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98 Cards in this Set

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Describe the structure of cyanocobalamin (vitamin B12)

What are the 2 coordination positions for Co+?
-Planar ring structure called a corrin ring system with 4 pyrrole rings and a central cobalt ion Co+.

-Nucleotide like structure with ribose, phosphate and 5,6 dimethylbenzamidazole

Co+ has 2 cordination positions that are not part of the ring structure: link to a CN group above, and link to a dimethylbenzamidazole below.
What are the 4 forms of cobalamin?

What does this depend on?

Which 2 are used as coenzymes?
Cyanocobalamin-CN-Cbl
Hydroxocobalamin (OH-Cbl)
Adenosylcobalamin (AdoCbl)
Methylcobalamin (MeCbl)

These depend on the groups coordinated to cobalt ABOVE the plane of the corrin ring system.

AdoCbl and MeCbl are used as coenzymes in the liver and blood respectively.
Give some food sources of cobalamin.

What is the daily dietary requirement for cobalamin?

In which organs is it most found?
Meat, liver, fish, eggs. milk

2-5 ug.

Liver and kidneys
What are the 2 human enzyme cobalamin dependent reactions?

Which pathology results if cobalamin is deficient?
1. Ado Cbl-dependent methylmalonyl CoA mutase (Propionoic acid metabolism)

2. MeCbl-dependent methyltransferase
(generation of Met from homocysteine)
Pathology: megaloblastic anemia (resulting from inhibition of DNA synthesis in red cell production).
Outline the role of cobalamin in propionic acid metabolism.
Propionic acid is metabolized to methylmalonyl CoA. This involves oxidation of a fatty acid. B12 is required to put a methyl group on the main chain and to straighten this out.

Methylmalonyl CoA is converted to succinyl CoA (4 C) by the enzyme methylmalonyl CoA mutase.

Enzyme: adocbl-dependent methylmalonyl coA mutase, in which cobalamin acts as a coenzyme.

OVERALL: 3C propionic acid --> propionyl coA --> carboxylase forming me-malonyl CoA (4 C). Racemerization gives me-methylmalonyl CoA as a different isomer. Then the enzyme Ado-Cbl-memalonyl CoA mutase converts the me-malonyl CoA to succinyl CoA, which can be fed into the Kreb's cycle.
Outline the generation of methionine (Met) from homocysteine.

What does the methylfolate trap refer to?
MeCbl (methylcobalamin) is combined with Homocysteine (homocys) to give Cbl and Met, with MeCbl serving as the methyl donor.

This converts homocysteine to methionine (CH2-CH2-SH to CH2-CH2-S-CH3).

N5-methyltetrahydrofolate is converted to tetrahydrofolate (FH4) to restore Cbl to MeCbl. (Aside: N2O depletes MeCbl).

No B12 slows the synthesis of Met and blocks conversion of N5-methylFH4 to FH4 (the methylfolate trap). When this occurs, FH4 cannot be used to generate N5,10-methylene FH4, impairing the conversion of dUMP to DTMP.

Enzyme thymidylate synthase is hindered, and the supply of dTMP (thymidylate nucleotide reqiuired for DNA synthesis) is compromised.

With increasing dUMP, there is misincorporation of U into DNA, changing structure, but not affecting RNA synthesis. QED: megaloblastic anemia = mature cytoplasm and immature nucleus...like a teenager!
What nervous system damage can occur in cobalamin deficiency?
-Swelling of myelinated fibers (indicating impaired synthesis of myelin due to methylmalonyl CoA accumuation and altered fatty acid synthesis...as fatty acids are made using acetyl CoA and malonyl CA).
What is intrinsic factor?

What is pernicious anemia?

What is extrinsic factor?
Instrinsic factor: a glycoprotein required for absorption of cobalamin.

Megaloblastic anemia caused by defective secretion of intrinsic factor by gastric cells.

Extrinsic factor = cobalamin
What is transcobalamin II?

What do transcobalamin I and III do?
Transcobalamin II: plasma protein transporting absorbed cobalamin in blood - recently absorbed! Important in cobalamin UPTAKE

Transcorbalamin I or hepatocorrin carry the longer lasting cobalmin, and may prevent loss of B12 in urine and sweat.
How is pernicious anemia treated?
Doses of cyanocobalamin until anemia responds, then 1 mg injected 1x/month/life.
What is the role of folic acid?

What inhibits folate synthesis in bacteria?

Food source of folic acid?
Folic acid (folate) = carrier of 1 C fragments, also causes megaloblastic anemias.

