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173 Cards in this Set
- Front
- Back
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Growth Factors
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-small proteins that are secreted locally (autocrine or paracrine)
-Cannot enter a cell but bind surface receptor and activate signal transduction to alter gene transcription -As cell differentiate they leave the cell cycle and cannot divide anymore (terminal differentiation) Ex: 1) PDGF (platlet-derived growth factor) - used by MSC division and wound repair 2) Epidermal Growth Factor (epidermal division and tooth differentiation) 3) Bone Morphogenetic Proteins - bone, cartilage and tooth differentiation |
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Growth Factor Receptors
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1) Serine/Threonine Kinase Receptors - Bound by TGF and BMP and directly activate TF that go to the nucleus. No relay involved
2) Receptor Tyrosine Kinase - bound by EGF, PDGF, and FGF) -act through G-protein and protein kinase cascade. Depending on the kinase you get different gene transcriptin -Works by dimers which are separate with a extracellular ligand domain, transmembrane, and intracellular tyrosine kinase activity. 1) When a ligand binds the RTK dimerize, change conformation, and autophosphorylate the tyrosines 2) The P-tyrosine act as docking sites for relay protein (GRB2) and adaptor protein (SOS) 3) SOS s a guanine nucleotide exchange factor (GEF) and it displaced the GDP from Ras and adds a GTP 4) Ras is a single subunit GTPase which is NOT a protein kinase. It does not add P to a protein, rather it is active when GTP bound and uses energy from phosphate to accomplish goal 5) Ras starts a protein kinase cadcade (MAPK) by activating Ser/Thr kinase Raf 6) Raf phosphorylates MEK, MEK phosphorylates ERK, and ERK translocates to the nucleus to activate TF ELK-1 for DNA synthesis |
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What Determines GF Response
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1) Cells must have a receptor for the GF
2) Cells have various MAP kinases and TF that the ligands activate 3) A signal can be modified by multiple growth factors, receptors, and kinases at the same time |
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Cell Division
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-allows a cell to increase in size, replicate DNA, and divide into 2 daughter cells
-Cell cycle can take hours (epithelial cells) or years (hepatocytes) Interphase G1: Cell grows and prepares for mitosis to have enough material for 2 cells S: DNA synthesis results in sister chromatids G2: Further growth M: Mitosis - condensation, nuclear envelope breakdown, chromosome segregation G0 - Cell withdraws from cycle due to lack of GF or to differentiate |
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Mitosis
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-interphase is long while mitosis is short
1) Prophase - Chromosome condensation 2) Metaphase - Chromosome attach to spindle at equator 3) Anaphase - Sister chromatids separate 4) Telophase - Chromosome repackage into 2 separate nuclei 5) Cytokinesis - Cleave of cell into 2 daughter cells |
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Regulation of Mitosis
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-Mitosis is tightly regulated so that a cell only divides when it has the requirements to ensure a successful copy
-This is done by phos/de-phos, gene expression, and protein degredation -To go from G1/S the cell needs to be an adequate size and there must be a favorable environment with lots of nutrients and GF To go from G2/M the DNA must be replicated and the size sufficient -To go from metaphase-to-anaphase the chromosomes must be attached to a spindle |
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Cell Cycle Control by Kinases
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-Cell cycle is controlled by the expression of certain proteins at different stages
-The main players are cyclin and cyclin-dependent kinase (CDK). Different variations of these molecules are coordinately up/down regulated before each stage of the cell cycle -Before a cell cycle stage CDK active site is blocked by a T-loop. Right before the stage transition, specific cyclins are upregulated and these bind CDK and moves the T-loop. After this CAK phosphorylate CDK to finish activation -Between G1 and S you have G1/S-cyclin and G1/S-Cdk. G1/S-cyclin leaves at the beginning of S -During S there is S-cyclin and S-CDK. S-cyclin disappears before mitosis -Between G2/M you have M-CDK and M-cyclin. M-cyclin goes way at the end of mitosis |
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Role of CDK
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-CDK phosphorylate the proteins necessary for that stage of the cell cycle
M-CDK - activate chromsomal proteins, microtubule associated proteins, and cyclin degradation molecules -CDK are inhibited by errors in mitosis like lack of growth factors, DNA damage, unreplicated DNA, and chromosomes that are not attached to the spindle -Also, CDK are inhibited by degredation of cyclines b APC/C -During metaphase if everything is going correctly inactive APC/C will be activated by Cdc20. This molecule is a ubiquitin ligase that catalyzes ubiquitin formation. Ubiquitin will destroy S and M cyclins via the 26S proteosome |
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Role of Growth Factors in all of this
