycemic coma. Happy all the time- producing alcohol- problem- bleeding glucose needed for brain into alcohol. Yeast make ethanol. Can be further fermented to acetic acid.
This does not happen in people in real life- made up test question.
30: energetics.
32: Your body only gets 33% of work out to ATP production. Rest goes to generate heat.
33: Repeat slide. Know this.
Allosteric- any site except catalytic site to which something binds. Hexokinase has more than one site for binding of ATP.
35: Pyruvate comes from amino acids.
39: What biotin does.
40: Reaction involves GTP.
42: Mammals have glucose 6-phosphatase only in liver.
43: Cori cycle- lactate to liver to convert to pyruvate and glucose. Alanine cycle gets amine to liver in chronic starvation, breaking down muscle protein.
44: Cori cycle.
45: Alanine cycle. Nitrogen converted to urea in liver.
46: Substrate cycling is not the same as futile cycling.From pyruvate to glucose, need 4 ATP.
52: Why both functions on same enzyme? one function can be turned on, the other off.
Few minutes of review:
on 1-26 on handout. Most are statements of what he expects us to know.
4. What are 2 functions of pentose phosphate pathway that can be carried out by glycogenolysis.
What are 3 irreversible steps in glycolysis?
7. Which enzyme forms NADH in glycolysis? G3PdH.
8. Name 2 enzymes.
9. Where does lactate go in man vs. yeast? What happens to NADH?
11. No enzyme to bypass pyruvate decarboxylase.
12. Name steps.
13. What is role of biotin?
14 and 15- irreversible steps.
16. Why is lactate released from muscle during anaerobic exercise, alanine during starvation?
Turn back to 51b.
What form is energy captured in? ion gradients. What are they used to do? Pump substances against their gradients or make ATP.
Why can you convert creatine phosphate to ATP but not Glucose 1-p to ATP? phosphoryl potential.
Digestion in gut does what for metabolism?
What is difference between use of NADPH and NADH?
Where do the major reactions of metabolism listed occur in glycolysis? (not ligation)
How do we regulate metabolism?
1. amount substrate
2. allosteric modification or covalent modification by phosphorylation
3. synthesis and/or degradation to control the amount of enzyme
Be familiar with glycolysis and gluconeogenesis.
Tuesday, November 28, 2006
Fain 51
Fain's slides and his handouts in class are definite and clear.
Remember structures:
Methane CH4, methanol CH3OH, formaldehyde CH2=O, formic acid CH=OOH. Each has less energy than the one before it.
Handout- heavy type is the keynotes he will cover. Covered in slides.
51B is what is on powerpoints and what you will be tested on. Review of metabolism on page 1 and the 26 review questions and statements at the end of the handout are tested material. He will not be here Friday for review session, but will go over at end of lecture if he has time.
3 simple sugars- glucose, fructose, galactose.
Complex- in polymers. Polyhydroxyaldehydes and ketones.
4. Glyceraldehyde is simplest sugar.
8.All proteins are made of L amino acids. All sugars are D.
12.Noncyclized sugars are reactive and dangerous.
22. Why not store glucose free? osmotic problem. Stored in liver and muscle as glycogen.
24 With only one reducing end, less likely to react.
31 Ois universal donor- without glycoprotein on outside of cell, not antigenic.
AB can accept any type.
33 glycoprotein 120,000 is target for HIV (binding site)
38 We oxidize compounds for energy.See handout.
39 Making- anabolic. Breaking-catabolic.
40- good question for exam.
41 PEP formed during glycolysis has highest energy,
G1P is formed by glycogen breakdown.
42 Creatine phosphate is a storage form for ATP (20x more creatine phosphate in muscle than ATP). Reacts via creatine kinase.
All life uses ATP.
43 energy yields to go to CO2. When we burn methane, get all energy at once. Biology- get same end product stepwise to get ATP at each step.
46 nucleoside diphosphokinase interconverts GTP and ATP.
Carriers: NADH, NADPH, FADH. NADPH used for reductive biosynthesis. FADH and NADH used to generate ATP.
48 Allosteric is second site (beside catalytic one) on an enzyme.
Regulated by
1. amount of substrate
2. activity by allosteric regulation or covalent modification
3. synthesis/degradation
See slides and handouts.
Remember structures:
Methane CH4, methanol CH3OH, formaldehyde CH2=O, formic acid CH=OOH. Each has less energy than the one before it.
Handout- heavy type is the keynotes he will cover. Covered in slides.
51B is what is on powerpoints and what you will be tested on. Review of metabolism on page 1 and the 26 review questions and statements at the end of the handout are tested material. He will not be here Friday for review session, but will go over at end of lecture if he has time.
3 simple sugars- glucose, fructose, galactose.
Complex- in polymers. Polyhydroxyaldehydes and ketones.
4. Glyceraldehyde is simplest sugar.
8.All proteins are made of L amino acids. All sugars are D.
12.Noncyclized sugars are reactive and dangerous.
22. Why not store glucose free? osmotic problem. Stored in liver and muscle as glycogen.
24 With only one reducing end, less likely to react.
31 Ois universal donor- without glycoprotein on outside of cell, not antigenic.
AB can accept any type.
33 glycoprotein 120,000 is target for HIV (binding site)
38 We oxidize compounds for energy.See handout.
39 Making- anabolic. Breaking-catabolic.
40- good question for exam.
41 PEP formed during glycolysis has highest energy,
G1P is formed by glycogen breakdown.
42 Creatine phosphate is a storage form for ATP (20x more creatine phosphate in muscle than ATP). Reacts via creatine kinase.
All life uses ATP.
43 energy yields to go to CO2. When we burn methane, get all energy at once. Biology- get same end product stepwise to get ATP at each step.
46 nucleoside diphosphokinase interconverts GTP and ATP.
Carriers: NADH, NADPH, FADH. NADPH used for reductive biosynthesis. FADH and NADH used to generate ATP.
48 Allosteric is second site (beside catalytic one) on an enzyme.
Regulated by
1. amount of substrate
2. activity by allosteric regulation or covalent modification
3. synthesis/degradation
See slides and handouts.
Marion1
He will cover the immune system. Today’s lecture introduces the way it functions, originates, is organized, and provides background so that you can read and learn on your own. He will post on blackboard his notes. Much of lecture will be drawn on the blackboard in the room.
What is system for?
1. major function is to protect against pathogens(protection from infection)- sometimes must kill cells of self to eliminate virus.
2. surveillance to eliminate tumorigenic cells
3. tolerance to self (protects against autoimmunity)
4. self- control (protects against over-inflammation, over-multiplication of immune cells)
To protect against infection, must recognize potential pathogens. (See slide with graph of number of bacteria vs. time)
There is innate recognition and acquired recognition. Receptors allow the cell to know there is a pathogen around. Our health depends on both. Without innate immunity, the mouse (or a person) is completely susceptible to infection without ability to get rid of it. With innate but without acquired immunity, you can deal for a while, but eventually get overwhelmed (bubble boy).
Normal function- organism grows for a bit, but we get rid of it eventually.
How do the 2 systems work?
Tomorrow- innate immune function.
Way innate works- see paper. Pathogens are detected as a class. Recognition based on expression of molecules necessary to pathogen’s function, mostly on surface. Example in viruses, dsRNA. This response is fast. Receptors are all encoded in germ line and expressed on every immune cell of a particular type. Germ line receptors do not involve somatic manipulation. Non-clonotypic- every phagocyte has same types of receptors and same ability to respond. This response is quick. Innate response initiates adaptive immune response and maintains immune tolerance.
Disadvantage of innate response:
not efficient enough at clearing an infection. Adaptive immunity can focus the response initiated by innate immune response.
limited specific recognition because receptors are not designed for specifics- designed for pattern recognition. Microorganisms change over time. (limited diversity)
What does adaptive immunity have that innate does not? immunological memory. This is most important difference. Why important? secondary response. Enables us to eliminate diseases with vaccines.
Adaptive immunity- major functional difference from innate immunity- how receptors are generated. Innate encoded in germ line. Adaptive are somatically derived from same genetic information everyone inherits- used differently in each cell making receptors, resulting in incredible diversity. Receptors are highly specific to target a single epitope within a molecule. Also clonotypic- any given immune cell has only one type of receptor, and all its derivatives will be specific for one specific determinant.
Adaptive immunity focuses innnate immune response.
Has memory to maintain protective immunity.
Ablity to generate response to any antigen includes determinants that are self. Immune system culls cells specific to self.
Disadvantages of adaptive immunity:
autoimmunity
time (slow- takes time to clone cell)
memory is specific, so if pathogen changes, you don’t remember it (flu)
How and where immune cells and tissues develop:
1. hematopoiesis:
He will post diagram on blackboard. Schematic of hematopoeisis:
in neonate, hematopoiesis in liver. Embryo- in yolk sac in blood islands in chicken. This pattern exists in mammals. In embryo, develop in fetal liver. Then shifts to bone marrow at birth.
Precursor: HSC- hematopoietic stem cell. It divided to generate precursors and another of itself. 2 forms: long term HSC (used to reconstitute immunodeficient individual through bone marrow transplant- gives rise to short-term. Usually quiescent. Signals only partially understood – at surface of bone marrow stromal cells and cytokines push differentiation) and short term HSC. CLP- common lymphoid progenitor. Short-term HSC differentiates into common lymphoid or common myeloid progenitor.
CLP can diff into B lymphocyte, T lymphocyte, or NK (natural killer). Lymphocytes are responsible for adaptive immunity.
B lymphocyte produces antibody.
T lymphocyte can differentiate to cytotoxic T cell (kills virus-infected cells), helper T cells ( 2 types: helper T cell and inflammatory T cell.Differ in induced response).
NK cell- innate immune cells. No clonotypic receptors. receptors recognize self molecule on cells and are triggered by decreased expression or absence of self molecules.
Generation of these occurs in bone marrow. B cells in mammals come out of bone marrow fully mature. T cells come out as pre-T cells which must migrate to thymus to undergo differentiation and selection. Bone marrow and thymus are central lymphoid organs. Function is to generate functional lymphoid cells. Derivation of term T cells and B cells is from early studies of development in chickens. Discovered in 1940s. B cells mature in bursa of fabricius (outpocket of cloaca) in chickens. T cells mature in thymus.
B cells produce antibody (Ab).
Common myeloid progenitor yields MEP(megakaryocyte/erythrocyte progenitor) and GMP(granulocyte/ monocyte progenitor).
Granulocytes are PMNs. Monocytes become macrophages in tissues. Eosinophils, basophils , and maybe mast cells come from GMP. These are innate immune cells. All carry innate immune receptors and have potential to eliminate pathogens. Neutrophils,Monocytes, Eosinophild are phagocytic "to eat cells"- they engulf pathogens by opsonization.
Basophils- function still obscure. Mast cells live in tissues and make you miserable. Mediate inflammation through histamine. Important to mobilize innate and adaptive cells to deal with pathogen.
Inflammation is an innate immune response that mobilizes innate and adaptive immune cells. When innate cells encounter an infection, they cause leaky vessels, microclots, accumulation immune cells and fluid, flushes into afferent lymphatics to push into peripheral lymphoid organs to see if need to mount an immune response.
Another term
cytokine- soluble protein secreted by innate and adaptive immune cells to alter function of other cells. Cell-to cell communication about response.
interleukin or IL are a kind of cytokine. Mostly between lymphocytes, though monocytes use them, too.
CD antigen- means cluster of differentiation. They are cell surface proteins used as markers to ID a particular kind of cell.
CD34,CD45, ckit markers identify HSC.
C19, CD20, CD40, Ig identify B cells.
CD34, CD45, CD2, CD25, CD3, CD4, CD8, CD128, CD154 can identify T cell or T cell precursor.
CD11, CD80, CD86 ID monocyte macrophage or dendritic cells (from GMP- not effector cells for destruction- they stimulate adaptive immune response)
What happens to cells after they are produced?
Thymus is located just over heart in chest cavity. T cells differentiate there and enter peripheral lymphoid system.
Peripheral system: spleen, lymph nodes (sample fluid from Afferent lymphatics(open fluid-collecting vessels)) Lymphoid cells are exposed to potential antigens in lymph nodes. Maximizes exposure of lymph cells to antigen.
Why is immune system designed so the antigen is delivered to lymphocytes in lymph node? Why not deal with it at the site of infection? Site of memory cells is lymph node. The process would be more destructive with more imflammation if handled at site of infection. Activated cells leave lymph node via efferent vessels which coalesce at thoracic duct to reenter circulation. Effector cells can go into infected tissue via circulation.
First time cells enter lymph nodes- enter via high endothelial venule. (HEV)- receptors for non-activated cells.Activated- lose receptors on surface and leave. Lymph nodes filter body fluid (lymph). New ones come in an artery, then HEV, then go to specific areas. T cells go to paracortex. B cells go to cortex and form follicles. T cells migrate in and out. B cells stuck until activated, then migrate out to bone marrow as plasma cell and produce antibody for long periods of time.T cells recirculate.
Dendritic cells display antigens from pathogens to T cells in paracortical area of lymph node. B cells sample fluid and are activated with help of T cells.
Spleen similar- blood filter. Few lymphatics here, but filters blood instead of lymph. Blood in through arterioles into marginal sinus and red pulp. Pools into venous sinuses in spleen. Leaves spleen through venous circulation. Lymphocytes are organized around arteriole and there is region called PALS (Periarteriolar lymphoid sheath-T cell zone)-adjacent are follicles with B cells, then surrounded by marginal sinus or marginal zone with macrophages and dendritic cells to sample fluid. Most lymphocyte migration is within white pulp. Activated T cells can enter circulation. Only B cells leaving would be activated ones. See paper and slides for details. There are some details on blackboard as well.
What is system for?
1. major function is to protect against pathogens(protection from infection)- sometimes must kill cells of self to eliminate virus.
2. surveillance to eliminate tumorigenic cells
3. tolerance to self (protects against autoimmunity)
4. self- control (protects against over-inflammation, over-multiplication of immune cells)
To protect against infection, must recognize potential pathogens. (See slide with graph of number of bacteria vs. time)
There is innate recognition and acquired recognition. Receptors allow the cell to know there is a pathogen around. Our health depends on both. Without innate immunity, the mouse (or a person) is completely susceptible to infection without ability to get rid of it. With innate but without acquired immunity, you can deal for a while, but eventually get overwhelmed (bubble boy).
Normal function- organism grows for a bit, but we get rid of it eventually.
How do the 2 systems work?
Tomorrow- innate immune function.
Way innate works- see paper. Pathogens are detected as a class. Recognition based on expression of molecules necessary to pathogen’s function, mostly on surface. Example in viruses, dsRNA. This response is fast. Receptors are all encoded in germ line and expressed on every immune cell of a particular type. Germ line receptors do not involve somatic manipulation. Non-clonotypic- every phagocyte has same types of receptors and same ability to respond. This response is quick. Innate response initiates adaptive immune response and maintains immune tolerance.
Disadvantage of innate response:
not efficient enough at clearing an infection. Adaptive immunity can focus the response initiated by innate immune response.
limited specific recognition because receptors are not designed for specifics- designed for pattern recognition. Microorganisms change over time. (limited diversity)
What does adaptive immunity have that innate does not? immunological memory. This is most important difference. Why important? secondary response. Enables us to eliminate diseases with vaccines.
Adaptive immunity- major functional difference from innate immunity- how receptors are generated. Innate encoded in germ line. Adaptive are somatically derived from same genetic information everyone inherits- used differently in each cell making receptors, resulting in incredible diversity. Receptors are highly specific to target a single epitope within a molecule. Also clonotypic- any given immune cell has only one type of receptor, and all its derivatives will be specific for one specific determinant.
Adaptive immunity focuses innnate immune response.
Has memory to maintain protective immunity.
Ablity to generate response to any antigen includes determinants that are self. Immune system culls cells specific to self.
Disadvantages of adaptive immunity:
autoimmunity
time (slow- takes time to clone cell)
memory is specific, so if pathogen changes, you don’t remember it (flu)
How and where immune cells and tissues develop:
1. hematopoiesis:
He will post diagram on blackboard. Schematic of hematopoeisis:
in neonate, hematopoiesis in liver. Embryo- in yolk sac in blood islands in chicken. This pattern exists in mammals. In embryo, develop in fetal liver. Then shifts to bone marrow at birth.