Sulfonamides (antibiotics) and anti-folates (methotrexate)-works by binding and inhibiting DHFR.

Spinach, asparagus, broccoli, lettuce, liver, kidney, yeast, mushrooms.
Describe the structure of folic acid.
Contains pteridine
p-aminobenzoic acid
glutamate.

Folate can also be called pteryoylglutamate.
What are the 2 forms folates are found in?

Where is each found?
Mono or polyglutamates. Glutamates linked by peptide bonds.

Intracellular glutamates are usually polyglutamates, plasma folate is usually in N5-metyhlFH4.
What form must F be in?

Which enzyme catalyzes this reaction? Which reducing agent is used?

At what sites does reduction occur?
Must be converted from F to FH2 then FH4.
Catalyzed by dihydrofolate reductase (DHFR). NADPH used as a reducing agent.

N5, C6, C7, N8
Give examples of the groups attached to FH4 which will donate a 1 C fragment.
-CHO, CH2, CH2OH, CH=, CH3, CHNH. These are all refered to as C-FH4.
What is the daily dietary requirement for folate? How long will stores last?
50ug. 4 months.
Outline folate metabolism.
FH4 donates 1-C fragments by accepting these, donating them in enzyme reactions and then receiving these again.

Polyglutamate is the active form.
How doe C-FH4 contribute to thymidylate synthesis?
Serine +FH4 --> N10 hydroxymethyl FH4 + glycine.

N10-hydroxymethyl FH4 --> N5,10 methylene FH4 + H20

N5,10 methylene FH4 + dUMP --> FH2 _ dTMP

FH2 + NADPH + H+ --> FH4 + NADP+ (DHFR).
What are the consequences of folic acid deficiency?

What are 2 other reactions folate is involved in?
Impairs dTMP and therefore DNA synthesis.

Histidine catabolism, purine synthesis
Where does folate absorption occur? Which enzyme is involved?

What does this enzyme do?
In jejunum using enzyme conjugase.

Conjugase removes polygutamate resides from dietary folates via hydrolysis.
How can megaloblastic anemia occur without respect to cobalamin and folate deficiency?
Antimetabolite drugs interfere with DNA synthesis. 6-mercaptopurine will inhibit purine synthesis, 5-fluoro-2deoxyuridine inhibits pyrimidine synthesis.
What is a porphyrin?
A compound bearing N in 4 covalently-lined, substituted pyrroles. Often have a central metal ion (e.g. Fe).
Outline the structure of heme.
-Protoporphyrin IX
-Central ferrous ion (F+2)

Classified as a type II porphyrin.
Heme is a prosthetic group (not composed of amino acids) bound to the protin to serve as the active component in Hb, Mb and cytochromes.
What are the porphyrias?
These refer to genetic mutations in the 8 enzymes required for the synthesis of protoporphyrin IX. = Genetic disorders where enzymes in path to heme synthesis are partly or completely deficient.
Outline what happens to heme iron after Hb is degraded.

What is jaundice?
Heme iron is transferred around the body via transferrin or stored with ferritin.

The porphyrin component of heme is degraded (cyclic tetrapyrrole is coverted to a linear tetrapyrole) and bilirubin is created in the process.

Jaundice: yellowing of skin and tissues caused by increased levels of plasma bilirubin. Mild detected at 2-2.5 mg/dL
What are the 3 causes of jaundice?

What is the difference between bile pigments and bile acids?
Pre-hepatic: coming form hemolytic anemia, -extracorpuscular: antibodies in plasma
-membrane: mutant forms of red cell membrane proteins, -cystolic: hemoglobinopathies i.e. HbS
-enzymopathies (G-6-P dehydrogenase deficient hemolytic anemias)

Hepatic: coming from true liver problem, e.g. infectious hepatitis, cirrhosis.

Post-hepatic: from obstruction such as a stone in the CBD or CA @ pancreatic head.
-extramural: CA at head of pancreas
mural: tumor in CBD
intramural: gall stone
Bile pigments - linear tetrapyrroles, acids are derived from cholesterol and are sterols used in intestinal dietary fat digestion.
Where does heme catabolism occur?

What happens to the protein (globin) portion?

What happens to the iron?

What happens to the heme?
In macrophages in reticuloendothelial cells.

The protein portion is degraded to amino acids which are recycled.

The iron is removed and conserved.

The heme porphyrin ring is opened by an enzyme: HEME OXYGENASE which converts the cyclic tetrapyrrole into a linear tetrapyrrole using molecular oxygen. This produces BILIVERDIN (green bile pigment) and CO. Biliverdin is enzymatically reduced to bilirubin.
Outline how bilirubin is transported.
Liver: biliburbin is bound to albumin and transported to liver. Antibiotics can compete for bilirubin binding sites on albumin and displace it, representing side effects.