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1) GF binds receptor leading to a signaling cascade and TF activation
2) TF activate cyclin and CDKs 3) Cyclin and CDKs activate TF like E2F 4) Enzymes like E2F activate proteins involved in various stages of mitosis |
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Mitogen Control of Cell Division (more detail of previous slide)
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-Mitogen is a GF involved in mitosis
1) Mitogen is involved in activating G1. They bind to a receptor and stimulate the RAS/MAPK pathways leading to transcription of immediatete early genes like Myc 2) Immediate early gene Myc increases expression of delayed response genes like G1 cyclins that lead to G1/CDK formation 3) G1/CDK phosphorylazes Rb can now releases and activates E2F 4) E2F stimulates transcription of G1/S and S-cyclins which leads to S/CDK and DNA synthesis |
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Turning off Growth Factors
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Turn off:
1) Growth factor dissociate from receptor 2) Ras can no longer bind/activate kinase when its GTPase cleaves GTP to GDP 3) Phosphatases are activated which remove phosphate and inactivate kinases 4) Cyclin production can be blocked so CDK no longer active What happens if this cascade doesn't work? -if genes involved in cell cycle control are mutated then control is lost Oncogenes: Genes that promote cell cycle are proto-oncogenes because their mutation can lead to cancer 1) Mutated receptors remain on 2) Mutated GTPase leaves Ras on 3) Cyclin over production leaves CDK on Tumor Suppressors: These genes normally turn off or inhibit cell division 1) Rb - If mutated it will fail to block cell division |
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Effect of DNA Damage on Cell Cycle
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-DNA damage leads to blocked cell cycle progression until the damage is repaired
1) DNA damage leads to upregulation of protein kinase ATM 2) ATM phos protein kinase Chk1/2 3) Chk1/2 phos p53 (major tumor suppressor mutated to 50% of cancer) 4) Active p53 will upregulate p21, a CDK inhibitory protein 5) p21 will inactivat the G1/S-CDK and S-CDK complexes arresting the cell in G1 |
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Cyclin Clarified
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-it appears as if the CDK activate production of cyclin for next cycle and activate degradation enzymes for their cyclin
-G1-CDK => G1/S cyclin=> G1/S-CDK => S-cyclin => S-CDK => M-cyclin => M-CDK |
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Bone Morphogenetic Proteins
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-GF important in bone and tooth development
-implanted ground-up demineralize bone component that leads to ectopic bone -Over 20 BMPs but only a few have osteoinductive properties. Results still under debate -indice pre-osteogenic (osteoprogenitor) cell to differentiate 1) BMP binding to receptor leads to multimerization 2) Autophosphorylation of Ser/Thr residues 3) Phos of Smad1/5 transcription factor 4) Smad 1/5 complexes with Smad 4 and this translocates to the nucleus to activate gene transcription needed for bone formation |
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Take Home Lesons
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-Cyclins synthesized in response to growth factors and degraded when no longer needed
-Cell cycle checkpoint include inadequate nutrient supply, absense of GF, failture to replicate DNA, DNA damage Proto-Oncogene - proteins that POSITIVELY regulate GF pathway (receptors, G-proteins, cyclins). Gain of function => cancer Tumor Suppressor - negatively regulate GF pathway. Loss of function mutation => cancer |
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Apoptosis Overview
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-tightly regulated intracellular process leading to cell death
-proteins are activated that degrade DNA and other proteins, however, the membrane remains intact and apoptotic bodies are made -apoptosis is required for development (fingers) and to eliminate threatening cells (cancerous/infected) 3 types: 1) Physiological - eliminate unwanted cells and maintain a steady # in tissue. =Example is development of fingers 2) Pathologic - Remove cells beyond repair 3) Iatrogenic - Drug induced |
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Physiologic Apoptosis
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1) Hormone-dependent Involution - Removal of hormones leads to cell death. Example is uterus and menstration
2) Death of cells that already served their purpose 3) Delete old cells in constant proliferating population (skin) 4) Get rid of cells not passing mitosis checkpoint due to abnormal DNA or DNA damage 5) Host Defense: Either T-cells or NK cells killing infected cells or autoreactive lymphocytes being targeted before maturation |
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Pathologic Apoptosis
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1) A cells that is exposed to toxins or stressed will undergo apoptosis instead of necrosis to avoid inflammation and further damage. Example is hepatocytes that are bombarded with toxins and if they died by necrosis our livers would fail
2) Too much apoptosis can lead disease like in Parkinson's with the neurodegenerative neuron death 3) Too little apoptosis can lead to disease like cancer Ex: lymphocyte tumor (follicocyte) Sqamous cell carcinoma |
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Iatrogenic Apoptosis