Precursor: HSC- hematopoietic stem cell. It divided to generate precursors and another of itself. 2 forms: long term HSC (used to reconstitute immunodeficient individual through bone marrow transplant- gives rise to short-term. Usually quiescent. Signals only partially understood – at surface of bone marrow stromal cells and cytokines push differentiation) and short term HSC. CLP- common lymphoid progenitor. Short-term HSC differentiates into common lymphoid or common myeloid progenitor.
CLP can diff into B lymphocyte, T lymphocyte, or NK (natural killer). Lymphocytes are responsible for adaptive immunity.
B lymphocyte produces antibody.
T lymphocyte can differentiate to cytotoxic T cell (kills virus-infected cells), helper T cells ( 2 types: helper T cell and inflammatory T cell.Differ in induced response).
NK cell- innate immune cells. No clonotypic receptors. receptors recognize self molecule on cells and are triggered by decreased expression or absence of self molecules.
Generation of these occurs in bone marrow. B cells in mammals come out of bone marrow fully mature. T cells come out as pre-T cells which must migrate to thymus to undergo differentiation and selection. Bone marrow and thymus are central lymphoid organs. Function is to generate functional lymphoid cells. Derivation of term T cells and B cells is from early studies of development in chickens. Discovered in 1940s. B cells mature in bursa of fabricius (outpocket of cloaca) in chickens. T cells mature in thymus.
B cells produce antibody (Ab).
Common myeloid progenitor yields MEP(megakaryocyte/erythrocyte progenitor) and GMP(granulocyte/ monocyte progenitor).
Granulocytes are PMNs. Monocytes become macrophages in tissues. Eosinophils, basophils , and maybe mast cells come from GMP. These are innate immune cells. All carry innate immune receptors and have potential to eliminate pathogens. Neutrophils,Monocytes, Eosinophild are phagocytic "to eat cells"- they engulf pathogens by opsonization.
Basophils- function still obscure. Mast cells live in tissues and make you miserable. Mediate inflammation through histamine. Important to mobilize innate and adaptive cells to deal with pathogen.
Inflammation is an innate immune response that mobilizes innate and adaptive immune cells. When innate cells encounter an infection, they cause leaky vessels, microclots, accumulation immune cells and fluid, flushes into afferent lymphatics to push into peripheral lymphoid organs to see if need to mount an immune response.
Another term
cytokine- soluble protein secreted by innate and adaptive immune cells to alter function of other cells. Cell-to cell communication about response.
interleukin or IL are a kind of cytokine. Mostly between lymphocytes, though monocytes use them, too.
CD antigen- means cluster of differentiation. They are cell surface proteins used as markers to ID a particular kind of cell.
CD34,CD45, ckit markers identify HSC.
C19, CD20, CD40, Ig identify B cells.
CD34, CD45, CD2, CD25, CD3, CD4, CD8, CD128, CD154 can identify T cell or T cell precursor.
CD11, CD80, CD86 ID monocyte macrophage or dendritic cells (from GMP- not effector cells for destruction- they stimulate adaptive immune response)
What happens to cells after they are produced?
Thymus is located just over heart in chest cavity. T cells differentiate there and enter peripheral lymphoid system.
Peripheral system: spleen, lymph nodes (sample fluid from Afferent lymphatics(open fluid-collecting vessels)) Lymphoid cells are exposed to potential antigens in lymph nodes. Maximizes exposure of lymph cells to antigen.
Why is immune system designed so the antigen is delivered to lymphocytes in lymph node? Why not deal with it at the site of infection? Site of memory cells is lymph node. The process would be more destructive with more imflammation if handled at site of infection. Activated cells leave lymph node via efferent vessels which coalesce at thoracic duct to reenter circulation. Effector cells can go into infected tissue via circulation.
First time cells enter lymph nodes- enter via high endothelial venule. (HEV)- receptors for non-activated cells.Activated- lose receptors on surface and leave. Lymph nodes filter body fluid (lymph). New ones come in an artery, then HEV, then go to specific areas. T cells go to paracortex. B cells go to cortex and form follicles. T cells migrate in and out. B cells stuck until activated, then migrate out to bone marrow as plasma cell and produce antibody for long periods of time.T cells recirculate.
Dendritic cells display antigens from pathogens to T cells in paracortical area of lymph node. B cells sample fluid and are activated with help of T cells.
Spleen similar- blood filter. Few lymphatics here, but filters blood instead of lymph. Blood in through arterioles into marginal sinus and red pulp. Pools into venous sinuses in spleen. Leaves spleen through venous circulation. Lymphocytes are organized around arteriole and there is region called PALS (Periarteriolar lymphoid sheath-T cell zone)-adjacent are follicles with B cells, then surrounded by marginal sinus or marginal zone with macrophages and dendritic cells to sample fluid. Most lymphocyte migration is within white pulp. Activated T cells can enter circulation. Only B cells leaving would be activated ones. See paper and slides for details. There are some details on blackboard as well.
Saturday, November 25, 2006
Calcium and Phosphate Regulation
Regulation of Calcium and Phosphate
Most Ca in our bodies is in bone-reserve if we need it.
PTH activates vitamin D.
Oxalates in diet may bind Ca so it is not free to be absorbed.
Gut absorbs 700 mg and secretes 600 mg.
Bone breakdown releases Ca and phosphate.
Remember the terminology.
Forms of Ca:
Normal plasma Ca in 3 units:
mg/dL is total Ca content. 3 forms:
1. bound to plasma protein
2. complexed with anions
3. free Ca2+, Ca++, or free Ca.
Bound to protein is not filtered- the other 2 forms are.
Phosphate- less is bound to protein. More is complexed to cations. Know the numbers for phosphate and how it is different. Plasma phosphate varies- doubles in adults in the afternoon- nobody knows why. Also double listed value in children. Why important to regulate:
Muscle tetany. The ionized Ca is what affects things.
Other units- 10 mg/dl=2.5 mM or 5 mEq/L.
Kidneys are where Ca concentration can be regulated.
Phosphates are used as preservatives, so intake can be quite high. People who drink colas with phosphate can have high intake.
4 parathyroid glands. PTH protects against hypocalcemia. It is a protein with about 84 AAs. Many fragments circulate in plasma. For valid measurement of this hormone, need 2-site immunoassay for C and N terminal ends so the assay does not react for just one end, picking up a fraction in fragmented inactive form.
Graph in our handout is deceptive. PTH and calcitonin- slopes should be steeper. People with chronic low Ca- system regultes PTH- it becomes more sensitibve. Gland senses ionized Ca.
PTH is regulated by decreased degradation of hormone in gland.Gland makes and breaks down PTH a lot.
People who do not convert vitamin D can be treated with very high doses vitamin D and it will work.
Calcitonin or thyrocalcitonoin comes from thyroid. Weak hormone- probably almost no effect under most circumstances. To study- infuse Ca. That can be dangerous, so inject pentagastrin to look at secretion.
Response of PTH calciferolaxis to hypocalcemia graph- induced hypocalcemia- lowers serum Ca levels. PTH goes up 2.5x. Ca back up, PTH falls. PTH stimulates the activation of vitamin D, with a delay of 12-24 hours. Urinary cAMP spikes as well.
Other influences:
excess glucocorticoids, estrogens keep bones strong.
Text image- osteoBLasts build bone. Some get trapped inside and are called osteocytes. They connect to osteoblasts on surface by tight junctions. Can ship out Ca. OsteoCLasts are multinucleated cells which cause bone breakdown.
Osteocalcin secreted by osteoblasts. Osteocytes have most rapid cycling Ca into and out of bone. between cells and bone is bone fluid, high in Ca and phosphate. Ca can be sent out from osteocytes via osteoblasts.
Osteoclasts- no PTH receptors. Signal probably comes from osteoblasts through cytokine signalling. This signaling is inhibited by estrogens, which is how they protect bone from breakdown. Calcitonin also inhibits osteoclasts.
Ca pump from osteoblast sends Ca out to extracellular fluid.
See fig 4 for summary.
Kidneys:
Regulation in distal tubule is stimulated by PTH and maybe vitamin D. Excretion Ca decreased by PTH and vitamin D, unless a huge amount of calcium suddenly is coming in.
PTH acts on proximal tubule for phosphate to DECREASE reabsorption and flush it out. Broad range, highly variable.
Vitamin D precursor is cholesterol. B ring opened in vitamin D. 3 step activation: skin/diet, liver (calcidiol), kidney (calcitriol1- or 24- hydroxylation)
Liver uses enzyme 25-hydroxylase which is negatively regulated by 25-OH vitamin D.
What does vitamin D do? increases Ca and Phosphate absorption in the intestine.
Ca binding protein 2 functions: binds Ca absorbed to maintain gradient so more is absorbed. Ca levels kept low to not foul up metabolism.
2. increases activity of Ca pump to ECF. Vitamin D also promoted phosphate absorption by mechanisms nobody has paid attention to.
Notes on Problem Set:
Functions of Bone:
1. Support vs. gravity
2. movement muscles attach and work on
3. protection (skull, rib cage, pelvis)
4. Calcium reservoir (and phosphate)
5. blood cell formation in marrow
Other questions:
2 and 3 gone over in class today.
4. Too much PTH- primary hyperparathyroidism
A. Ca increased, because high PTH stimulates osteoclasts, resulting in bone breakdown and osteoblast Ca secretion. Kidneys increase reabsorption. Intestine- indirectly increases Ca absorption by increase in vitamin D. Urinary Ca excretion increases, esp chronically. Kidneys overwhelmed by increase in filtered load. Hypercalciuria happens.
B Phosphate: more from bone and intestine, decreased reabsorption in kidneys. increased excretion from kidneys. Plasma phosphate stays relatively constant, or slight decrease.
Likely to get kidney stones. Avoid stones- drink a lot of water.
C. Changes in bone metabolism:
demineralized bones, more likely to break.
5:
damage to proximal tubules AND glomeruli(add that part).
BSAP is from osteoblasts, stimulated by PTH.
proteinuria caused by protein leakage. Liver is fine. 1st step vitamin D activation probably OK. Step 2 activation of vitamin D is in proximal tubule- impaired 1,25 vitamin D formation. Less absorption Ca. Stimulates PTH.
A. low bone density.
B. 1,25 low.
Most Ca in our bodies is in bone-reserve if we need it.
PTH activates vitamin D.
Oxalates in diet may bind Ca so it is not free to be absorbed.
Gut absorbs 700 mg and secretes 600 mg.
Bone breakdown releases Ca and phosphate.
Remember the terminology.
Forms of Ca:
Normal plasma Ca in 3 units:
mg/dL is total Ca content. 3 forms:
1. bound to plasma protein
2. complexed with anions
3. free Ca2+, Ca++, or free Ca.
Bound to protein is not filtered- the other 2 forms are.
Phosphate- less is bound to protein. More is complexed to cations. Know the numbers for phosphate and how it is different. Plasma phosphate varies- doubles in adults in the afternoon- nobody knows why. Also double listed value in children. Why important to regulate:
Muscle tetany. The ionized Ca is what affects things.
Other units- 10 mg/dl=2.5 mM or 5 mEq/L.
Kidneys are where Ca concentration can be regulated.
Phosphates are used as preservatives, so intake can be quite high. People who drink colas with phosphate can have high intake.
4 parathyroid glands. PTH protects against hypocalcemia. It is a protein with about 84 AAs. Many fragments circulate in plasma. For valid measurement of this hormone, need 2-site immunoassay for C and N terminal ends so the assay does not react for just one end, picking up a fraction in fragmented inactive form.
Graph in our handout is deceptive. PTH and calcitonin- slopes should be steeper. People with chronic low Ca- system regultes PTH- it becomes more sensitibve. Gland senses ionized Ca.
PTH is regulated by decreased degradation of hormone in gland.Gland makes and breaks down PTH a lot.
People who do not convert vitamin D can be treated with very high doses vitamin D and it will work.
Calcitonin or thyrocalcitonoin comes from thyroid. Weak hormone- probably almost no effect under most circumstances. To study- infuse Ca. That can be dangerous, so inject pentagastrin to look at secretion.
Response of PTH calciferolaxis to hypocalcemia graph- induced hypocalcemia- lowers serum Ca levels. PTH goes up 2.5x. Ca back up, PTH falls. PTH stimulates the activation of vitamin D, with a delay of 12-24 hours. Urinary cAMP spikes as well.
Other influences:
excess glucocorticoids, estrogens keep bones strong.
Text image- osteoBLasts build bone. Some get trapped inside and are called osteocytes. They connect to osteoblasts on surface by tight junctions. Can ship out Ca. OsteoCLasts are multinucleated cells which cause bone breakdown.
Osteocalcin secreted by osteoblasts. Osteocytes have most rapid cycling Ca into and out of bone. between cells and bone is bone fluid, high in Ca and phosphate. Ca can be sent out from osteocytes via osteoblasts.
Osteoclasts- no PTH receptors. Signal probably comes from osteoblasts through cytokine signalling. This signaling is inhibited by estrogens, which is how they protect bone from breakdown. Calcitonin also inhibits osteoclasts.
Ca pump from osteoblast sends Ca out to extracellular fluid.
See fig 4 for summary.
Kidneys:
Regulation in distal tubule is stimulated by PTH and maybe vitamin D. Excretion Ca decreased by PTH and vitamin D, unless a huge amount of calcium suddenly is coming in.
PTH acts on proximal tubule for phosphate to DECREASE reabsorption and flush it out. Broad range, highly variable.
Vitamin D precursor is cholesterol. B ring opened in vitamin D. 3 step activation: skin/diet, liver (calcidiol), kidney (calcitriol1- or 24- hydroxylation)
Liver uses enzyme 25-hydroxylase which is negatively regulated by 25-OH vitamin D.
What does vitamin D do? increases Ca and Phosphate absorption in the intestine.
Ca binding protein 2 functions: binds Ca absorbed to maintain gradient so more is absorbed. Ca levels kept low to not foul up metabolism.
2. increases activity of Ca pump to ECF. Vitamin D also promoted phosphate absorption by mechanisms nobody has paid attention to.
Notes on Problem Set:
Functions of Bone:
1. Support vs. gravity
2. movement muscles attach and work on
3. protection (skull, rib cage, pelvis)
4. Calcium reservoir (and phosphate)
5. blood cell formation in marrow
Other questions:
2 and 3 gone over in class today.
4. Too much PTH- primary hyperparathyroidism
A. Ca increased, because high PTH stimulates osteoclasts, resulting in bone breakdown and osteoblast Ca secretion. Kidneys increase reabsorption. Intestine- indirectly increases Ca absorption by increase in vitamin D. Urinary Ca excretion increases, esp chronically. Kidneys overwhelmed by increase in filtered load. Hypercalciuria happens.
B Phosphate: more from bone and intestine, decreased reabsorption in kidneys. increased excretion from kidneys. Plasma phosphate stays relatively constant, or slight decrease.
Likely to get kidney stones. Avoid stones- drink a lot of water.
C. Changes in bone metabolism:
demineralized bones, more likely to break.
5:
damage to proximal tubules AND glomeruli(add that part).
BSAP is from osteoblasts, stimulated by PTH.
proteinuria caused by protein leakage. Liver is fine. 1st step vitamin D activation probably OK. Step 2 activation of vitamin D is in proximal tubule- impaired 1,25 vitamin D formation. Less absorption Ca. Stimulates PTH.
A. low bone density.
B. 1,25 low.
Sex Determination and Development
Sex Determination and Development
A person’s sex has 5 meanings- see first sheet.
Y chromosome has SRY gene- causes primordial gonads to become testes- otherwise they are ovaries.
TDF- testis determining factor makes primordial gonad become a testis. What is this factor? Not quite known.
figures 9 and 10 in Johnson, page 729 for illustration of how gonads become ovaries and testes. Female- Mullerian ducts predominate. Male- Wolffian.
Remove gonads- without testes- get female pattern. Remove one testis, get male gender on the intact side, female ducts on the other without ovary (testis removed).
MIS-Mullerian inhibiting substance- active to cause development of Wolffian ducts.