Liver has a carrier system on the sinusoidal surface. Transport occurs via facilitated diffusion, with a large capacity for bilirubin.

Hepatocytes will uptake bilirubin.
Outline 1) how jaundice can occur in a newborn, 2) a possible consequence of this, and 3) how the jaundice is treated.
1) Bilirubin can build-up in blood if liver is immature and cannot metabolize bilirubin effectively.

2) Bilirubin can cross the blood brain barrier and cause brain damage - KERNICTERUS.

3) Tx: UV light will metabolize bilirubin chemically.
How is bilirubin metabolized?
Enzyme glucuronyl transferase in ER of liver uses UDP-glucuronate to transfer 2 molecules of polar hydrophilic molecule glucuronate to non-polar, hydrophobic bilirubin.

Addition of polar glucuronate helps in makes excretion of modified bilirubin possible:

Bilirubin + UDP-glucuronate --> bilirubin monoglucuronate + UDP

Bilirubin monoglucuronate + UDP-glucuronate --> Bilirubin diglucaronate + UDP.

This modification = CONJUGATION.

Note: there are 2 isoforms of glucuronyl transferase to handle the seperate glucuronyl transfers.
What does conjugation of bilirubin result in?

What happens with this form of bilirubin?
Conjugation increases the water solubility of the hydrophobic bilirubin. This form is secreted from hepatocytes into the bile by ACTIVE transport. Liver facilitates movement of bilirubin from blood to bile, and bile enters duodenum by CBD.
Outline intestinal metabolism of bilirubin diglucuronide.

What occurs when high levels of bile pigment or a defective enterophepatic urobilinogen cycle are present?
Bilirubin diglucuronide is converted back to bilirubin by removal of the glucaronate resides by the bacterial beta-glucuronidases.

Bilirubin is REDUCED to form stercobilinogens (colourless, referred to urobilinogens in urine).

Small portion of the stercobilinogens are taken up in the terminal ileum and large intesting and excreted again by liver, = ENTEROHEPATIC UROBILINOGEN CYCLE. Stercobilinogens are oxidized by intestinal bacteria to form coloured stercobilins giving stools colour.

If defective pathways, stercobilinogen/urobilinogen may be excreted in the urine.
What is the Ehrlich reaction? What does it measure?
Occurs in the presence of methanol. Plasma bilirubin will react with diazotizied sulfanilic acid to form a reddish-purple product. This gives a measure of total plasma bilirubin.

Without methanol, a portion of the bilirubin reacts directly with the reagent, a fraction called direct-acting bilirubin. This is the more hydrophillic bilirubin diglucuronate (while unconjugated form required methanol for rxn).
How is free bilirubin calculated?

What is meant by hyperbilirubinemia?
Total bilirubin (+ methanol) - direct acting bilirubin (no methanol) = free bilirubin (Indirect bilirubin).

Hyperbilirubinemia: accumulation of bilirubin in the blood. (causes jaundice).
Outline the physiology of jaundice in hemolytic anemia and state the expected result on indirect bilirubin.

Repeat for obstructive jaundice.
Hemolytic anemia = excessive breakdown of rbc. Free bilirubin in plasma is carried to the liver, increasing the amount of indirect bilirubin in the plasma.

For obstructive jaundice, there is a build up of bile in the bile duct because of blockage. This would cause a back-up of conjugated bilirubin from the bile to be present in the blood, leading to high levels of plasma direct acting bilirubin.
Outline the expected results for serum bilirubin, urine urobilinogen, urine bilirubin and fecal urobilinogen in each of the following conditions:

a) Normal
b) Hemolytic anemia
c) Hepatitis
d) Obstructive jaundice
Normal:
SB: direct = 0.1-0.4mg/dL, indirect = 0.2-0.7 mg/dL.
UUB: 0-4 mg/24 h
UB: Nope
FU: 40-280 mg/24h

Hemlytic Anemia:
SB: indirect elevated
UUB: increased
UB: absent
FB: increased

Hepatitis:
SB: direct and indirect elevated
UUB: decreased
UB: present
FB: decreased

Obstructive Jaundice:
SB: direct elevated
UUB: absent
UB: present
FU: trace to absent.
What are 2 defining characteristics of stem cells?

What are the 3 types of stem cells?
Characteristics:
1. Self renewing
2. Able to differentiate into multiple cell layers.