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-induced both intentionally and unintensionally by drugs
1) Cytotoxic drugs or radiation to treat cancer 2) Drugs to inhibit autoimmune reactions 3) Drugs to prevent thyroid goiters |
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Apoptotic Morphogenesis
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-affect a single cells
-plasma membrane DOES NOT rupture -apoptotic cell removed by phagocyte -NO INFLAMMATION -Cell shrinks and the cytoplasm and nucleus condense -Chromatin condense, aggregate in the periphery and DNA fragments -Cell breaks into MEMBRANE bound blebs (apoptotic bodies) filled with cellular and DNA fragments which are eaten by phagocytes |
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Enzyme Involved in Apoptotis - Capases
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-cysteine proteases that cleaves after ASP residue
-exist as inactive precursor and are activated by auto-cleavage and dimerization -broken into initiator and effector caspases 1) Initiator - first activated and responsible for activating effector/downstream caspases 2) Effector - Cleave cell substrates |
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Enzymes Involved in Apoptosis - BCL-2 Protein Family
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-Regulate apoptosis via the mitochondria
-Two groups 1) Anti-apoptotic - BCL-2, BCL-XL 2) Pro-Apoptotic - BID, BAD, BAX -apoptosis determined by balance between the two 1) In a healthy cell there are more anti-apoptotic proteins that seal mitochondria pores 2) During apoptosis pro-apoptotic proteins are released or upregulated and these bring anti-apoptotic and replace them making the mitochondria leaky 3) Leaky mitochondria releases cytochrome C and apoptosis inducing factor which trigger the caspase cascade 4) Leaky Mitochondira cannot do oxidative phosphorylation, no ATP is made, oxygen radicals are created, and MT is not functional |
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Apoptotic Pathways: Intrinsic
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-the extrinsic, intrinsic, and execution pathways are usually all tied together
1) Extrinsic - initiated by death receptors Fas and TNFR1 bining to Fas-L. This leads to adaptor protein activation and caspase initiator cascade and execution pathway. Can occur with or without the mitochondria 2) Intrinsic Pathway - Involved mitochondrial permeabillity regulation and death inducers AIF and cytochrome C -Causes by growth factor withdrawl, DNA damage, or protein misfolding causing a change in the balance between pro/anti-apoptotic proteins 3) Execution Pathways - Intrinsic and Extrinsic pathway ultiimately lead to this which does the dirty work of creating apoptotic bodies -Caspase cascade is activated which degrade cellular components destroying nuclear scaffold and cytoskeleton |
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Differentiating Apoptosis and Necrosis
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-apoptosis affects a single cell while necrosis affects a region
-apoptosis has no inflammation while necrosis has inflammation -apoptosis is controlled while necrosis is not -apoptosis maintains cell membrane -Necrosis has random DNA fragmentation leading to a solid DNA gel while apoptosis has specfiic endonucleases clipping DNA between histones and a more patterned DNA gel |
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Stem Cell Achievements
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Evans - Isolated mouth embryonic stem cells
Cappecchi and Smithies - Homologous recombination and gene targeting |
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Stem Cell Properties
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1) Self renewal - maintain population
2) Differentiate into many specialized cell types. As they differentiate they loss potential but gain specialization -Stem cells commit towards a lineage and then go through specific stages until reaching final differentiated cell (progenitor/precursor) -Potential and differentiation are controlled at a transcriptional level 3) Undifferentiated |
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Stem Cell Hierarchy
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1) Totipotent - Inner/Extra embryonic tissue from a fertiziled egg. Can become any cell
2) Pluripotent - Inner embryonic tissue that gives rise to ESC that can give rise to most cell types 3) Multipotent - Can give rise to some cell types |
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Embryonic Stem Cells
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-from the inner mass of the blastocyst that differentiate during development
-They have a high potential for differentiation and can form all adult tissue and therefore have a high potential for therapy -Tons of ethical concerns -These are also considered pluripotent and can give rise to ALL adult types and tissues but cannot do so unless mixed with normal embryonic cells |
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Adult Stem Cells
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-These are maintained in tissue specific niches throughout life and are limited in their diff. potential to that tissue and a few other cell types (multipotent)
-They have no major ethical concerns and can be used for limited and specific tissue therapy -These undiff. cells among diff. cells are used for tissue maintainence, repair, and regeneration -Quiescent until triggered -You can distinguish a adult stem cell, progenitor, and diff. cell through cellular activity (function), morphology, and genetic markets expressed (most popular) -In bone marrow there exist hematopoietic stem cells that make RBC/WBC and bone marrow stromal stem cells (mesenchymal) that make osteoblast, muscle, chondrocyte, and adipocyte |