Muscle growth in men is induced by androgens. Steroids influence gonadotrpoin secretion.
General pattern of Hormonal control:
Hypothalamus secretes GnRH to Anterior pituitary. Gonadotrophs secrete LH and FSH. LH and FSH are the same male and female. Cell type A in males and females synthesizes and secretes androgens (M-testosterone, females testosterone and other androgens). The androgens diffuse to affect type B along with FSH to promote development of germ cells. Type B tends to secrete inhibin which feeds back negatively to the pituitary gland to reduce secretion of FSH. Gonads also release substances that affect skin, brain , muscles in stimulatory or inhibitory ways.
Effect of androgen- affects pituitary(Reduces LH, and reduces FSH at high doses) and hypothalmus (affects GnRH) in a negative way. Feedback inhibition. gonadotrophs under inhibition respond less to what GnRH they do get than they usually would.
Male Reproductive System
for testes to produce sperm, they function lower than abdominal temperature. Temperature is maintained about 1- ½ degrees C lower by testicular movement, hair (to insulate to stay warm) and sweat glands, circulatory system. Humans cannot pull in testes completely (rats can).
Ram (diagram in notes)- big temperature differential. Countercurrent heat exchanger. Warmer blood of arteries gives up heat to cooler venous blood. This occurs in the pampiniform plexus. Arteries and veins are highly coiled and intertwined.
Semen is the product of testes plus accessory glands. Most volume from seminal vesicles (60%),then prostate, then sperm.
Testes- Leydig cells produce testosterone. See diagram from text, figure 1 page 721. Sperm are produced at edges of tubules. Interstitum has Leydig cells. Tubular lumen- Sertoli or nurse cells facilitate development of sperm from primary spermatogonia, to spermatocyte, to spermatid, to spermatozoa, to mature sperm.
High concentration of testosterone are due to LH and GnRH. (2 indirect hormones)
Why is there a high concentration of testosterone? Produced by Leydig cells and diffuses across to Sertoli cells. These respond to FSH and testosterone to enhance production of sperm. If there are no Leydig cells, a really high dose of testosterone is necessary to produce sperm.
Tight junctions keep materials in circulation from reaching sperm. Protects sperm from materials in blood.
Sertoli- nourish development of sperm.
blood-testis barrier is a barrier to chemicals. It protects sperm from harm to som extent.
ABP (androgen binding protein) binds testosterone and carries along with sperm. Sertoli cells phagocytose defective sperm. Size of cytoplasm is greatly reduced during sperm development. Sertoli cells do this. Not all defective sperm are detected.
Testosterone shows some diurnal variation, but it is considered pretty steady compared to monthly cycling of females.
Some testosterone must be activated to be effective. DHT activation occurs in target cells. DHT binds receptor, goes to nucleus, affects gene transcription.
Testosterone can be converted to estrogen by aromatase. This happens in brain, pituitary, liver, breast, adipose tissue. Obese boys get apparent mammary gland development from higher estrogens from adipose tissues promoting fat deposition in mammary areas.
Degradation:
1. 5B reductase.
2. 17-OH->17keto->17-ketosteroids
3. conjugation with sulfate or glucoronate
4. urinary excretion- measure 17-KS in urine.
17-KS in person with liver disease decreases. Adipose tissue may convert excess testosterone to estrogens.
Do not have to know or replicate diagram of synthesis.
All steroids start with cholesterol. Androstenediol and dione are weak androgens. Most potent is testosterone. Enzyme making DHT is 5-alpha reductase. 5-beta reductase inactivates testosterone. Aromatase makes estradiol. All can be converted to water-soluble metabolites.
Tables indicate steroids in adult men and cycling women. Do not know numbers- look at patterns of similarity and units.
Most testosterone is bound, but only free can get out to target tissues.
Androstenedione and testosterone in both genders affect sex drive.
Men and women have PRL. May act on testes.
Other table- adult men. Most testosterone from testes. Most DHEA from adrenal glands- chief adrenal androgen.
Transport testosterone: see paper.
Know the lists for testosterone, estrogen, progesterone.
Testerone does not cause baldness. Genes do. Test allows it to happen. i and j are controversial.
General Pattern diagram:
A. Leydig cell. B-Sertoli cell.
Female Reproductive system:
See outline on paper.
Average menstrual cycle about 28 days long. day 0 is 1st day menstruation. Menstruation lasts 4-5days. Cells are sloughed off. Repairs and grows first half of cycle- ends at ovulation. Variations in length of cycle are due to variation in first part of cycle. Growth in new follicles is stimulated by FSH at end of previous cycle. In the middle, high estrogen causes midcycle surge of LH and FSH- LH triggers ovulation. then 2nd half.
Estrogen is high twice in the cycle, progesterone high only in second half.
As cells grow, they die and slough off. Midcycle with high estrogen- cornification- cells look like corn flakes. Sign that vagina has seen high levels of estrogen.
Estrogens are antiatherogenic. Increase HDL/LDL ratio. Estrogen increases HDL and decreases LDL. Estrogens
10f: increases suppleness, collagen content, water content of skin. Estrogens are added to some skin care products because of this.
Progesterone and progestigins :
5. BBT is basal body temperature. Ways to raise body temperature- muscle activity, coffee.
BBT measured same time every day, first thing in morning. BBTin mid -cycle dips, then rises. Higher temperature second half of cycle during increase of progesterone.
Ovary-
Both ovaries, each cycle- some primordial follicles start growing. dominant one on one ovary goes to surface. Mature follicle bulges, ruptures, egg released. The remaining granulosa and thecal cells turn into corpeus luteum (yellow from lipid)- makes progesterone and estrogen. Antrum is fluid- high in estrogen. Thecal cells produce testosterone, which gets turned into estrogen.
We still do not know what substance inhibits follicular growth. Follicle becomes atretic.
Inhibin decreases FSH secretion near the end of the follicular phase.
Another factor- GnSAF or GnSIF- gonadotropin cycle surge attenuating/inhibiting factor. Inhibits until dominant follicle is ready to release ovum.
To increase number of follicles, give FSH. Get multiple children. To get ovulation- treat with LH-like hormone.
Corpus luteum starts to develop as progesterone starts to rise.
hCG is human chorionic gonadotropin. It comes from placenta. Blastocyst implants and develops placenta- chorion produces chorionic gonadotropin to keep corpus luteum from regressing- continues to secrete estrogen and progesterone for successful pregnancy.
hCG goes up before end of 2 weeks to keep corpus luteum from degenerating. See text.
Ovary is key in regulation. Ovary regulates by estrogen it secretes. Need one functional ovary, portal connection, listed things in C.
Hypogonadal- low estrogen and progesterone. Hypogonadotropic- no pulsatile GnRH. Treat with pulses GnRH, stimulates ovaries. GnRH must be administered in pulses.
Reproductive Aging
Know difference hCG(LHlike) and hMG(FSH like)
Thelarche (pronounced "thelarkee") is beginning of breast development. Adrenarche or pubarche= hair growth. First period is after these. First ovulation may be around 13-14.
Boys- increase in FSH, testicular size, LH up, testosterone up, other events start at same time.
Pregnancy
1. egg drawn into open end of oviduct. See paper for the rest.
hCG keeps corpus luteum working first 2 months. Estrogen and progesterone increase throughout pregnancy- from placenta after 1st 2 months. hPL- human placental lactogen or hCS- comes from chorion- acts like prl and GH. Gets mammary gland ready.
Page with physical changes- lots of systems altered in pregnancy. You do not have to memorize these.
Please note that a LOT of the notetaking for this involved drawing arrows in little blanks on the paper he handed out in class. See paper for someone who was there.
A person’s sex has 5 meanings- see first sheet.
Y chromosome has SRY gene- causes primordial gonads to become testes- otherwise they are ovaries.
TDF- testis determining factor makes primordial gonad become a testis. What is this factor? Not quite known.
figures 9 and 10 in Johnson, page 729 for illustration of how gonads become ovaries and testes. Female- Mullerian ducts predominate. Male- Wolffian.
Remove gonads- without testes- get female pattern. Remove one testis, get male gender on the intact side, female ducts on the other without ovary (testis removed).
MIS-Mullerian inhibiting substance- active to cause development of Wolffian ducts.
Muscle growth in men is induced by androgens. Steroids influence gonadotrpoin secretion.
General pattern of Hormonal control:
Hypothalamus secretes GnRH to Anterior pituitary. Gonadotrophs secrete LH and FSH. LH and FSH are the same male and female. Cell type A in males and females synthesizes and secretes androgens (M-testosterone, females testosterone and other androgens). The androgens diffuse to affect type B along with FSH to promote development of germ cells. Type B tends to secrete inhibin which feeds back negatively to the pituitary gland to reduce secretion of FSH. Gonads also release substances that affect skin, brain , muscles in stimulatory or inhibitory ways.
Effect of androgen- affects pituitary(Reduces LH, and reduces FSH at high doses) and hypothalmus (affects GnRH) in a negative way. Feedback inhibition. gonadotrophs under inhibition respond less to what GnRH they do get than they usually would.
Male Reproductive System
for testes to produce sperm, they function lower than abdominal temperature. Temperature is maintained about 1- ½ degrees C lower by testicular movement, hair (to insulate to stay warm) and sweat glands, circulatory system. Humans cannot pull in testes completely (rats can).
Ram (diagram in notes)- big temperature differential. Countercurrent heat exchanger. Warmer blood of arteries gives up heat to cooler venous blood. This occurs in the pampiniform plexus. Arteries and veins are highly coiled and intertwined.
Semen is the product of testes plus accessory glands. Most volume from seminal vesicles (60%),then prostate, then sperm.
Testes- Leydig cells produce testosterone. See diagram from text, figure 1 page 721. Sperm are produced at edges of tubules. Interstitum has Leydig cells. Tubular lumen- Sertoli or nurse cells facilitate development of sperm from primary spermatogonia, to spermatocyte, to spermatid, to spermatozoa, to mature sperm.
High concentration of testosterone are due to LH and GnRH. (2 indirect hormones)
Why is there a high concentration of testosterone? Produced by Leydig cells and diffuses across to Sertoli cells. These respond to FSH and testosterone to enhance production of sperm. If there are no Leydig cells, a really high dose of testosterone is necessary to produce sperm.
Tight junctions keep materials in circulation from reaching sperm. Protects sperm from materials in blood.
Sertoli- nourish development of sperm.
blood-testis barrier is a barrier to chemicals. It protects sperm from harm to som extent.
ABP (androgen binding protein) binds testosterone and carries along with sperm. Sertoli cells phagocytose defective sperm. Size of cytoplasm is greatly reduced during sperm development. Sertoli cells do this. Not all defective sperm are detected.
Testosterone shows some diurnal variation, but it is considered pretty steady compared to monthly cycling of females.
Some testosterone must be activated to be effective. DHT activation occurs in target cells. DHT binds receptor, goes to nucleus, affects gene transcription.
Testosterone can be converted to estrogen by aromatase. This happens in brain, pituitary, liver, breast, adipose tissue. Obese boys get apparent mammary gland development from higher estrogens from adipose tissues promoting fat deposition in mammary areas.
Degradation:
1. 5B reductase.
2. 17-OH->17keto->17-ketosteroids
3. conjugation with sulfate or glucoronate
4. urinary excretion- measure 17-KS in urine.
17-KS in person with liver disease decreases. Adipose tissue may convert excess testosterone to estrogens.
Do not have to know or replicate diagram of synthesis.
All steroids start with cholesterol. Androstenediol and dione are weak androgens. Most potent is testosterone. Enzyme making DHT is 5-alpha reductase. 5-beta reductase inactivates testosterone. Aromatase makes estradiol. All can be converted to water-soluble metabolites.
Tables indicate steroids in adult men and cycling women. Do not know numbers- look at patterns of similarity and units.
Most testosterone is bound, but only free can get out to target tissues.
Androstenedione and testosterone in both genders affect sex drive.
Men and women have PRL. May act on testes.
Other table- adult men. Most testosterone from testes. Most DHEA from adrenal glands- chief adrenal androgen.
Transport testosterone: see paper.
Know the lists for testosterone, estrogen, progesterone.
Testerone does not cause baldness. Genes do. Test allows it to happen. i and j are controversial.
General Pattern diagram:
A. Leydig cell. B-Sertoli cell.
Female Reproductive system:
See outline on paper.
Average menstrual cycle about 28 days long. day 0 is 1st day menstruation. Menstruation lasts 4-5days. Cells are sloughed off. Repairs and grows first half of cycle- ends at ovulation. Variations in length of cycle are due to variation in first part of cycle. Growth in new follicles is stimulated by FSH at end of previous cycle. In the middle, high estrogen causes midcycle surge of LH and FSH- LH triggers ovulation. then 2nd half.
Estrogen is high twice in the cycle, progesterone high only in second half.
As cells grow, they die and slough off. Midcycle with high estrogen- cornification- cells look like corn flakes. Sign that vagina has seen high levels of estrogen.
Estrogens are antiatherogenic. Increase HDL/LDL ratio. Estrogen increases HDL and decreases LDL. Estrogens
10f: increases suppleness, collagen content, water content of skin. Estrogens are added to some skin care products because of this.
Progesterone and progestigins :
5. BBT is basal body temperature. Ways to raise body temperature- muscle activity, coffee.
BBT measured same time every day, first thing in morning. BBTin mid -cycle dips, then rises. Higher temperature second half of cycle during increase of progesterone.
Ovary-
Both ovaries, each cycle- some primordial follicles start growing. dominant one on one ovary goes to surface. Mature follicle bulges, ruptures, egg released. The remaining granulosa and thecal cells turn into corpeus luteum (yellow from lipid)- makes progesterone and estrogen. Antrum is fluid- high in estrogen. Thecal cells produce testosterone, which gets turned into estrogen.
We still do not know what substance inhibits follicular growth. Follicle becomes atretic.
Inhibin decreases FSH secretion near the end of the follicular phase.
Another factor- GnSAF or GnSIF- gonadotropin cycle surge attenuating/inhibiting factor. Inhibits until dominant follicle is ready to release ovum.
To increase number of follicles, give FSH. Get multiple children. To get ovulation- treat with LH-like hormone.
Corpus luteum starts to develop as progesterone starts to rise.
hCG is human chorionic gonadotropin. It comes from placenta. Blastocyst implants and develops placenta- chorion produces chorionic gonadotropin to keep corpus luteum from regressing- continues to secrete estrogen and progesterone for successful pregnancy.
hCG goes up before end of 2 weeks to keep corpus luteum from degenerating. See text.
Ovary is key in regulation. Ovary regulates by estrogen it secretes. Need one functional ovary, portal connection, listed things in C.
Hypogonadal- low estrogen and progesterone. Hypogonadotropic- no pulsatile GnRH. Treat with pulses GnRH, stimulates ovaries. GnRH must be administered in pulses.
Reproductive Aging
Know difference hCG(LHlike) and hMG(FSH like)
Thelarche (pronounced "thelarkee") is beginning of breast development. Adrenarche or pubarche= hair growth. First period is after these. First ovulation may be around 13-14.
Boys- increase in FSH, testicular size, LH up, testosterone up, other events start at same time.
Pregnancy
1. egg drawn into open end of oviduct. See paper for the rest.
hCG keeps corpus luteum working first 2 months. Estrogen and progesterone increase throughout pregnancy- from placenta after 1st 2 months. hPL- human placental lactogen or hCS- comes from chorion- acts like prl and GH. Gets mammary gland ready.
Page with physical changes- lots of systems altered in pregnancy. You do not have to memorize these.
Please note that a LOT of the notetaking for this involved drawing arrows in little blanks on the paper he handed out in class. See paper for someone who was there.
Naren 5 and 6
Naren Lecture 5:
Morning vesicular trafficking secretion, afternoon endocytosis.
Will test on objective 4 from the Morning’s lecture.
See red box slide 4. We are in Golgi- proteins destined to go outside cell or to lysosome.
"Regulated" means you need a signal for vesicles to secrete out.
"Constitutive" means protein is secreted constantly.
Albumin is an example of a constitutively-secreted protein.
There are differences between constitutive protein and regulated protein.
Regulated ones come from a common pool of vesicles. Selective protein aggregated in electron- dense packing. Express unique proteases.