3 Types:

1. Totipotent: able to generate ANY type of cell body, including extra-embryonic membranes like placenta

2. Pluripotent-able to generate any type of cell in the body, except extra-embryonic membranes. E.g. Embryonic stem cells from inner mass of blastocyst, embryonic germ cells from embryonic gonad precursors, and embryonic carcinoma cells from teratocarcinomas.

3. Multipotent: makes multiple mature cell types, but only those of a particular tissue. Found in adults. E.g. hemopoietic stem cells.
What are 3 attributes characterizing hematopoietic stem cells?
1. Ability to self-renew.
2. Ability to differentiate into ALL mature blood lineages.
3. Ability to reconstitute the hematopoietic system in a lethally radiated host.

Rare, and non-cycling at steady state, but this will increase in hematopoietic stress (bleeding, infection, chemo).
Where are hemopoietic stem cells derived from?

Where do cells migrate from?
Mesoderm patterned along dorsal-ventral axis in gastrulation after being directed here by bone morphogenetic proteins (BMP) and transforming growth factor (TGF-beta).

Cells migrate from extra-embryonic yolk sac.
Outline primitive hematopoiesis.

What regulates migration in late fetal stage?
Occurs in the aortic-gonadal-mesonephros of the embryo, then HSC expands and migrates to the fetal liver and spleen. In late fetal stage, HSC migrate to the bone marrow.

Migration is regulated by Chemokine receptors (SDF-CXCR) and beta-intigrins.
What are the 3 possible actions of hemopoietic stem cells.
3 actions: self renew, differentiate, or apoptosis. These are determined by intrinsic and extrinsic control factors.
What are the 4 forms of extrinsic regulation?
Cytokines, hedgehog protein, bone morphogenic proteins (BMP), and NOTCH signaling.
How are cytokines involved in renewal of HSC?
Cytokines are involved in self-renewal of HSC. These include: -----Stem cell Factor (and the stem cell factor receptor KIT)
-FLT3 ligand (Flat 3 ligand) -a tyrosine receptor
-Thrombopoietin (enhancing megakaryocytic proliferation and differentation, Mpl (a TPO receptor)

Without these, HSC have reduced ability to reconstitute hematopoiesis when transplanted.
How do hedgehog proteins regulate HSC?

Sonic?
Hedgehog proteins are glycoproteins involved in mesoderm organization. Without these, stem cells do not migrate along the dorsal axis.

These up-regulate bone morphogenetic proteins which induce primitive hematopoiesis.

Sonic - a subtype that increases proliferation of HSC.
How do Bone Morphogenetic Proteins regulate HSC?
Induce primitive hematopoiesis and are members of transforming growth-factor beta-family.

Receptors for BMP are found in bone marrow, cord blood, and peripheral blood.
What are NOTCH proteins?

What are the 2 types and their functions?
NOTCH is a transmembrane receptor found on HSC.

2 types: Jagged and Delta. Delta - signaling increases generation of HSC, and maintains ability to differentiate into all subtypes. Jagged - decreases proliferation.
How do intrinsic factors within HSC determine if a cell will self-renew or differentiate?

Give examples.
Accomplished by expression of genes that encode for the decision.

Examples:
-Stem cell leukemia gene
-Lim-only 2 protein - expressed at hemangioblast level, required for primitive and definitive hematopoiesis.
-AML1
MYB - involved in definitive hematopoesis.
-GATA2-involved in regeneration of HSC
IKAROS-involved in primitive and definitive hematopoiesis. Maybe be involved in survival, regeneration and proliferation. Involved in commitment to lymphoid lineage.
Outline how homeobox genes are involved in HSC regulation.
These include HOC and non-HOX families, while under-expression of HOX genes leads to poor granulopoiesis and lymphopoiesis in mice. Over expression may lead to leukemia.
How does epigenetic regulation influence HSCs?
Modification of histone proteins via methylation, acetylation, phosphorylation, ubiquitrination or ADP ribosylation affects the structure and encodes for gene regulation.

This can enhance or restrict differentiation.
How is apoptosis controlled in HSCs?
Apoptosis results from over-expression of BCL-2 gene.
What surface markers are found on HSCs?
CD34 positive, Kit SCF receptor positive, but are CD38 negative.
How are HSC's identified?
1. Dye efflux: HSc pumps out dyes, so undyed cells = stem cells.