9
Vesicle buds- brings bound ligand into cell.
Recycles.
How do we study pathways?
10:
Label and follow thorugh pathways. They use a temperature sensitive protein. With a high temperature folding defect, the protein gets stuck in ER. If you reduce the temperature, the protein folds properly. By 180 min- protein goes to the to cell surface. Graph- compare how much is where. Add cycloheximide- suppresses further synthesis so you can view trafficking.
Proteins receive further sugar modification in Golgi.
How to discriminate sugars- SDS Page- if the protein has added sugars, molecular weight shifts upwards (slide 16). Form higher in gel is sensitive to endoglycosidase D. You can follow which proteins are in the sensitive form by how they migrate before and after digestion. Densitometric scan tells you how much protein is coming out.
18:
wild type yeast secrete normal protein. Mutant yeast- identify pathway from ER to Golgi (cis, medial, trans) to vesicles to surface.
19:
Classical experiment- N-acetylglucosamine transferase present in wild type. Without it other residues not added on.
Took medial Golgi from cells without VSV infection. G protein and infected mutant cells (without the NAG transferase) and mixed. Found addition of NAG to G protein.
21
Coat has 2 principal functions:
1. concentration of specific membrane proteins into patches to help select protein in the vesicle for transport.
2. Assembly of coat proteins into curved lattice defines vesicle of uniform size for its type
22
Coat proteins in green. Fusion- coat falls off and snare protein bring membranes close together to fuse.
24:
Kinds of coats:
COPI, COPII, Clathrin.
Concentrate on COPII.
25:
How do we know coat is formed?
Purified ER membrane and saw electron dense areas (26).
What is molecular switch (on test): Slide 27. SAR 1 is switch. Binds Sec12, GTP exchanges for GDP, Coat forms, Coat excised off by hydrolysis of GTP to GDP.
How does assembly take place?
SEC24 has binding to cargo protein (slide 28). Hydrolysis- N terminal tail withdraws from membrane..
How do we know GTP is required?
Add as on slide 29. GTP gammaS is a nonhydrolysable form of GTP. In presence of this substance, the coat accumulates and can’t come off.
30:
Know circled area.
Know SAR1, GTP exchange, Sec23 promotes hydrolysis of Sar1.
Transport can be Anterograde (Forward) and retrograde (Back).
Why is there retrograde transport?
Things can get lost and come back. If a protein gets mis-sorted, some receptors bind it and it goes to COPI pathway and comes back.
PDI binds KDEL receptor and signals to go back.
35:
How does fusion take place?
Vis vesicle snare, t is target snare.
Snare hypothesis- 1991- there are SNARES that bind to each other and lead to the fusion process. Has been revised.
37
fused SNARE complex is interacting in parallel 4-helix bundle. 2 from SNAP25, 1 from syntaxin , 1 from VAMPII. V snare and t snare were all that was required for fusion.
NSF and SNAP have ATPase activity and can disassemble the SNARE complex.
40 Snare well studied in neurotransmission.
42- synaptotagmin is Ca sensor.
Botox acts on snare protein. Botulinum toxins and tetanus toxins can act on proteins.These toxins have been shown to inhibit neurotransmission
Review: docking and fusion, Rab, assembly of SNARES, membrane fusion, disassembly.
How do membranes fuse- highly debated.
47
Hemagluttinin can bind to sialic acid at globular domain. In endosome, pH is low. Conformational change and globular domain falls off as helical domain enters membrane.
It is like a spring-loaded gun.Membranes come close together and open up. SNARES may act like this, too.
50.
Summary
Review Role of SAR1 in assembly and disassembly of COPII coats. 5 steps.1. GTP exchange
2.Coat assembly
3. cargo protein
4 GTP hydrolysis
5. coat disassembly. Then review docking fusion, SNAREs.
He will post review slides.
Lecture 6-Thank You to Daniel!!!
Danielnotes—"The backup’s backup!"TM
Cell and Molecular Biology
A.P. Naren Ph. D
Lecture 6: Vesicular Traffic, Secretion, and Endocytosis
Note: the order he went in was different that the order the slides are arranged in the handout. Numbers in parentheses (ie. (6)) indicate the slide number from the PowerPoint that’s on Blackboard. I only reproduce here what was not on the slides themselves. A slide without any comment indicates that Dr. Naren added nothing that is not on the slide.
BOLD headings mean the slide is on the examination
(1) Title. Endocytosis is the internalizing of the outside of the cell. We will also talk about lysosomal traffic today.
(8) Objectives: only objective 4, receptor mediated endocytosis, is on the test
(2) Both exocytosis and endocytosis happen together as a constant process. Exocytosis secretes cargo out. Endocytosis internalizes cargo-containing vescicles.
(10)
(11) Clathrin is a protein that is a major constituent of vesicles in endocytosis
(9) Clathrin forms a cage around vesicles
(12) Clathrin forms the Triskelion structure
(13) The sizes of each chain are on the slide and were emphasized.
(14) 10 Triskelion form the assembly intermediate
(15) A movie on clathrin assembly. On Blackboard, perhaps?
(17) Dynamin, a GTPase, performs a pinching action, pinching off the vesicles
(18) Dynamin acts as a noose, pinching off the vesicle in response to GTP hydrolysis
(19) GTP hydrolysis is critical for this process
(20) In the flies in this example, there is a dynamin mutation. Protein is stuck in the membrane at 30 degrees C. At 20 degrees C, the protein folds normally, and escapes the membrane without incident
(Not in power point) What determines lysosome targeting?
(22) Manose 6 phosphate determines lysosome targeting via a two-step reaction process
(23)
(24)
(25) He spoke about Hurler’s syndrome and I-cell disease, the difference between the two being the lack of M6P in I-cell disease.
(26) A sugar enzyme is missing in Fabry’s disease. It also leads to mental retardation and death
(27)
(28) Transcytosis—both endocytosis and exocytosis in combination. In this diagram, the apical (luminal) side is on the right, and the basal (blood) side, on the left
(29) A good example of transcytosis is the transmission of maternal IgG antibodies from ingested breast milk to the neonatal blood stream via intestinal lumen absorption
(30)
(31)
(32) This topic is ON TEST. Receptor mediated endocytosis—the budding of vesicles from the plasma membrane, bringing specific bound ligands into the cell
(33) LDL particles hit LDL receptors in an LDL binding domain. It binds at pH 7 with high affinity.
(34) NPXY is the sorting signal (the X meaning any amino acid) and corresponds to YXXphi. The sorting signal binds to the AP2 complex. Then clathrin is recognized and the cage is formed
(35) LDL-ferritin here is electron dense
(36) LDL-ferritin here is inside the vesicle
(37) Don’t forget that dynamin is needed for this process
(39) Especially noted was the step where the clathrin coat is shedded
(38) When the vesicle fuses to the endosome, the endosome has a lower pH than the lumen
(41) All this occurs at pH 7.0
(42) All this occurs at the lower pH of the endosome, pH 5.0. The LDL particle is released
(40) Dark spots here have been labeled with gold
(39) After delivering the LDL to the endosome, the receptor is returned to the cell surface via another vesicle. The LDL goes into the lysosome for recycling and use.
(Not in powerpoint) Endocytosis can regulate the number of G-protein cell receptors at the cell surface
(44) When activated, the G-protein receptor is phosphorylation and other bits bind to it. These bits pull it into the cell to desensitize the surface, thus preventing hypersensitivity
(45) Number 2, autophagic enzymes digest bits of the cell itself in starvation type situations
(46) Only proteins that are slightly deranged are pulled in via the autophagic mechanism
(47) For example, viruses are internalized. The key here is the replicated viruses that bud outward
(48) ESCRT complex—ubiquitin binds to it. It is ATP dependent. It either internalizes cargo via multivesicles, or causes budding of cargo-containing vesicles
(49) (b) here is a case with a mutant ESCRT, so no budding can occur
(50) Important to remember from here are:
Clathrin, Adaptors, and dynamin in endocytosis
Sorting signal (YXXphi)
Dissociation (importance of pH)
(53) A schematic overview of the receptor mediated endocytosis pathway. Note Sorting signal, dissociation, and lysosome here.
(54)
At this point, Dr. Naren opened a new slideshow for exam review. It mainly consisted of slide 51 from our powerpoint, with a few alterations, which served to concisely summarize all his lectures. The slide he showed read the following :
REVIEW1. The importance of ER in protein translocation
What is cotranslational and post-translational translocation?
How are proteins inserted into ER membranes?
2. Glycosylation, disulfide bond formation
- Proper folding, and degradation of misfolded protein
3. Nuclear transport: Import (NLS signal) and export
4. Mitochondrial and peroxisomal protein transport
5. Vesicular traffic, secretion/exocytosis: Sar1 and COPII, docking and fusion of vesicles
6. Vesicular traffic, Secretion and Endocytosis: Receptor mediated endocytosis
Dr. Naren then gave a brief overview of each lecture that he had given. This provided no new information than that which he already gave in previous lectures, save that, for point 3, the RAN cycle and exchange factor are important.
Daniel Taylor
11/19/2006
Morning vesicular trafficking secretion, afternoon endocytosis.
Will test on objective 4 from the Morning’s lecture.
See red box slide 4. We are in Golgi- proteins destined to go outside cell or to lysosome.
"Regulated" means you need a signal for vesicles to secrete out.
"Constitutive" means protein is secreted constantly.
Albumin is an example of a constitutively-secreted protein.
There are differences between constitutive protein and regulated protein.
Regulated ones come from a common pool of vesicles. Selective protein aggregated in electron- dense packing. Express unique proteases.
9
Vesicle buds- brings bound ligand into cell.
Recycles.
How do we study pathways?
10:
Label and follow thorugh pathways. They use a temperature sensitive protein. With a high temperature folding defect, the protein gets stuck in ER. If you reduce the temperature, the protein folds properly. By 180 min- protein goes to the to cell surface. Graph- compare how much is where. Add cycloheximide- suppresses further synthesis so you can view trafficking.
Proteins receive further sugar modification in Golgi.
How to discriminate sugars- SDS Page- if the protein has added sugars, molecular weight shifts upwards (slide 16). Form higher in gel is sensitive to endoglycosidase D. You can follow which proteins are in the sensitive form by how they migrate before and after digestion. Densitometric scan tells you how much protein is coming out.
18:
wild type yeast secrete normal protein. Mutant yeast- identify pathway from ER to Golgi (cis, medial, trans) to vesicles to surface.
19:
Classical experiment- N-acetylglucosamine transferase present in wild type. Without it other residues not added on.
Took medial Golgi from cells without VSV infection. G protein and infected mutant cells (without the NAG transferase) and mixed. Found addition of NAG to G protein.
21
Coat has 2 principal functions:
1. concentration of specific membrane proteins into patches to help select protein in the vesicle for transport.
2. Assembly of coat proteins into curved lattice defines vesicle of uniform size for its type
22
Coat proteins in green. Fusion- coat falls off and snare protein bring membranes close together to fuse.
24:
Kinds of coats:
COPI, COPII, Clathrin.
Concentrate on COPII.
25:
How do we know coat is formed?
Purified ER membrane and saw electron dense areas (26).
What is molecular switch (on test): Slide 27. SAR 1 is switch. Binds Sec12, GTP exchanges for GDP, Coat forms, Coat excised off by hydrolysis of GTP to GDP.
How does assembly take place?
SEC24 has binding to cargo protein (slide 28). Hydrolysis- N terminal tail withdraws from membrane..
How do we know GTP is required?
Add as on slide 29. GTP gammaS is a nonhydrolysable form of GTP. In presence of this substance, the coat accumulates and can’t come off.
30:
Know circled area.
Know SAR1, GTP exchange, Sec23 promotes hydrolysis of Sar1.
Transport can be Anterograde (Forward) and retrograde (Back).
Why is there retrograde transport?
Things can get lost and come back. If a protein gets mis-sorted, some receptors bind it and it goes to COPI pathway and comes back.
PDI binds KDEL receptor and signals to go back.
35:
How does fusion take place?
Vis vesicle snare, t is target snare.
Snare hypothesis- 1991- there are SNARES that bind to each other and lead to the fusion process. Has been revised.
37
fused SNARE complex is interacting in parallel 4-helix bundle. 2 from SNAP25, 1 from syntaxin , 1 from VAMPII. V snare and t snare were all that was required for fusion.
NSF and SNAP have ATPase activity and can disassemble the SNARE complex.
40 Snare well studied in neurotransmission.
42- synaptotagmin is Ca sensor.
Botox acts on snare protein. Botulinum toxins and tetanus toxins can act on proteins.These toxins have been shown to inhibit neurotransmission
Review: docking and fusion, Rab, assembly of SNARES, membrane fusion, disassembly.
How do membranes fuse- highly debated.
47
Hemagluttinin can bind to sialic acid at globular domain. In endosome, pH is low. Conformational change and globular domain falls off as helical domain enters membrane.
It is like a spring-loaded gun.Membranes come close together and open up. SNARES may act like this, too.
50.
Summary
Review Role of SAR1 in assembly and disassembly of COPII coats. 5 steps.1. GTP exchange
2.Coat assembly
3. cargo protein
4 GTP hydrolysis
5. coat disassembly. Then review docking fusion, SNAREs.
He will post review slides.
Lecture 6-Thank You to Daniel!!!
Danielnotes—"The backup’s backup!"TM
Cell and Molecular Biology
A.P. Naren Ph. D
Lecture 6: Vesicular Traffic, Secretion, and Endocytosis
Note: the order he went in was different that the order the slides are arranged in the handout. Numbers in parentheses (ie. (6)) indicate the slide number from the PowerPoint that’s on Blackboard. I only reproduce here what was not on the slides themselves. A slide without any comment indicates that Dr. Naren added nothing that is not on the slide.
BOLD headings mean the slide is on the examination
(1) Title. Endocytosis is the internalizing of the outside of the cell. We will also talk about lysosomal traffic today.
(8) Objectives: only objective 4, receptor mediated endocytosis, is on the test
(2) Both exocytosis and endocytosis happen together as a constant process. Exocytosis secretes cargo out. Endocytosis internalizes cargo-containing vescicles.
(10)
(11) Clathrin is a protein that is a major constituent of vesicles in endocytosis
(9) Clathrin forms a cage around vesicles
(12) Clathrin forms the Triskelion structure
(13) The sizes of each chain are on the slide and were emphasized.
(14) 10 Triskelion form the assembly intermediate
(15) A movie on clathrin assembly. On Blackboard, perhaps?
(17) Dynamin, a GTPase, performs a pinching action, pinching off the vesicles
(18) Dynamin acts as a noose, pinching off the vesicle in response to GTP hydrolysis
(19) GTP hydrolysis is critical for this process
(20) In the flies in this example, there is a dynamin mutation. Protein is stuck in the membrane at 30 degrees C. At 20 degrees C, the protein folds normally, and escapes the membrane without incident
(Not in power point) What determines lysosome targeting?
(22) Manose 6 phosphate determines lysosome targeting via a two-step reaction process
(23)
(24)
(25) He spoke about Hurler’s syndrome and I-cell disease, the difference between the two being the lack of M6P in I-cell disease.
(26) A sugar enzyme is missing in Fabry’s disease. It also leads to mental retardation and death
(27)
(28) Transcytosis—both endocytosis and exocytosis in combination. In this diagram, the apical (luminal) side is on the right, and the basal (blood) side, on the left
(29) A good example of transcytosis is the transmission of maternal IgG antibodies from ingested breast milk to the neonatal blood stream via intestinal lumen absorption
(30)
(31)
(32) This topic is ON TEST. Receptor mediated endocytosis—the budding of vesicles from the plasma membrane, bringing specific bound ligands into the cell
(33) LDL particles hit LDL receptors in an LDL binding domain. It binds at pH 7 with high affinity.
(34) NPXY is the sorting signal (the X meaning any amino acid) and corresponds to YXXphi. The sorting signal binds to the AP2 complex. Then clathrin is recognized and the cage is formed
(35) LDL-ferritin here is electron dense
(36) LDL-ferritin here is inside the vesicle
(37) Don’t forget that dynamin is needed for this process
(39) Especially noted was the step where the clathrin coat is shedded
(38) When the vesicle fuses to the endosome, the endosome has a lower pH than the lumen
(41) All this occurs at pH 7.0
(42) All this occurs at the lower pH of the endosome, pH 5.0. The LDL particle is released
(40) Dark spots here have been labeled with gold
(39) After delivering the LDL to the endosome, the receptor is returned to the cell surface via another vesicle. The LDL goes into the lysosome for recycling and use.