2. Growing cell cultures - placed in an environment with enhancers, may favour myloid or lymphoid conditions to differentiate into a given lineage.
What are some sources of HSC?
1. Bone marrow
2. Peripheral blood - easier to harvest from here, have faster neutrophil and platelet recovery, but may increase the risk of G vs. Host Dx.
3. Cord Blood - useful for smaller patients, as this has the slowest recovery of hematopoiesis.
How do embryonic and adult stem cells differ in plasticity?
Embryonic stem cells are derived from the developing blastocyst which are self-renewing and can differentiate into all germ layers (endo, meso, ecto).

Adult stem cells are derived from tissues in the fetal/post natal stage, were originally thought to differentiate into only the same germ layer, but there is increasing evidence of plasticity.
Typical blood volume in male? Female?

Normal Hemoglobin values?

Hematocrit?

Distribution of cell types in blood?
Male: 5.3 L, F: 3.8 L

Hb: Male: 155 g/L, F: 140 g/L

(%): Male: 47%, F: 42%

Distribution: plasma = 55%, buffy coat = 1%, red cells = 45%
What does the shape and flexibility of a red blood cell depend on?

Describe rbc membrane.

Survival time?
Cytoskeleton - composed of 3 proteins spectrin, ankryn, actin.

Membrane: lipid bilayer with transmembrane proteins - protein 3 and glycophorins.

120 day survival time.
What is polycythemia? Outline primary and secondary causes.
Polycythemia: too many rbc.
Primary causes: myeloproliferative disorder, e.g. due to a JAK-2 tyrosine kinase mutation.
Secondary causes: hypoxia (heart/lung dx, altitude, hb abnormality), increased EPO production (e.g. CA, uterine fibroids, renal cysts, renal artery stenosis, EPO doping, androgen abuse).
Outline granulocyte structure and function.

Survival time?

Give 5 main activities of granulocytes.
Nucleus with 2-5 lobes, contains granules. Has enzymes for phagocytosis and destruction and inflammatory reactions.

Circulate in blood for ~10 hours, migrate into tissue and survive there for 2-3 days.

Activities: 1. phagocytosis, 2. cytotoxic to alter permeability of invaders!, 3. generate free radicals from Fe bound to bacteria, 4. digest killed bacteria, 5. create respiratory burst.
What is granulocytopenia?

How does this occur as a result of decreased production? (6 conditions)

How does this occur as a result of increased destruction? (3 conditions)
Decreased numbers of granulocytes.

Decreased production:
1.defective differentiation (not enough neutrophils to differentiate properly, ie. aplastic anemia)
2. substrate deficiency (B12 or folate)
3. myeloptheisis (i.e. marrow infiltration - no space!)
4. toxicity to precursors (e.g. medications, infections)
5. increased apoptosis (e.g. chronic benign neuropenia).
6. congenital problems (increased apoptosis - Kostmann's syndrome)

Increased destruction:
1. hypersplenism (Felty's syndrome)
2. immune - autoimmune, or connective tissue disorders (e.g. lupus)
3. alloimmune - via medication of viruses.
What is granulocytopenia?

How does this occur? (3 reasons)
Increased numbers.

Occurs due to: 1. demargination (from endothelium to circulating blood) in face of stress,exercise, pain, etc.

2. Reactive - increased production due to infection, CA, inflammation.

3. Primary: chronic myelogenous leukemia, immature granulocyte forms seen in smear.
Describe lymphocytes.

Where are they found?

What are the 3 types?
Mononuclear, little cytoplasm, small number of granules w. lysosomal enzymes.

Found in lymphoid tissue and systemic circulation.

2 types: B lymphocytes - receptors for antigens, make antibodies, memory B cells. T lymphocytes - have CD4 (helper) which stimulate B cells to make antigens and CD8 (cytotoxic) destroy antigen on cell. Natural killer: destroy cells without needing antigen recognition.
When does lymphocytosis occur?
Occurs in response to viral infections (mono, CMV), stress (MI), smoking, or in malignant situations, e.g. lymphoma.
Describe eosinophils.

Function?
Eosinophils: 2-3 lobes, orange/red granules, short time in circulation and localize to the skin, GI and genital tract, lungs. Contain 3 types of granules: primary, secondary (form crystal shapes), and small granules.

Function: cytotoxic, inflammatory reactions, hypersensitivity reactions. Major role: to go to tissues and cause infection/mediate allergic reaction, parasite destruction.
Describe basophils.

Under what conditions does basophilia occur?
Contain purple/black granules which contain heparin, chondroitin sulfates, and HISTAMINE. React with IgE to mediate hypersensitivity and chronic inflammatory conditions. Key - not a killer of things

Basophilia: cuased by hypersensitivity reactions - or in myeloproliferative disorders.
Describe monocytes.

What are they involved in?

When does monocytosis occur?
Horseshoe shaped nucleus. Grey/blue cytoplasm with reddish granules, circulate briefly and migrate into tissues. Most action is in TISSUES, mature into macrophages here.

Involved in: phagocytosis and debris, APC, acute inflammation.

Monocytosis: in chronic infections -e.g. TB, syphilis, chronic inflammatory conditions, and malignancy (acute and chronic leukemias).
What are the 3 main functions of platelets in clotting?

What do they contain?
1. aggregation, 2. vasoconstriction, 3. tissue repair.

Contents: dense body granules contain agonists - ADP, ATP, serotonin and Ca++; alpha granules contain adhesive proteins like fibrinogen, VWF, growth modulators, and coagulation factors (Factor V).
What is pseudothrombocytopenia?

Give examples of thrombocytopenia as a result of decreased production.

Increased production?

As a reactive measure?
False appearance of low platelets numbers due to 1. clumping and 2. satelitism.

Increased production: consumption, immune, or sequestration (i.e. in hypersplenism).

Decreased production: defective differentiation (e.g. acquired or congenital), substrate deficiency, myelophtisis, or toxic.
When does thrombocytosis occur?
May be REACTIVE, PRIMARY, or RESDISTRIBUTIVE.

Reactive: infection, inflammation, iron deficiency, cancer, bone marrow infiltration. May be primary, e.g. a myeloproliferative disorder or redistributive, e.g. post spenectomy.
What is hematopoiesis?

What are the 3 main compartments?
Generation of a spectrum of highly specialized, differentiated cell types from a population of pluripotent, self-renewing stem cells. This may be under steady sate or increased demand.

3 compartments:

1. Primitive self-sustaining pluripotent stem cells
2. Progenitor cells with various degree of lineage restriction and little self-renewal capacity
3. Morphologically identifiable, lineage restricted, maturing cells

E.g. stem cells --> colony forming units = progenitor cells such as myeloid stem cells and lymphoid stem cells --> myloblasts, megakaryocytes, etc.
What is bone marrow?

How does this change with age?

Structure?
Site of origin of all peripheral blood and hemapoietic elements, start developing at 15-20 weeks, have a daily production rate to meet demand.

Filled with hematopoietic cells at birth, replaced by adipose cells with age such that it is eventually only found in the cavities of centrally located bones.

Bone has solid cortical and trabecular structure, supplied by a nutrient artery and by periosteal capillaries, with capillary-venous sinuses for blood exit.
Describe the structure of the sinus wall and 2 major components.
Consists of endothelial cells covered by a basement membrane. 2 major components = stromal cells, which adhere to the surface and provide framework, regulatory factors, adhesion molecules, proteins to stimulate hematopoiesis and recruit precursor cells, etc., and hematopoietic cells - with mature cells released into capillary venous sinus.
Describe the scaffolding matrix of bone marrow.
Contains - stromal cells (e.g. fibroblasts, endothelial cells, etc., accessory cells (monocytes, T cells, etc), and the extracellular matrix (produced by the stromal cells) providing specificity, adhesion molecules.
How do stromal cells regulate the environment?

How do the cell populations produced contribute?
1. Produce cytokines - e.g. GM-CSF, G-CSF, IL-1,3,6,11, KIT-ligand, FLT-3 ligand and fibroblastic growth factor. These influence the growth of marrow progenitors.

2. They also produce negative regulators - TGF-beta, interferon-gamma, TNF.

3. Paracrine reactions - cell/cell adhesion which allow hematopoietic cells to stay in environment.

Accessory cell contribution:

T-cells make Il-3, TNF, IFN-y
Macrophages express VCAM for adhension
Macrophages phagocytose dead cells
Where do the following occur?

1. Erythropoiesis
2. Granulopoiesis
3. Megakaryopoiesis
Erythropoiesis: in the erythroid island surrounding the central macrophage

Granulopoiesis: adjacent to boney trebeculae

Megakaryopoiesis: adjacent to sinue endothelium
Outline the process of granulopoiesis.
Stem cell becomes a CFU-GM at 4 weeks, neutrophils appear in second trimester. In adults, myeloblast divides into promyelocyte, which contains primary granules containing lysosmal enzymes.

Promyelocytes divide ever 20 hours to give myelocytes. This is the last myeloid precursor able to divide. These mature and give secondary (pink) granules. These lack myeloperoxidase, and contain lysozyme, collagenase, plasminogen activator, aminopeptidase and lactoferrin.