(Not in powerpoint) Endocytosis can regulate the number of G-protein cell receptors at the cell surface
(44) When activated, the G-protein receptor is phosphorylation and other bits bind to it. These bits pull it into the cell to desensitize the surface, thus preventing hypersensitivity
(45) Number 2, autophagic enzymes digest bits of the cell itself in starvation type situations
(46) Only proteins that are slightly deranged are pulled in via the autophagic mechanism
(47) For example, viruses are internalized. The key here is the replicated viruses that bud outward
(48) ESCRT complex—ubiquitin binds to it. It is ATP dependent. It either internalizes cargo via multivesicles, or causes budding of cargo-containing vesicles
(49) (b) here is a case with a mutant ESCRT, so no budding can occur
(50) Important to remember from here are:
Clathrin, Adaptors, and dynamin in endocytosis
Sorting signal (YXXphi)
Dissociation (importance of pH)
(53) A schematic overview of the receptor mediated endocytosis pathway. Note Sorting signal, dissociation, and lysosome here.
(54)
At this point, Dr. Naren opened a new slideshow for exam review. It mainly consisted of slide 51 from our powerpoint, with a few alterations, which served to concisely summarize all his lectures. The slide he showed read the following :
REVIEW1. The importance of ER in protein translocation
What is cotranslational and post-translational translocation?
How are proteins inserted into ER membranes?
2. Glycosylation, disulfide bond formation
- Proper folding, and degradation of misfolded protein
3. Nuclear transport: Import (NLS signal) and export
4. Mitochondrial and peroxisomal protein transport
5. Vesicular traffic, secretion/exocytosis: Sar1 and COPII, docking and fusion of vesicles
6. Vesicular traffic, Secretion and Endocytosis: Receptor mediated endocytosis
Dr. Naren then gave a brief overview of each lecture that he had given. This provided no new information than that which he already gave in previous lectures, save that, for point 3, the RAN cycle and exchange factor are important.
Daniel Taylor
11/19/2006
Wednesday, November 22, 2006
Notes will be posted
Sorry for the delay, folks. I have notes for Naren 5 and 6 and the lectures this week for systems. My cat has become very ill and will have to be euthanized today(Wednesday). In addition I have 8 guests coming for Thanksgiving. Naturally this is delaying my postings somewhat, but they will happen by next Monday at the latest. Thank you for your patience.
Saturday, November 18, 2006
Naren 5 and 6
O.K., y'all. I was not there for lecture 6, so these will not be posted until someone returns the favor and ships me some notes, or I borrow a recording, or I reconstruct the notes from text, or some combination of the above happens. I was in an auto accident Monday and had to go take care of unpleasant business. I'm not hurt, so it could have been worse.
Diabetes Lecture Notes
These are my notes, but I REALLY recommend using his from Blackboard.
Insulin Actions in diabetes
He is posting a text from which he worked as well as the lecture.
Slide3:
Structure:
note disulfide bonds. A chain, B chain , C peptide. Insulin is a highly conserved hormone among all species. See figure 9 in Johnson, page 644
Oral glucose is more effective than IV glucose for inducing insulin release.
Insulin goes from pancreas to liver (portal circulation). Diabetics can only administer insulin subcutaneously, so it is hard to recreate release of insulin into the portal circulation.
7:
Insulin is key anabolic regulator. Supports protein synthesis in muscle, inhibits gluconeogenesis in liver. Other actions are general. Production of ketone bodies is important in diabetic ketoacidosis.
Important:glucose uptake, gluconeogenesis, ketogenesis in diabetes.
Type I Diabetes:
No phenotype (symptoms) for patient until beta cells are wiped out. Metabolic symptoms: hyperglycemia. Hormone sensitive lipase is activated in diabetes- Fatty acids go to liver, ketogenesis.
Historically- Langerhans defined the islets.
Oskar Minkowski demonstrated that removing pancreas in dog caused diabetes. Tasted dog’s urine-sweet. Postulated a hormone produced by the pancreas, but was unable to purify it. Banting and Best did purify it in 1921. Administered to dogs and were able to restore blood sugar. Provided first "cure" for previously fatal type I. 1923- Nobel prize.
Energy needs met in uncontrolled diabetes:
Alanine and alpha-ketoglutarate come from muscle to liver. There is no synthesis of fatty acids in adipose tissue.
Much of damage from diabetes is due to high blood sugar. Proteins get glycosylated inappropriately. Vision, circulatory problems in periphery, kidney problems, CV trouble.
Glucose transport:
There are 7 different GLUT transporters. Liver has bidirectional non-insulin dependent transporter (GLUT 2). Glut 4 is unidirectional.Absence of insulin- less transporter in muscle.
GLUT4 – 12 membrane spanning helices. Glut 4 in muscle has insulin-responsive glucose uptake.
They are sequestered in vesicles until insulin binds receptor and triggers movement to membrane through signalling cascade.
Capacity of muscles to take up glucose is increased 30 fold in the presence of these transporters (see Johnson chapter 41).
Muscle diagram
Exercise increases insulin-independent transport.
Adipose tissue: insulin stimulates lipoprotein lipase. Brings in fats for storage. Insulin also stimulates horm-sensitive lipase. Absence of insulin- free fatty acids are released and go to liver.
Liver diagram:
amino acids come in and gluconeogenesis is strongly activated in Type I diabetes.
Fatty acids enter TCA cycle and exit as ketone bodies.
Type I:
insulin imperative. Diet and exercise help to control the damage.
Problems: kinetics of insulin release hard to replicate because treatment involves peripheral administration instead of portal release.
Exercise and diet can increase insulin sensitivity and make it easier to maintain blood sugar levels.
Pumps use only rapid insulin. Needle is in abdomen. Increase rate of infusion before meals and dial it down at night. Trying to maintain levels as close to normal as possible.
Patient comes in in coma to hospital- give fluid and glucose. If in insulin coma- will revive. Can give insulin later. Adverse reactions to insulin administration include HYPOglycemia, insulin resistance, and problems at injection site.
Type II is the most common form of diabetes. Being overweight does not mean you have type II diabetes- just a greater likelihood of developing it.
Treated with dietary and lifestyle changes first. Exercise and losing weight generally work.
With insulin resistance, B cells compensate by putting out more insulin. Increased gluconeogenesis contributes to hyperglycemia. Diabetes has decreased insulin secretion.
Graph slide- numbers don’t matter. Principle does.
McDonald’s diet, especially with children, is highly associated with Type II diabetes. High-fat, high sugar diets.
Metformin inhibits gluconeogenesis.
TZDs are calles glitazones. They improve glucose sensitivity.
Remember rosiglitazone. TZDs do not affect insulin release. They bind to DNA and express genes to improve insulin sensitivity. Drug started in clinic before they realized it was ligand for PPARgamma. Receptor is high in adipose tissue. Patients tend to gain weight.
Review:
diabetes type I II
high blood glucose Y Y
ketoacidosis Y N
obesity N Y
insulin resistance N(some) Y
pancreatic failure Y Ninitially, eventually Y
juvenile onset Y recently Y
genetic component classically no Y
Fat rats are used to model impaired insulin signalling. Remember most overweight people are not diabetic.
Insulin binds to transmembrane spanning receptor with tissue-specific effects. See slide.
How do you get from surface to every compartment of cell with a single hormone signal?
All growth-factor receptors are membrane spanning. Receptors are monomers. In case of EGF they dimerize and a tyrosine kinase allows for autophosphorylation.
Insulin binds, causing a conformational change of receptor- signal transferred to B subunit- tyrosine autophosphorylation- signal at membrane. The receptor needs an intact ATP binding domain. Tyrosines on subunits interact with other proteins. Know about the autophosphorylation of Tyrosine. Phosphorylation of Ser/Thr is inhibited.
Insulin signalling- cascade of protein-protein interaction is involved. IRS-1 is important. Kahn used a phosphotyrosine antibody within a minute of insulin admin. IRS=insulin receptor substrate.
IRS is a huge protein. 3 domains- PH (pleckstrin homology) used by many proteins. Recognizes polar lipids- keeps protein associated with membrane.
IRS gets phosphorylated by subunit.
Remember plextrin homology, PTB, and SH2 and SH3. Everything is physical interactions.
SH2/SH3 recognize phosphorylated Tyr and a short amino acid sequence in IRs (insulin receptors). protein plugs in in physical association.Advantages of IRS:
signal amplification, IRS dissociation insulin signalling from the internalization of the receptor, expansion of the number of signals possible.
IRS interacts with PI3 kinase. PI3 kinase generates PI3 inositol-> PIP3-> multiple signals.
PIP3 is important here. PI3kinase is activated by many growth factors. Insulin activates specifically through IRS.
Insulin can also activate RAS/RAF pathways, but main metabolic actions thru PKB pathways
Insulin Actions in diabetes
He is posting a text from which he worked as well as the lecture.
Slide3:
Structure:
note disulfide bonds. A chain, B chain , C peptide. Insulin is a highly conserved hormone among all species. See figure 9 in Johnson, page 644
Oral glucose is more effective than IV glucose for inducing insulin release.
Insulin goes from pancreas to liver (portal circulation). Diabetics can only administer insulin subcutaneously, so it is hard to recreate release of insulin into the portal circulation.
7:
Insulin is key anabolic regulator. Supports protein synthesis in muscle, inhibits gluconeogenesis in liver. Other actions are general. Production of ketone bodies is important in diabetic ketoacidosis.
Important:glucose uptake, gluconeogenesis, ketogenesis in diabetes.
Type I Diabetes:
No phenotype (symptoms) for patient until beta cells are wiped out. Metabolic symptoms: hyperglycemia. Hormone sensitive lipase is activated in diabetes- Fatty acids go to liver, ketogenesis.
Historically- Langerhans defined the islets.
Oskar Minkowski demonstrated that removing pancreas in dog caused diabetes. Tasted dog’s urine-sweet. Postulated a hormone produced by the pancreas, but was unable to purify it. Banting and Best did purify it in 1921. Administered to dogs and were able to restore blood sugar. Provided first "cure" for previously fatal type I. 1923- Nobel prize.
Energy needs met in uncontrolled diabetes:
Alanine and alpha-ketoglutarate come from muscle to liver. There is no synthesis of fatty acids in adipose tissue.
Much of damage from diabetes is due to high blood sugar. Proteins get glycosylated inappropriately. Vision, circulatory problems in periphery, kidney problems, CV trouble.
Glucose transport:
There are 7 different GLUT transporters. Liver has bidirectional non-insulin dependent transporter (GLUT 2). Glut 4 is unidirectional.Absence of insulin- less transporter in muscle.
GLUT4 – 12 membrane spanning helices. Glut 4 in muscle has insulin-responsive glucose uptake.
They are sequestered in vesicles until insulin binds receptor and triggers movement to membrane through signalling cascade.
Capacity of muscles to take up glucose is increased 30 fold in the presence of these transporters (see Johnson chapter 41).
Muscle diagram
Exercise increases insulin-independent transport.
Adipose tissue: insulin stimulates lipoprotein lipase. Brings in fats for storage. Insulin also stimulates horm-sensitive lipase. Absence of insulin- free fatty acids are released and go to liver.
Liver diagram:
amino acids come in and gluconeogenesis is strongly activated in Type I diabetes.
Fatty acids enter TCA cycle and exit as ketone bodies.
Type I:
insulin imperative. Diet and exercise help to control the damage.
Problems: kinetics of insulin release hard to replicate because treatment involves peripheral administration instead of portal release.
Exercise and diet can increase insulin sensitivity and make it easier to maintain blood sugar levels.
Pumps use only rapid insulin. Needle is in abdomen. Increase rate of infusion before meals and dial it down at night. Trying to maintain levels as close to normal as possible.
Patient comes in in coma to hospital- give fluid and glucose. If in insulin coma- will revive. Can give insulin later. Adverse reactions to insulin administration include HYPOglycemia, insulin resistance, and problems at injection site.
Type II is the most common form of diabetes. Being overweight does not mean you have type II diabetes- just a greater likelihood of developing it.
Treated with dietary and lifestyle changes first. Exercise and losing weight generally work.
With insulin resistance, B cells compensate by putting out more insulin. Increased gluconeogenesis contributes to hyperglycemia. Diabetes has decreased insulin secretion.
Graph slide- numbers don’t matter. Principle does.
McDonald’s diet, especially with children, is highly associated with Type II diabetes. High-fat, high sugar diets.
Metformin inhibits gluconeogenesis.
TZDs are calles glitazones. They improve glucose sensitivity.
Remember rosiglitazone. TZDs do not affect insulin release. They bind to DNA and express genes to improve insulin sensitivity. Drug started in clinic before they realized it was ligand for PPARgamma. Receptor is high in adipose tissue. Patients tend to gain weight.
Review:
diabetes type I II
high blood glucose Y Y
ketoacidosis Y N
obesity N Y
insulin resistance N(some) Y
pancreatic failure Y Ninitially, eventually Y
juvenile onset Y recently Y
genetic component classically no Y
Fat rats are used to model impaired insulin signalling. Remember most overweight people are not diabetic.
Insulin binds to transmembrane spanning receptor with tissue-specific effects. See slide.
How do you get from surface to every compartment of cell with a single hormone signal?
All growth-factor receptors are membrane spanning. Receptors are monomers. In case of EGF they dimerize and a tyrosine kinase allows for autophosphorylation.
Insulin binds, causing a conformational change of receptor- signal transferred to B subunit- tyrosine autophosphorylation- signal at membrane. The receptor needs an intact ATP binding domain. Tyrosines on subunits interact with other proteins. Know about the autophosphorylation of Tyrosine. Phosphorylation of Ser/Thr is inhibited.
Insulin signalling- cascade of protein-protein interaction is involved. IRS-1 is important. Kahn used a phosphotyrosine antibody within a minute of insulin admin. IRS=insulin receptor substrate.
IRS is a huge protein. 3 domains- PH (pleckstrin homology) used by many proteins. Recognizes polar lipids- keeps protein associated with membrane.
IRS gets phosphorylated by subunit.
Remember plextrin homology, PTB, and SH2 and SH3. Everything is physical interactions.
SH2/SH3 recognize phosphorylated Tyr and a short amino acid sequence in IRs (insulin receptors). protein plugs in in physical association.Advantages of IRS:
signal amplification, IRS dissociation insulin signalling from the internalization of the receptor, expansion of the number of signals possible.
IRS interacts with PI3 kinase. PI3 kinase generates PI3 inositol-> PIP3-> multiple signals.
PIP3 is important here. PI3kinase is activated by many growth factors. Insulin activates specifically through IRS.
Insulin can also activate RAS/RAF pathways, but main metabolic actions thru PKB pathways
Thursday, November 16, 2006
Naren November 16
Sorting to Mitochondria and Peroxisomes
Lecture 3 has been reposted. He is attempting to post the movies.
Not testing on this, but it is interesting.
3:
talking about transport of cytosolic protein into peroxisome or mitochondria.
Mitochondrial Import
mitochondrial inner membrane has large surface area. Inner membrane is nearly 75% protein. Everything goes through active transport. Matrix is not hollow- has lots of proteins.
There is a unique feature to the mitochondrial targeting sequence- an amphipathic helix.
11:
What it looks like.
Experiment to find the sequence- slide 13.
Mitochondrion has 2 sets of pores- Tom complex, TIM23, TIM22, OXA.
23: 1. keeps protein unfolded.
18:
If protein is kept unfolded, introduce a linker, molecule penetrates membrane. Add methatrexate, binds active site DHFR- folds and gets stuck. 50 AAs was best linker. Labelled using immunogold.
Targeting sequence:
table slide 32.
Peroxisomes defined:
35.
Peroxisomes are strictly eukaryotic.
Peroxisome has one bilayer. Present largely in liver cells for detoxification.
37: PTS2 is not well defined.