SO......Promyelocyte --> myelocyte --> metamyelocytes --> band forms --> neutrophils

Neutrophils stay in the marrow as reserve available in increased demand.
Outline the life-course of neutrophils.

What factor increases neutrophil survival in circulation?

What factor is responsible for migration?
Circulate in peripheral blood for a few hours, then migrate into tissue.

GM-CSF prolongs circulation.

CSCR1, 2 are chemokine receptors responsible for migration of neutrophils in response to chemokines (e.g. IL-8, granulocyte chemoattractant protein-2, growth related oncogene)
What factors are required to make eosinophils?

At what stage do these develop?

What do their granules contain?'

What attracts these to migrate into tissues? When is it produced?
Factors: IL-5, GM-CSF, IL-3

Develop at: PROMYELOCYTE stage

Graules are peroxidase positive with a crystalloid core of phosphoplipids, melanin, arginine, and lysine holding off the enzymes peroxidase and arylsulfatase.

Migrate into tissues with CCR3 (chemokine receptor) is stimulated by eotaxin.
Where do basophils originate from?

What is required to make a basophil?

What is contained in the granules?

What other cell-type originates from the same precursor?
From CFU-GEMM.

Require IL-3 and KIT ligand

Granules contain heparin, histamine, lipid, and proteoglycan, NOT proteolytic enzymes.

Tissue mast cells originate from the same precursor.
Where do monocytes arise from?

What stimulates monocyte development?

What is the stage progression to arrive at a monocyte?

What are the functions of monocytes?

What activates monocytes?

Circulation time?
From CFU-GM

Stimulated from M-CSF, GM-CSF, IL-3

Progression: CFH-GM --> monoblasts --> promonocytes --> monocytes.

Functions: migration, chemotaxis, phagocytosis, NADPH-dependent oxidase, respiratory burst capacity, etc.

Activated by GM-CSF, M-CSF, TNF, IFN-y

Circulate in blood for 3 days, migrate into tissues for 3 months.
What is the major stimulant of monocytes?

What are the major roles of monocytes?
Stimulated by M-CSF

3 Roles: phagocytic activity, immune recongition (dendric cells), and cytokine production for action in hematopoiesis, inflammatory activity, and immune reactions.
What are dendritic cells?

What cell type do these stimulate?

What other cell types can these arise from?
APC's which lack phagocytotic activity, found in bone marrow.

Stimulate primary T cell response

These can also arise from lymphoid or myeloid precursors when stimulated by GM-CSF and TNF respectively
What are osteoclasts?

What are they stimulated by?
Cells of the bone marrow which differentiate from CFU-GM involved in resorption of bone.

Stimulated by M-CSF, IL-1,6, vitamin D, calcitonin, parathryroid hormone, etc.
Outline erythropoiesis in fetal development.
Develops in yolk sac, then paraortic region, liver, and bone marrow at 14-19th days (start). These have primative Hb. BFU-E (burst forming units) circulate in the liver. Liver is the major source of hematopoiesis from 3-6 months, and accumulation of fetal hemoglobin (HbF) is prevalent (vs. gower and portland).

Erythropoiesis in bone marrow starts at 5 months. Cytokines (KIT ligand, IL-3, GM-CSF, and EPO) stimulate.

EPO initiates CFU-E (or pro-erythroblast stage).
What does the proerythroblast stage give?

What is the last stage able to undergo mitosis?

What does this stage give?
Proerythroblast stage initiates ribosome assembly and Hb synthesis. THis gives basophilic erythroblast, which accumulate RNA and hb, and divide into polychromatophilic erythroblasts.

Last mitosis: polychromatophilic erythroblasts.

Give orthrochromatic erythroblasts which give the reticulocyte. The nucleus is ejected and digested by macrophages.

So...BFU-E --> Proerythroblast --> basophilic erythroblast --> polychromatophilic erythroblast --> orthrochromatic eryhtroblast --> reticulocyte.
How long do reticulocytes circulate for?

Outline the role of EPO.
2 days, during which they continue Hb synthesis and the mc and ribosomes dissociate.

EPO- a glycoprotein produced by renal cells in response to hypoxia, bind to cell surface receptors that are in greatest numbber on erythroid progenitor cells and absent on mature erythrocytes. Negative feedback = Heme and TNF.
Where do megakryocytes develop from?

Outline progression.

What is this regulated by?
From stem cells through own lineage: BFU-MK to CFU-MK (burst forming unit to colony forming unit). Have a dividing compartment and a non-dividing compartment which has ongoing DNA synthesis, but no mitosis = nuclear lobation!.

Megakaryoblast --> promegakaryocyte (develops granules), to megakaryocyte. Acquire surface glycoproteins (VWF, Gp1b/2b), cytoplasmic granules develop, and platelets bud off to systemic circulation.

Regulated by thrombopoietin produced in BM stromal cells, hepatocytes and proximal tubular epithelial cells of kidney.
How long do each of the following survive? RBC, WBC, Platelet?
RBC - 120 days, WBC = 3-4 hours, Platelet = 10 days.
What do NK cells do?

What do they arise from?
Function: MHC non-restricted cytotoxicity. Produce cytokines that influence hematopoiesis and immunity (IL1,2,4, CSF, interferons). These are large cells with cytoplasmic granules of cytoxic enzymes.

Arise from common lymphocyte progenitor giving T, B and NK cells.
What are the 2 heme proteins?

What do both contain?

Describe the heme structure.
2 proteins: myoglobin and hemoglobin. Mb - heme protein in muscle cells, released in muscle injuries creaiting myoglobulinuria. MI will also release Mb. Hb - red blood cells.

Both contain a prosthetic group (not composed of amino acids) that is tightly bound to the protein.

The heme structure is a ring structure of tetrapyrrole protoporphyrin IX and one FERROUS iron. There are 4 bonds between N's in the porphyrin ring and the iron, and 2 more potential bonds that can be formedn by the iron on either side of the planar ring. The 5th position = binds His residue with globin, and 6th coordination can bind oxygen.
What drugs can influence Hb function?

What happens when Fe is in the ferric form?
CN-, CO, sulfonamides.

Ferric forms - ability to bind oxygen is lost, these are called MetHb and MetMb and bind water instead of oxygen at 6th coordinate position.
Describe the structure of Mb.
Single polypeptide with one heme group. Made up of 8 alpha helical regions. Numbered and designated by letters, e.g. E-7, F-8.

Globular protein, spherical in shape, soluble in water and charged polar aa are on surface, non-polar are in interior. Heme is in hydrophobic part between His E-7 and His-F-8. Proximal His-F-8 binds globin protein to heme group by binding with ferrous iron.
Describe the structure of Hb.
Composed of 4 polypeptide chains with 2 subunits - alpha and beta. Held in spherical shape by non-covalent forces. One heme group per subunit and 4/tetramer = 4 molecules of O2 transported.
What happens when O2 binds to Hb?

What happens when O2 is unbound from Hb?
Shift in the position of the dimers relative to each other, producing the R or relaxed form which is more open.

In deoxyHb, the structure is in the T or taut form.
Describe the shape of the O2 binding curve associated with Mb.
Hyperbolic. Has a > affinity for O2 and quickly approaches sat at low PO2. Has a hyperbolic velocity vs. substrate curve.
Describe the shape of the O2 binding curve with Hb.
Sigmoid shaped curve, requiring higher PO2 to reach full saturation. This indicates cooperatively (accounted for by structural changes). The Fe moves to the other side of the ring and pulls His F-8 with it, relaxing the configuration.
What does low PO2 promote? High?
Low PO2 promotes unloading, high PO2 promotes loading.
What effect do the following have on Hb-O2 binding curve?

Low pH
2,3-Bisphosphoglycerate
Chronic anoxia
CO2
Low pH shifts RIGHT, assisting in UNLOADING from oxyHb. = Bohr effect, and picup of protons is greater, stabilizing the deoxyHB.

2,3 DPG: generated in breakdown of glucose for E. Binds with deoxyHb and pulls subunits more closely, stabilizing T conformation. This lower O2 affinity, acting as an allosteric regulator for O2.

Chronic anoxia: 2,3 BPGA concentration in increased, (e.g. altitude, emphysema), favouring unloading. Blood stored in acid-citrate-dextrose as an anticoagulant decreases 2,3 BPGA, unloading oxygen poorly.

CO2: lowers pH and can react with N-terminal end of amino acid to form charged carbamates that promote more salt-bridging of deoxyHb, stabilizing Hb and decreasing O2 affinity.
Outline the differences in the following types of Hb:

HbA
HbA2
HbAIc
HbF
HbS

How many globin genes code for each the alpha and beta chain?
HbA: Adult Hb, (a2b2), major adult form.

HbA2: minor component, with 2 delta-subunits/tetramer.

HbAlc: Glycosalated, occurs in response to prolonged levels of circulating glucose.

HbF: a2y2, major Hb of fetal circulation to 8th month, binds Hb 2,3 BPGA poorly, facilitating transfer at placenta.

HbS: sickle.

4 genes on CHR 16 to code for alpha. 2 genes on 11 to code for beta.