Lecture 3 has been reposted. He is attempting to post the movies.
Not testing on this, but it is interesting.
3:
talking about transport of cytosolic protein into peroxisome or mitochondria.
Mitochondrial Import
mitochondrial inner membrane has large surface area. Inner membrane is nearly 75% protein. Everything goes through active transport. Matrix is not hollow- has lots of proteins.
There is a unique feature to the mitochondrial targeting sequence- an amphipathic helix.
11:
What it looks like.
Experiment to find the sequence- slide 13.
Mitochondrion has 2 sets of pores- Tom complex, TIM23, TIM22, OXA.
23: 1. keeps protein unfolded.
18:
If protein is kept unfolded, introduce a linker, molecule penetrates membrane. Add methatrexate, binds active site DHFR- folds and gets stuck. 50 AAs was best linker. Labelled using immunogold.
Targeting sequence:
table slide 32.
Peroxisomes defined:
35.
Peroxisomes are strictly eukaryotic.
Peroxisome has one bilayer. Present largely in liver cells for detoxification.
37: PTS2 is not well defined.
Nutting Notes November 16
Growth hormone was once thought to be secreted constantly. Male rats have major periods of secretion. Episodic every 3 hours or so. Humans also have episodic secretion, less regular than rat. Ours when we go into deep sleep.
Finishing table of pituitary hormones- FSH and LH are gonadotropins. Sertoli cells promote spermatogenesis and follicular growth. Inhibin does not affect libido- inhibits FSH secretion.
Feeding vs. Fasting page:
Look from body’s perspective.
Objectives: The carbohydrates we eat fill out glycogen stores. Glycogen requires water, so we don’t store much glycogen. We lose glucose through kidneys if glucose in blood goes too high. TAG are stored. AAs may be used for gluconeogenesis, most stored as protein.
Reduction of gluconeogenesis and glycogenolysis results in an increase of glycogen stores.
Ketogenesis also declines when we eat. Insulin induces these things.
Between meals:
Need to keep up glucose for CNS. Muscle and other tissues burn fats for energy. Other sparable proteins can be broken down for gluconeogenesis.
FFA stands for free fatty acids (circulating, not esterified to glycerol).
Time-course of hormone action:
Primary mechanism by which short-acting hormones work- they change function of existing enzymes or transport proteins. They change enzyme activity or sensitivity to regulators.
Long acting hormones turn on mRNA and ribosomes to make more protein, mostly. Change amounts of enzymes.
Hormones in feeding vs. fasting
see chart
Big changes come from plasma insulin concentration and increase in anabolic processes. and decrease in catabolic processes, except glycolysis and TCA cycle.
Cortisol tends to try to keep glycogen steady, but effect is slight.
See chart for rest.
Between meals-see chart. If you fast many days, the levels of hormones that are otherwise constant will change.
High levels of GH or cortisol could exaggerate effects.
Lipolytic triggers are catecholamines.
Glucagon physiologically acts only in liver.
Insulin and Glucagon are from pancreas-see paper.
Figure 1, page 639, Johnson: Bulk of Islets are insulin-producing B cells. Somatostatin acts as a paracrine agent in these islets.
GIP gastric inhibitory peptide is now glucose-dependent insulinotropic peptide. Promotes response to glucose.
See figure 20 page 654- main stimulus of beta cells is glucose and amino acids. Inhibitory- somatostatin, epi, NE.
Permissive stimuli are GH, Cortisol. T3 increases sensitivity of beta cells to glucose and amino acids.
Glucagon secretion
3. Other stimuli- see text figure 19 page 653.
If you eat a low carbohydrate meal- insulin spikes. You become hypoglycemic without glucagon.
Normal meal: increased insulin, no change in glucagon. Liver may see more glucagon than peripheral tissue. Peripheral levels of glucagon do not change much. Liver uses it up.
Enough insulin gets past liver to affect muscle and fat cells.
Insulin stimulates glucose uptake . Utilization means oxidation or storage as glycogen or lipid.
Nerve cells require glucose, but insulin does not stimulate brain or nerve cells.
Glucose uptake- see figure 12 for how insulin signals transport of glucose receptors to membrane.
Glucagon actions:
promotes release from liver. Phosphorylase stim glycogen breakdown.
Decreases glycogen synthesis.
PK is pyruvate kinase.
Take home: glucagon influences activity of preformed enzymes to produce glucose from glycogen and spare glucose.
Actions of insulin:
most potent hormone.
Hormone-sensitive lipase is sensitive to catecholamines.
Last figure is intense. All the processes are shown together. Glycogen, fat, protein are the three storage forms. Insulin stimulates protein synthesis, amino acid uptake in muscle, inhibits protein breakdown in muscle. Glycogenesis increased by insulin. Insulin inhibits glycogen breakdown and decreases ability for catecholamines to stimulate glycogen breakdown. Glycolysis- insulin inhibits fat breakdown and stimulates lipogenesis. Catcholamines stimulate lipoysis and phopshorylase. Try to diagram out where all the previously listed things act.
Finishing table of pituitary hormones- FSH and LH are gonadotropins. Sertoli cells promote spermatogenesis and follicular growth. Inhibin does not affect libido- inhibits FSH secretion.
Feeding vs. Fasting page:
Look from body’s perspective.
Objectives: The carbohydrates we eat fill out glycogen stores. Glycogen requires water, so we don’t store much glycogen. We lose glucose through kidneys if glucose in blood goes too high. TAG are stored. AAs may be used for gluconeogenesis, most stored as protein.
Reduction of gluconeogenesis and glycogenolysis results in an increase of glycogen stores.
Ketogenesis also declines when we eat. Insulin induces these things.
Between meals:
Need to keep up glucose for CNS. Muscle and other tissues burn fats for energy. Other sparable proteins can be broken down for gluconeogenesis.
FFA stands for free fatty acids (circulating, not esterified to glycerol).
Time-course of hormone action:
Primary mechanism by which short-acting hormones work- they change function of existing enzymes or transport proteins. They change enzyme activity or sensitivity to regulators.
Long acting hormones turn on mRNA and ribosomes to make more protein, mostly. Change amounts of enzymes.
Hormones in feeding vs. fasting
see chart
Big changes come from plasma insulin concentration and increase in anabolic processes. and decrease in catabolic processes, except glycolysis and TCA cycle.
Cortisol tends to try to keep glycogen steady, but effect is slight.
See chart for rest.
Between meals-see chart. If you fast many days, the levels of hormones that are otherwise constant will change.
High levels of GH or cortisol could exaggerate effects.
Lipolytic triggers are catecholamines.
Glucagon physiologically acts only in liver.
Insulin and Glucagon are from pancreas-see paper.
Figure 1, page 639, Johnson: Bulk of Islets are insulin-producing B cells. Somatostatin acts as a paracrine agent in these islets.
GIP gastric inhibitory peptide is now glucose-dependent insulinotropic peptide. Promotes response to glucose.
See figure 20 page 654- main stimulus of beta cells is glucose and amino acids. Inhibitory- somatostatin, epi, NE.
Permissive stimuli are GH, Cortisol. T3 increases sensitivity of beta cells to glucose and amino acids.
Glucagon secretion
3. Other stimuli- see text figure 19 page 653.
If you eat a low carbohydrate meal- insulin spikes. You become hypoglycemic without glucagon.
Normal meal: increased insulin, no change in glucagon. Liver may see more glucagon than peripheral tissue. Peripheral levels of glucagon do not change much. Liver uses it up.
Enough insulin gets past liver to affect muscle and fat cells.
Insulin stimulates glucose uptake . Utilization means oxidation or storage as glycogen or lipid.
Nerve cells require glucose, but insulin does not stimulate brain or nerve cells.
Glucose uptake- see figure 12 for how insulin signals transport of glucose receptors to membrane.
Glucagon actions:
promotes release from liver. Phosphorylase stim glycogen breakdown.
Decreases glycogen synthesis.
PK is pyruvate kinase.
Take home: glucagon influences activity of preformed enzymes to produce glucose from glycogen and spare glucose.
Actions of insulin:
most potent hormone.
Hormone-sensitive lipase is sensitive to catecholamines.
Last figure is intense. All the processes are shown together. Glycogen, fat, protein are the three storage forms. Insulin stimulates protein synthesis, amino acid uptake in muscle, inhibits protein breakdown in muscle. Glycogenesis increased by insulin. Insulin inhibits glycogen breakdown and decreases ability for catecholamines to stimulate glycogen breakdown. Glycolysis- insulin inhibits fat breakdown and stimulates lipogenesis. Catcholamines stimulate lipoysis and phopshorylase. Try to diagram out where all the previously listed things act.
Wednesday, November 15, 2006
endocrinology lectures 1 and 2
Intro to Endocrinology
Today we will cover the basics. Covering only some hormones and glands. Know only level we cover in class for the ones he just barely mentions; read book sections or chapters on those he emphasizes.
What are endocrine glands and what are hormones?
endocrine glands are part of 3 regulatory systems in body:
1. nervous- communication with direct wiring (nerves). Generally operate 1:1 (or 1:100) with direct connections.
2. endocrine involves chemical transfer through bloodstream and diffusion to interact with tissues.
In some cases systems merge to form neuroendocrine glands. Nerve cells secrete a product to bloodstream. Operate by dumping into bloodstream. Hormones affect only the tissues with appropriate receptors.
3. maybe immune as well.
List of hormones we will cover is under A2. Endocrinology- know acronyms.
What is a hormone?
chemical substance which is released in small quantities from certain glands (endocrine glands). These substances travel through the circulatory system to elicit response in certain tissues(target tissues or cells)
include neurohormones, but not CO2 or intracellular messengers like cAMP.
A chart here in my notes did not copy properly.
Major glands:
hypothalamus affects anterior pituitary through circulatory system.
Posterior pituitary
thyroid gland in neck –regulates metabolism
4 glands on surface are parathyroid- secrete PTH
abdomen- kidneys have some endocrine function. top each is adrenal gland. The adrenal gland has 2 distinct endocrine tissues- medulla in center part is neuroendocrine- secretes epinephrine (epi) and norepinephrine(NE). outside is cortex, secreting cortisol and aldosterone.
Pancreas- GI related with islets for insulin and glucagon. Has exocrine tissue and endocrine tissue. GI tract also secretes GI hormones. Affect digestive processing, motility, secretion pancreatic hormones.
reproductive tissues: ovaries, placenta during pregnancy, testes in male.
Neuroendocrine hormones:Nervous systems affecting hormones
Epinephrine controlled directly by nervous systems. So are vasopressin and oxytocin.
Hypothalamic releasing hormones
Hormones affecting nervous systems:
epinephrine- causes anxiety and fear.
regulation by sex hormones of sex activity- (no testes- males lose interest in sex)
3 categories of hormones: listed under B
Why categories- listed below the categories.
Liver disease can foul up endocrine systems by not producing enough plasma proteins to bind hormones in blood.
Renal disease- lose plasma proteins in leaky kidney.
Metabolism also malfunctions in renal/liver diease.
2 pages tables in handout- glands and hormones. fill in as we go.
See chart
tropic=growth.
FSH is active in males and females. So is LH.
T4- 4 iodines. T3-3 iodines. Body converts T4 to T3.
Gland makes mostly T4.
calcitonin also called thyrocalcitonin.
On table- know acronyms and other names, major functions, details of regulatory functions
adrenal medulla- regulation organic metabolism- stimulates glycogen and fat breakdown
chemical nature of epi and NE- catecholamines
major stimuli to increase or decrease secretion:
increase- stress (fear), hypoglycemia, fight or flight response.
what hormone down in hypoglycemia- insulin
opposite inhibits secretion.
4 categories in Table 2: chemical nature of hormones:
see table 2. progesterone promotes gestation.
steroids, amino acid derivatives, proteins, peptides are the categories.
Catecholamines have catechol (benzine ring with 2 adjacent OHs on the ring) R-NH2 is an amine.
Proteins arbitrarily have molecular weight more than 8000-9000 . About 70 amino acids. Proteins made in anterior pituitary, kidney, placenta.
Dopamine is not a peptide- it is a catecholamine.
GHRH- growth hormone releasing hormone.
GHIH- growth hormone release inhibiting hormone (somatostatin) statin- means hold steady.
There may be a prolactin releasing hormone. PRL suppressed most of the time by dopamine. Dopamine relased almost continuously and keeps PRL suppressed. PRL is stimulated by suckling- stop dopamine secretion. PRL goes to mammary glands for milk production. Oxytocin causes milk release. Keep these straight. Oxytocin is also stimulated by suckling.
Will not ask which are proteins and which are peptrides.
Smallest one is TRH.
Structure of steroids- all synthesized from cholesterol. All retain 4 rings ABCD. Some of side chain lopped off and you get steroids with 21 or 19 carbons. Cortisol has 21 carbons .
Estradiol- 18 Cs. There is one OH in the 17 beta position of estradiol, hence the name. Estradiol has aromatic ring. Aldosterone is different because aldehyde group on C-18. Specific enzymes make modifications. These hormones operate on specific tissues.
Synthetic pathway- see handout. Do not have to learn enzymes that make conversions. Specific tissues have specific enzymes. The point is that if a person has a genetic deficiency of enzyme, his or her ability to synthesize the final product is not there. Drugs can also block enzymes. The body senses the lack of final product and tries to make more. The first processes are driven until the blocked step. Intermediates build up and may be weakly active. Some effects may happen, but they will be weak. Some substances have different action altogether.
Mineralocorticoid- aldosterone.
Catecholamine chart- another example of hormone synthesis
4 enzymes in sequence. Dopamine is produced in hypothalamus and adrenal glands- medulla. Most converted to NE,then to Epi by PNMT. PNMT enzyme requires cortisol or corticosterone for activity. Only tissue with high enough concentration of corticoids is adrenal gland. Drainage from cortex passes through medulla before entering veins, sees high cortisol.
Do not have to draw. Know precursor and organs and what it takes to get to epi.
Hormones in blood:
Biologically important form of any hormone is unbound. Bound can’t get out of capillaries. Little peptides need to be bound to prevent filtering out in kidneys.
thyroid hormone is small.
TeBG is also called sex hormone binding globulin, or testosterone-estrogen binding globulin.
There is always equilibrium between free and bound hormone. For many hormones, the equilibrium is shifted toward bound form. Some free leaves, more jumps off proteins. Keeps free level where it should be. If you lose proteins, you’ll have problems.
3. Total concentration of hormone in plasma= free+ bound (most bound)
4. Active hormone
a. on target cells-free
b. on feedback sensors controlling secretion- free.
Biologically active form is sensed by cells regulating and producing hormone secretions.
C. Steps in Life of Hormone-
Regulators stimulate, inhibit, or have both competing. Other hormones also influence processes in endocrine cells themselves. Hormione is secreted, diffuses to plasma, bound, free or combination. Most assays measure both bound and free protein. In plasma- some excreted, inactivated, further activated,
Target cells have receptors. Receptors give specificity. How do they act(genes, transcription, 2nd messengers). Action of hormone may be feedback. Ex: insulin lowers glucose. Takes away stimulation for secretion insulin. Reaction of target tissue is also influenced by other factors. Any step shown can get messed up.
Given a scenario- think of places with potential problem or places for intervention.
Cellular mechanism of action-
receptors. High affinity receptors, mostly.
changing number of receptors will influence reactivity of target tissue. More receptors, more response.
If insulin is high for a while, body tissues react by reducing number of insulin receptors. Means bigger doses of insulin.
Receptors can change sensitivity and response capacity of tissue. Same maximum response when sensitivity changes.
Changing response capacity (see graphs of dose-response curves, page 567, text):
increase capacity- same concentration gives same half maximum response. Total response changes.
increase sensitivity-response shifts to left, meaning a lower concentration is needed for the same response.
Lots of tissues have spare receptors- hardly ever are all used.
Some hormones diffuse across the cell membrane and get to nucleus to receptor in nucleus- interact with genetic material to give response. These tend to be steroid and thyroid hormones. All other hormones act at surface to generate 2nd messengers. These
1. amplify signal of hormone-receptor complex at surface (signal cascade)
2. disperse hormone signal to variety of places in cell.
Secretory pattern of hormones- most not steady. See paper.
May be diurnal, circadian, ultraradian.
Measuring hormone does not necessarily measure activity. If data on hormone concentration does not match activity, something may be odd.
Most hormones are secreted episodically.
Cortisol stimulated by ACTH appears to be secreted once a day. See graphs. More sensitive assays- peaks early in morning, but also present at other times. Really ultradian rhythm, but it may be characterized as diurnal rhythm.
Other hormones not affected. Other patterns of secretion- testosterone in adult man does not change a lot, but has a rhythm.
LH=pulsatile secretion- changes about 50-60%.
Stimulation- induced secretion – PRL stimulated by suckling.
E and F are summary tables.
Remember there are lots of places hormone secretion, synthesis, action can be affected. Essay questions abound.
Lecture 2:
Hormonal Pathways
2. PTH goes to kidneys and stimulates activation of vitamin D to target in bone and small intestine.
examples of 3 glands and 3 hormones will be covered in lecture. Here is one:
RH to pituitary trophic hormone to peripheral glands to secrete their hormones.
Fig. 2 in handout on G. Hormonal Pathways page:
Endocrine gland inputs can be stimulatory(+) or inhibitory(-).
fig 3
2 nd gland can influence how well 1st gland stimulates to release or inhibit hormone.
One other concept:
"permissiveness"-permissive effect of hormone. Hormone allows another hormone or other signals to work much better. Ex: lipolysis. Put a fat cell in a flask. Add thyroid hormones such as T3- see nothing obvious- no release. Take next bunch and add Epi and NE- lipolytic- small amounts fatty acids released. .3rd group add T3, then epi, get large amount fatty acids released. T3 allows epi to be more potent in stimulating lipolysis.
Can’t add both hormones to flask at same time – thyroid hormone has to go to nucleus and modify gene expression. Modifying gene expression takes time. We have some T3 around all the time- makes other hormones more effective.
2 important concepts:
feedback regulation
2types: +/- feedback.
1. positive feedback- during labor and delivery. Oxytocin does not usually stimulate initial contraction. Contraction begins, then oxytocin does its task. Signal ends up in post pituitary. Baby delivered-stimulus gone. Note diagram(like on page 769, text-not in handout)- loop will have all +s.
Other + stimulus- suckling. impulse to nervous system- post pituitary- oxytocin- mammary gland- let down- suckling continues until baby is full. Other stimuli may start process. Process may begin with baby crying or if mom knows it is scheduled time to feed and begins to prepare.
2. negative feedback is far more common. End response negates stimulus that started it. 2 examples: blood glucose regulation by insulin and glucagon.
insulin and glucose-A. glucose uptake lowers blood glucose until it goes back to normal. glucagon stimulated by drop in glucose. See paper. Each is separate negative feedback system because regulation leads to negation of stimulus that started it. Counterregulatory systems like insulin and glucagon give smooth control.
Glucose tolerance test is done because it is hard to see something unless you push the system.
Initial conditions: fasting and minimally stressed. (Adapt animal to procedure). Normal animal- fasting glucose 90 mg/dL. Challenge at t=0. Plasma insulin starts at 10 microunits per mL. Goes up 5 fold. Removal of glucose removes stimulus. Insulin also turns off body’s release of glucose from glycogen stores. Insulin is rapidly cleared. Total time course is 5 hours. No insulin- blood sugar starts a lot higher, goes up. comes back to a high level because it is excreted. Be able to sit down and explain this on the test. We drew this on paper-check with somebody.
hypothalamus and pituitary:
see paper.
Above pituitary is hypothalamus.
Look at figures one and two, pages 574-575, and 4 on page 580 (All references are to the Johnson text).
neurohypophysis- is the bottom of hypothalamus. Know anterior and posterior.
Bottom of hypothalamus has capillary bed- where RH are released into "long" portal veins- probably less than 1 cm long. Short portal vessels go front to back of pituitary. 2 capillary beds separated by a blood vessel make what is known as a portal system. RH are not used up in pituitary- go to heart, and get diluted too much to be effective. Must be high concentration to regulate. Blood goes in through superior and inferior arteries. Blood leaves into venous sinuses. If you need to measure RH levels- neurosurgerons can sample venous sinuses for the anterior lobe of the pituitary.
median eminence- axon terminals release RH carried by LPV to anterior pituitary(see figures 1 and 2, pp574-575).
Anterior lobe figure 2- arterial blood picks up RH- through long portal vein- stimulates release of anterior pituitary hormone- picked up in second capillary bed- out to distribution.
Posterior gland- cell bodies synthesize and pack post-pituitary hormones. Vesicles travel down axon to post pituitary. Go by axoplasmic flow through nerve axon. Stored in post pituitary gland at nerve terminals. Cell body fires action potentials fast- axoplasmic flow is slow. Vesicles stored are released like neurotransmitters are released. Stimuli summate to give a string of action potentials- just like release of neurotransmitter at neuromuscular junction or between 2 nerves.
Vasopressin is ADH. Both nuclei make both hormones. What if you cut infundibular stalk and sealed with wax? No secretion of vasopressin and oxytocin. see p585 Johnson. After a while, excessive drinking slows. Blood vessels and capillaries grow around wound and pick up hormone at a higher anatomical place. Produces diabetes insipidus.
2 terms to know:
diabetes mellitus- type I or II – sweet urine. May have large volume due to sugar drawing water out for excretion.
diabetes insipidus- urine is very dilute. Low urea and salt. Large volume, dilute.
nonapeptide- 9AAs. Both have a disulfide bridge. ADH and oxytocin have overlapping biological activity- do similar things. ADH has some weak oxytocin-like activity. Tiny moelcules are synthesized as larger molecules. Stored, cut out. Neurophysin is other cut part. Secreted at post- pituitary nerve terminals. Neurophysins have no function as hormones as far as we know (see 583 Johnson for putative function).
Oxytocin increases contractility of myoepithelial cells in mammary glands. Also stimulates uterine muscle. Myoepithelial cells surround alveoli containing milk. Oxytocin comes from blood and stimulates cells to contract to force milk out to capillaries and ducts. Epithelial cells make milk. Myoepithelial cells move it.
PVN and SON of hypothalamus send action potentials down for milk ejection or letdown. Also effects utierine smooth muscle.
ADH increases contractility of arterial smooth muscle (pressor effect to increase blood pressure).
ADH also effects kidneys to increase water reabsorption in collecting duct and distal tubule. Antidiuretic action.
E. control of secretion
ADH 2 main stimuli- decreased plasma volume or increased plasma osmolarity. osmolarity receptors in hypothalamus.
See fig 4 on extra sheet.. X on bottle- not enough water. Without water, water concentration decreases. Osmolarity increases.ADH increases. ADH increases water permeability and increases water reabsorption in kidney. Decreases water excretion.
Fig 5 more water in –see arrows.
Fig 6- baroreceptor mediate pathway.
Note sensible relations of stimuli to cause increase or decrease in secretion. intermediate lobe- not discussing.
Anterior pituitary gland
see sheet.
Most are tropic hormones- cause growth. Somatotropin causes body to grow. TSH increases size of thyroid. LH and FSH male and female gonads.
ACTH is peptide.
POMC is synthesized as a larger molecule and cut down. ACTH activity- cut from C to N terminal end and measure activity- only need 20 of 39 amino acids.N terminal region is biological activity. C terminal end is where immunologic activity is (antibodies will be toward C end). If abnormality is at the N end, immunoassay will say the protein is normal.
alpha MSH structure is within ACTH- melanocyte stimulating hormone in 1st 13 AAs ACTH. Take frog, put in aquarium with a little water, bright room- frog skin is light. inject with ACTH- turns dark. Just like if frog goes from sand to leafy background. In nature the change happens via MSH. Possible human applications. Humans with high ACTH have complexion changes.
Cell types producing ACTH can be determined by staining. It varies. May be acidophilic 50% (GH. PRL from somatotrophs, lactotrophs), basophilic 20-25% (TSH, LH, FSH- thyrotrophs, gonadotrophs) chromophobes(10-15%- corticotrophs).
Histology:
see paper.
hCG produced during the 1st trimester of pregnancy. human chorionic gonadotropin.
Tests need to measure beta subunit.
EPT tests do this for B subunit hCG.
Actions and secretion- see chart.
See fig 7 and fig 8.
ACTH provokes actions of cortisol.
Remove adrenals- no cortisol. Treat with cortisone- adrenal glands decrease in size. Glands do not synthesize as much cortisol due to atrophy- later, when it is needed. it can’t be made. To get around this problem, you taper the dosage or give ACTH. Drawback to a protein like ACTH-must be given by injection.
Lots of notes were taken on paper by drawing up and down arrows on charts. You need to get those from someone who was there.
Today we will cover the basics. Covering only some hormones and glands. Know only level we cover in class for the ones he just barely mentions; read book sections or chapters on those he emphasizes.
What are endocrine glands and what are hormones?
endocrine glands are part of 3 regulatory systems in body:
1. nervous- communication with direct wiring (nerves). Generally operate 1:1 (or 1:100) with direct connections.
2. endocrine involves chemical transfer through bloodstream and diffusion to interact with tissues.
In some cases systems merge to form neuroendocrine glands. Nerve cells secrete a product to bloodstream. Operate by dumping into bloodstream. Hormones affect only the tissues with appropriate receptors.
3. maybe immune as well.
List of hormones we will cover is under A2. Endocrinology- know acronyms.
What is a hormone?
chemical substance which is released in small quantities from certain glands (endocrine glands). These substances travel through the circulatory system to elicit response in certain tissues(target tissues or cells)
include neurohormones, but not CO2 or intracellular messengers like cAMP.
A chart here in my notes did not copy properly.
Major glands:
hypothalamus affects anterior pituitary through circulatory system.
Posterior pituitary
thyroid gland in neck –regulates metabolism
4 glands on surface are parathyroid- secrete PTH
abdomen- kidneys have some endocrine function. top each is adrenal gland. The adrenal gland has 2 distinct endocrine tissues- medulla in center part is neuroendocrine- secretes epinephrine (epi) and norepinephrine(NE). outside is cortex, secreting cortisol and aldosterone.
Pancreas- GI related with islets for insulin and glucagon. Has exocrine tissue and endocrine tissue. GI tract also secretes GI hormones. Affect digestive processing, motility, secretion pancreatic hormones.
reproductive tissues: ovaries, placenta during pregnancy, testes in male.
Neuroendocrine hormones:Nervous systems affecting hormones
Epinephrine controlled directly by nervous systems. So are vasopressin and oxytocin.
Hypothalamic releasing hormones
Hormones affecting nervous systems:
epinephrine- causes anxiety and fear.
regulation by sex hormones of sex activity- (no testes- males lose interest in sex)
3 categories of hormones: listed under B
Why categories- listed below the categories.
Liver disease can foul up endocrine systems by not producing enough plasma proteins to bind hormones in blood.
Renal disease- lose plasma proteins in leaky kidney.
Metabolism also malfunctions in renal/liver diease.
2 pages tables in handout- glands and hormones. fill in as we go.
See chart
tropic=growth.
FSH is active in males and females. So is LH.
T4- 4 iodines. T3-3 iodines. Body converts T4 to T3.
Gland makes mostly T4.
calcitonin also called thyrocalcitonin.
On table- know acronyms and other names, major functions, details of regulatory functions
adrenal medulla- regulation organic metabolism- stimulates glycogen and fat breakdown
chemical nature of epi and NE- catecholamines
major stimuli to increase or decrease secretion:
increase- stress (fear), hypoglycemia, fight or flight response.
what hormone down in hypoglycemia- insulin
opposite inhibits secretion.
4 categories in Table 2: chemical nature of hormones:
see table 2. progesterone promotes gestation.
steroids, amino acid derivatives, proteins, peptides are the categories.
Catecholamines have catechol (benzine ring with 2 adjacent OHs on the ring) R-NH2 is an amine.
Proteins arbitrarily have molecular weight more than 8000-9000 . About 70 amino acids. Proteins made in anterior pituitary, kidney, placenta.
Dopamine is not a peptide- it is a catecholamine.
GHRH- growth hormone releasing hormone.
GHIH- growth hormone release inhibiting hormone (somatostatin) statin- means hold steady.
There may be a prolactin releasing hormone. PRL suppressed most of the time by dopamine. Dopamine relased almost continuously and keeps PRL suppressed. PRL is stimulated by suckling- stop dopamine secretion. PRL goes to mammary glands for milk production. Oxytocin causes milk release. Keep these straight. Oxytocin is also stimulated by suckling.
Will not ask which are proteins and which are peptrides.
Smallest one is TRH.
Structure of steroids- all synthesized from cholesterol. All retain 4 rings ABCD. Some of side chain lopped off and you get steroids with 21 or 19 carbons. Cortisol has 21 carbons .
Estradiol- 18 Cs. There is one OH in the 17 beta position of estradiol, hence the name. Estradiol has aromatic ring. Aldosterone is different because aldehyde group on C-18. Specific enzymes make modifications. These hormones operate on specific tissues.
Synthetic pathway- see handout. Do not have to learn enzymes that make conversions. Specific tissues have specific enzymes. The point is that if a person has a genetic deficiency of enzyme, his or her ability to synthesize the final product is not there. Drugs can also block enzymes. The body senses the lack of final product and tries to make more. The first processes are driven until the blocked step. Intermediates build up and may be weakly active. Some effects may happen, but they will be weak. Some substances have different action altogether.
Mineralocorticoid- aldosterone.
Catecholamine chart- another example of hormone synthesis
4 enzymes in sequence. Dopamine is produced in hypothalamus and adrenal glands- medulla. Most converted to NE,then to Epi by PNMT. PNMT enzyme requires cortisol or corticosterone for activity. Only tissue with high enough concentration of corticoids is adrenal gland. Drainage from cortex passes through medulla before entering veins, sees high cortisol.
Do not have to draw. Know precursor and organs and what it takes to get to epi.
Hormones in blood:
Biologically important form of any hormone is unbound. Bound can’t get out of capillaries. Little peptides need to be bound to prevent filtering out in kidneys.
thyroid hormone is small.
TeBG is also called sex hormone binding globulin, or testosterone-estrogen binding globulin.
There is always equilibrium between free and bound hormone. For many hormones, the equilibrium is shifted toward bound form. Some free leaves, more jumps off proteins. Keeps free level where it should be. If you lose proteins, you’ll have problems.
3. Total concentration of hormone in plasma= free+ bound (most bound)
4. Active hormone
a. on target cells-free
b. on feedback sensors controlling secretion- free.
Biologically active form is sensed by cells regulating and producing hormone secretions.
C. Steps in Life of Hormone-
Regulators stimulate, inhibit, or have both competing. Other hormones also influence processes in endocrine cells themselves. Hormione is secreted, diffuses to plasma, bound, free or combination. Most assays measure both bound and free protein. In plasma- some excreted, inactivated, further activated,
Target cells have receptors. Receptors give specificity. How do they act(genes, transcription, 2nd messengers). Action of hormone may be feedback. Ex: insulin lowers glucose. Takes away stimulation for secretion insulin. Reaction of target tissue is also influenced by other factors. Any step shown can get messed up.
Given a scenario- think of places with potential problem or places for intervention.
Cellular mechanism of action-
receptors. High affinity receptors, mostly.
changing number of receptors will influence reactivity of target tissue. More receptors, more response.
If insulin is high for a while, body tissues react by reducing number of insulin receptors. Means bigger doses of insulin.
Receptors can change sensitivity and response capacity of tissue. Same maximum response when sensitivity changes.
Changing response capacity (see graphs of dose-response curves, page 567, text):
increase capacity- same concentration gives same half maximum response. Total response changes.
increase sensitivity-response shifts to left, meaning a lower concentration is needed for the same response.
Lots of tissues have spare receptors- hardly ever are all used.
Some hormones diffuse across the cell membrane and get to nucleus to receptor in nucleus- interact with genetic material to give response. These tend to be steroid and thyroid hormones. All other hormones act at surface to generate 2nd messengers. These
1. amplify signal of hormone-receptor complex at surface (signal cascade)
2. disperse hormone signal to variety of places in cell.
Secretory pattern of hormones- most not steady. See paper.
May be diurnal, circadian, ultraradian.
Measuring hormone does not necessarily measure activity. If data on hormone concentration does not match activity, something may be odd.
Most hormones are secreted episodically.
Cortisol stimulated by ACTH appears to be secreted once a day. See graphs. More sensitive assays- peaks early in morning, but also present at other times. Really ultradian rhythm, but it may be characterized as diurnal rhythm.
Other hormones not affected. Other patterns of secretion- testosterone in adult man does not change a lot, but has a rhythm.
LH=pulsatile secretion- changes about 50-60%.
Stimulation- induced secretion – PRL stimulated by suckling.
E and F are summary tables.
Remember there are lots of places hormone secretion, synthesis, action can be affected. Essay questions abound.
Lecture 2:
Hormonal Pathways
2. PTH goes to kidneys and stimulates activation of vitamin D to target in bone and small intestine.
examples of 3 glands and 3 hormones will be covered in lecture. Here is one:
RH to pituitary trophic hormone to peripheral glands to secrete their hormones.
Fig. 2 in handout on G. Hormonal Pathways page:
Endocrine gland inputs can be stimulatory(+) or inhibitory(-).
fig 3
2 nd gland can influence how well 1st gland stimulates to release or inhibit hormone.
One other concept:
"permissiveness"-permissive effect of hormone. Hormone allows another hormone or other signals to work much better. Ex: lipolysis. Put a fat cell in a flask. Add thyroid hormones such as T3- see nothing obvious- no release. Take next bunch and add Epi and NE- lipolytic- small amounts fatty acids released. .3rd group add T3, then epi, get large amount fatty acids released. T3 allows epi to be more potent in stimulating lipolysis.
Can’t add both hormones to flask at same time – thyroid hormone has to go to nucleus and modify gene expression. Modifying gene expression takes time. We have some T3 around all the time- makes other hormones more effective.
2 important concepts:
feedback regulation
2types: +/- feedback.
1. positive feedback- during labor and delivery. Oxytocin does not usually stimulate initial contraction. Contraction begins, then oxytocin does its task. Signal ends up in post pituitary. Baby delivered-stimulus gone. Note diagram(like on page 769, text-not in handout)- loop will have all +s.
Other + stimulus- suckling. impulse to nervous system- post pituitary- oxytocin- mammary gland- let down- suckling continues until baby is full. Other stimuli may start process. Process may begin with baby crying or if mom knows it is scheduled time to feed and begins to prepare.
2. negative feedback is far more common. End response negates stimulus that started it. 2 examples: blood glucose regulation by insulin and glucagon.
insulin and glucose-A. glucose uptake lowers blood glucose until it goes back to normal. glucagon stimulated by drop in glucose. See paper. Each is separate negative feedback system because regulation leads to negation of stimulus that started it. Counterregulatory systems like insulin and glucagon give smooth control.
Glucose tolerance test is done because it is hard to see something unless you push the system.
Initial conditions: fasting and minimally stressed. (Adapt animal to procedure). Normal animal- fasting glucose 90 mg/dL. Challenge at t=0. Plasma insulin starts at 10 microunits per mL. Goes up 5 fold. Removal of glucose removes stimulus. Insulin also turns off body’s release of glucose from glycogen stores. Insulin is rapidly cleared. Total time course is 5 hours. No insulin- blood sugar starts a lot higher, goes up. comes back to a high level because it is excreted. Be able to sit down and explain this on the test. We drew this on paper-check with somebody.
hypothalamus and pituitary:
see paper.
Above pituitary is hypothalamus.
Look at figures one and two, pages 574-575, and 4 on page 580 (All references are to the Johnson text).
neurohypophysis- is the bottom of hypothalamus. Know anterior and posterior.
Bottom of hypothalamus has capillary bed- where RH are released into "long" portal veins- probably less than 1 cm long. Short portal vessels go front to back of pituitary. 2 capillary beds separated by a blood vessel make what is known as a portal system. RH are not used up in pituitary- go to heart, and get diluted too much to be effective. Must be high concentration to regulate. Blood goes in through superior and inferior arteries. Blood leaves into venous sinuses. If you need to measure RH levels- neurosurgerons can sample venous sinuses for the anterior lobe of the pituitary.
median eminence- axon terminals release RH carried by LPV to anterior pituitary(see figures 1 and 2, pp574-575).
Anterior lobe figure 2- arterial blood picks up RH- through long portal vein- stimulates release of anterior pituitary hormone- picked up in second capillary bed- out to distribution.
Posterior gland- cell bodies synthesize and pack post-pituitary hormones. Vesicles travel down axon to post pituitary. Go by axoplasmic flow through nerve axon. Stored in post pituitary gland at nerve terminals. Cell body fires action potentials fast- axoplasmic flow is slow. Vesicles stored are released like neurotransmitters are released. Stimuli summate to give a string of action potentials- just like release of neurotransmitter at neuromuscular junction or between 2 nerves.
Vasopressin is ADH. Both nuclei make both hormones. What if you cut infundibular stalk and sealed with wax? No secretion of vasopressin and oxytocin. see p585 Johnson. After a while, excessive drinking slows. Blood vessels and capillaries grow around wound and pick up hormone at a higher anatomical place. Produces diabetes insipidus.
2 terms to know:
diabetes mellitus- type I or II – sweet urine. May have large volume due to sugar drawing water out for excretion.
diabetes insipidus- urine is very dilute. Low urea and salt. Large volume, dilute.
nonapeptide- 9AAs. Both have a disulfide bridge. ADH and oxytocin have overlapping biological activity- do similar things. ADH has some weak oxytocin-like activity. Tiny moelcules are synthesized as larger molecules. Stored, cut out. Neurophysin is other cut part. Secreted at post- pituitary nerve terminals. Neurophysins have no function as hormones as far as we know (see 583 Johnson for putative function).
Oxytocin increases contractility of myoepithelial cells in mammary glands. Also stimulates uterine muscle. Myoepithelial cells surround alveoli containing milk. Oxytocin comes from blood and stimulates cells to contract to force milk out to capillaries and ducts. Epithelial cells make milk. Myoepithelial cells move it.
PVN and SON of hypothalamus send action potentials down for milk ejection or letdown. Also effects utierine smooth muscle.
ADH increases contractility of arterial smooth muscle (pressor effect to increase blood pressure).
ADH also effects kidneys to increase water reabsorption in collecting duct and distal tubule. Antidiuretic action.
E. control of secretion
ADH 2 main stimuli- decreased plasma volume or increased plasma osmolarity. osmolarity receptors in hypothalamus.
See fig 4 on extra sheet.. X on bottle- not enough water. Without water, water concentration decreases. Osmolarity increases.ADH increases. ADH increases water permeability and increases water reabsorption in kidney. Decreases water excretion.
Fig 5 more water in –see arrows.
Fig 6- baroreceptor mediate pathway.
Note sensible relations of stimuli to cause increase or decrease in secretion. intermediate lobe- not discussing.
Anterior pituitary gland
see sheet.
Most are tropic hormones- cause growth. Somatotropin causes body to grow. TSH increases size of thyroid. LH and FSH male and female gonads.
ACTH is peptide.
POMC is synthesized as a larger molecule and cut down. ACTH activity- cut from C to N terminal end and measure activity- only need 20 of 39 amino acids.N terminal region is biological activity. C terminal end is where immunologic activity is (antibodies will be toward C end). If abnormality is at the N end, immunoassay will say the protein is normal.
alpha MSH structure is within ACTH- melanocyte stimulating hormone in 1st 13 AAs ACTH. Take frog, put in aquarium with a little water, bright room- frog skin is light. inject with ACTH- turns dark. Just like if frog goes from sand to leafy background. In nature the change happens via MSH. Possible human applications. Humans with high ACTH have complexion changes.
Cell types producing ACTH can be determined by staining. It varies. May be acidophilic 50% (GH. PRL from somatotrophs, lactotrophs), basophilic 20-25% (TSH, LH, FSH- thyrotrophs, gonadotrophs) chromophobes(10-15%- corticotrophs).
Histology:
see paper.
hCG produced during the 1st trimester of pregnancy. human chorionic gonadotropin.
Tests need to measure beta subunit.
EPT tests do this for B subunit hCG.
Actions and secretion- see chart.
See fig 7 and fig 8.
ACTH provokes actions of cortisol.
Remove adrenals- no cortisol. Treat with cortisone- adrenal glands decrease in size. Glands do not synthesize as much cortisol due to atrophy- later, when it is needed. it can’t be made. To get around this problem, you taper the dosage or give ACTH. Drawback to a protein like ACTH-must be given by injection.
Lots of notes were taken on paper by drawing up and down arrows on charts. You need to get those from someone who was there.
Naren Notes
Lecture 1: ER translocation
Start with protein secretion movie.
Movie is what we will talk about.
Imagine a cell. mRNA comes from nucleus. Protein synthesis happens in the cytosol. The ER transports proteins to go outside, or to organelles inside cell, or into nucleus. What determines the fate of protein?
Protein destined to be secreted is secreted into ER. Protein goes to Golgi, which add sugars, then passes thorugh trans golgi and out.
Sorting and targeting:
Protein sorting: protein can go through secretory pathway or cytosolic, or to peroxisome or mitochondria.
There is no well-defined ER signalling sequence. Contains lots of leucine, other hydrophobic residues.
Experimental proof of signal sequence:
Give cells in media without methionine or cysteine a pulse with radioactive 35S met/cys, grow 30-40 min, homogenize, treat with detergent and trypsonize or monitor protein.
Without microsomes in a cell-free system, protein is made, but signal sequence stays on. Add microsome afterward, protein deos not enter.
Add microsome at beginning- protein translocates as it is translated. This is cotranslational translocation. Know this.
1. signal sequence- SRP is a signal recognition particle.
3. translocon is a ligand-gated channel
Details on individual slides.
SRP is ribonuclear protein complex.
Converting type 2 to type 3: (page 7) If you mutate + to – on type II, protein flips as translocation is taking place, lacking interaction with translocon.
What starts in ER lumen winds up on outside cell.
Lecture 2:
Protein modification, folding, and quality control in the ER
As protein is folded, prcesses go through quality control. This is not on test.
Just study slides.
X on page 2 slide 1 is any amino acid except proline.
Look up J.Clin. Invest. volume 108 p. 1579-1582.
PDI is used as standard to monitor ER during organelle isolation.
Page 5 slide 3 hemaglutinin
Lecture 3: Nuclear Transport
Warning: This file got corrupted, so I reconstructed this part from memory and the text. Lodish pages 509-517. Reference in our little booklet is incorrect.
2:
You will see this slide several times. Today we are focusing on the red box.
3. This is the nuclear membrane structure. Notice the nuclear pores. Notice what needs energy to get through and what does not. An RNP is a ribonuclear protein complex, a complex of mRNA and associated proteins. See slides 15-17 for more detailed structure of nuclear pores.
5. In eukaryotes, DNA transcription and translation occur in the nucleus, but protein synthesis occurs in the cytoplasm.
Slide 10:
Note the roles of nuclear pores.
11: We will be tested over the nuclear localization signal. It is usually internal. Also know the experiment described on slides 12-14. Pyruvate kinase normally stays in cytoplasm, but can be localized to nucleus with NLS.
Slide 19: Digitonin acts like a detergent to break up cell membranes. If you permeabilize the cell membrane so the cytoplasm leaks out, but the nucleus and nuclear pore complexes are intact, and put in a protein with an NLS signal, it will not localize to the nucleus. With cell lysate, it will. The NLS is not sufficient on its own- there must be cytosolic components involved in localizing proteins to the nucleus.
Slide 23: Remember these proteins.
Slide 24: He will ask about the RAN cycle. Remember where it is associated with GDP and where with GTP. Slide 25 illustrates.
Slide 29: Fuse a Hela (human) cell with a xenopus cell in the presence of PEG. Their nuclei come close together. RNP-C stays localized in the Hela nuclei (no NES). RNP A1 is exported to the xenopus nucleus.
32: mRNA exporter is a protein that interacts with hydrophobic FG (rich in phenylalanine and glycine) repeats in the FG nucleoporins.
Slides 33-36: Balbiani ring mRNA codes for a glue that helps the larva stick to a leaf. It uncoils from the associated proteins as it enters the cytoplasm and is transcribed.
Slide 39: HIV Rev protein enables unspliced mRNA to be exported. Rev binds a rev response element on the mRNA and contains an NES that interacts with exportin 1 and Ran-GTP.
Slide 40 is a summary of the important things to remember.
Start with protein secretion movie.
Movie is what we will talk about.
Imagine a cell. mRNA comes from nucleus. Protein synthesis happens in the cytosol. The ER transports proteins to go outside, or to organelles inside cell, or into nucleus. What determines the fate of protein?
Protein destined to be secreted is secreted into ER. Protein goes to Golgi, which add sugars, then passes thorugh trans golgi and out.
Sorting and targeting:
Protein sorting: protein can go through secretory pathway or cytosolic, or to peroxisome or mitochondria.
There is no well-defined ER signalling sequence. Contains lots of leucine, other hydrophobic residues.
Experimental proof of signal sequence:
Give cells in media without methionine or cysteine a pulse with radioactive 35S met/cys, grow 30-40 min, homogenize, treat with detergent and trypsonize or monitor protein.
Without microsomes in a cell-free system, protein is made, but signal sequence stays on. Add microsome afterward, protein deos not enter.
Add microsome at beginning- protein translocates as it is translated. This is cotranslational translocation. Know this.
1. signal sequence- SRP is a signal recognition particle.
3. translocon is a ligand-gated channel
Details on individual slides.
SRP is ribonuclear protein complex.
Converting type 2 to type 3: (page 7) If you mutate + to – on type II, protein flips as translocation is taking place, lacking interaction with translocon.
What starts in ER lumen winds up on outside cell.
Lecture 2:
Protein modification, folding, and quality control in the ER
As protein is folded, prcesses go through quality control. This is not on test.
Just study slides.
X on page 2 slide 1 is any amino acid except proline.
Look up J.Clin. Invest. volume 108 p. 1579-1582.
PDI is used as standard to monitor ER during organelle isolation.
Page 5 slide 3 hemaglutinin
Lecture 3: Nuclear Transport
Warning: This file got corrupted, so I reconstructed this part from memory and the text. Lodish pages 509-517. Reference in our little booklet is incorrect.
2:
You will see this slide several times. Today we are focusing on the red box.
3. This is the nuclear membrane structure. Notice the nuclear pores. Notice what needs energy to get through and what does not. An RNP is a ribonuclear protein complex, a complex of mRNA and associated proteins. See slides 15-17 for more detailed structure of nuclear pores.
5. In eukaryotes, DNA transcription and translation occur in the nucleus, but protein synthesis occurs in the cytoplasm.
Slide 10:
Note the roles of nuclear pores.
11: We will be tested over the nuclear localization signal. It is usually internal. Also know the experiment described on slides 12-14. Pyruvate kinase normally stays in cytoplasm, but can be localized to nucleus with NLS.
Slide 19: Digitonin acts like a detergent to break up cell membranes. If you permeabilize the cell membrane so the cytoplasm leaks out, but the nucleus and nuclear pore complexes are intact, and put in a protein with an NLS signal, it will not localize to the nucleus. With cell lysate, it will. The NLS is not sufficient on its own- there must be cytosolic components involved in localizing proteins to the nucleus.
Slide 23: Remember these proteins.
Slide 24: He will ask about the RAN cycle. Remember where it is associated with GDP and where with GTP. Slide 25 illustrates.
Slide 29: Fuse a Hela (human) cell with a xenopus cell in the presence of PEG. Their nuclei come close together. RNP-C stays localized in the Hela nuclei (no NES). RNP A1 is exported to the xenopus nucleus.
32: mRNA exporter is a protein that interacts with hydrophobic FG (rich in phenylalanine and glycine) repeats in the FG nucleoporins.
Slides 33-36: Balbiani ring mRNA codes for a glue that helps the larva stick to a leaf. It uncoils from the associated proteins as it enters the cytoplasm and is transcribed.
Slide 39: HIV Rev protein enables unspliced mRNA to be exported. Rev binds a rev response element on the mRNA and contains an NES that interacts with exportin 1 and Ran-GTP.
Slide 40 is a summary of the important things to remember.
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