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Umbilical Cord Blood Stem Cells
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-a type of adult stem cells from the FETUS (not ESC)
-hematopoetic stem cells used to form platelets, RBC, and WBC -Better than BMSC cause fast/easy collection, more matches/available, lower risk of infection, reduced graft versus host -Not great to treat child where they came from because same genetic disorder present at birth |
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Dental Pulp Stem Cell
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-MSC isolated from the dental pulp and can make odontoblast and neurons and more
-They are easily accessible and can be cryopreserved |
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NIche
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-The stem cell location is critical to maintain their stemness and activate their differentiation and renewal
-The important parts of the niche is architectural space, location (near BV), and effectors -Effectors include secreted soluble factors (metabolic products), endocrine, paracrine factors), extracellular matrix, and integral membraner proteins for cell-cell contact -Maintanence of pluripotency AND differentiation is due to the tissue specific gene expression. This expression is controlled by outside signals in the niche whcih regulate the balance of TF |
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Induced Pluripotency
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-You can induce pluripotency by expressing certain genes (TF) to turn a fibroblast into a ESC
-Genes are oct4, sox2, among others -These are now called induced pluripotency stem cells or IPS cells |
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Using Stem Cells for Therapeutic Potential
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1) Establishing a stem cells line (collection and expansion) - includes purification
2) Maintain undifferentiated state - Make a artificial niche using a feeder layer of cells to provide cell-cell contact and secreted GF 3) Trigger Specific Differentiation - Control commitment and lineage by activating/silencing specific genes using GF and cell culture conditions 4) Making an organ 5) Control differentiation and migration after transplantation |
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Current Stem Cell Therapies
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1) Nuclear Transfer - After collecting cells in a biopsy you transfer the nucleus to a oocyte and reprogramming the cell with specific genes. Once grown to the blastocyst stage you collect the inner mass ESC and re-implant in the area needed
-The risks are development of tumors (teratoma) and a immune reaction |
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Ethics Involved in Stem Cells
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-reproductive cloning is banned while therapeutic cloning of specific cells and tissue is allowed
-major problem is that harvesting the blastocyst inner mass results in embryo destruction |
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ECM Overview
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-Tissue ECM is diverse and adapted for special functions of that tissue
1) Mechanical support (BL) 2) Separates compartments (BL) 3) Sequester GF 4) Pathway for migration - embryonic cell, wound healing matrix, innate immunity 5) Conveys info from the environment to the cell to direct division and differentiation (Dynamic Reciprocity) -Endothelial cells treated with VEGF only differentiate on matrigel |
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ECM Components
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1) Collagen - tensile strength
2) Elastin - Resilience 3) Proteoglycans - Compression resistance. Ex: Hyaluron 4) Fibronectin - Cell adhesion |
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Collagen
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-mostly made by fibroblast
-most abundant protein in CT is considered a glycoprotein 1) Fibril-forming - Col I (main), II, III (skin), and XI. Uninterrupted triple helix which makes it rigid 2) Fibril-Associated - Col IX, XII. Several non triple helical regions throughout that makes it a bit flexible 3) Network Forming - Col IV in BL -lots of non-triple helical regions making it very flexible -Collagen is a triple helix and the majority of the fibril is Gly-X-Y with X being proline and Y being hydroxyproline -The glyine is in the middle due to its size as are the peptide bonds so they are resistant to proteolysis -The Pro and Hypo are on the outside -Each strand for a polyproline type II helix |
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Collagen Synthesis
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-extensive co-post translational processing intra and extra cellular
1) Pre-Procollagen a-chain single polypeptide begins synthesis in the cytoplasm 2) N-terminus peptide targets protein to the ER for the secretory pathway. The N-terminus is cleaved as procollagen a-chain is made 3) In the rER 50% of the lysine and proline hydroxylated -Prolyl/Lysyl hydroxylase uses Fe+2 as a cofactor to add oxygen to these amino acids. To reduce Fe to the original state ascorbate is needed -Hydroxylated proline is involved in hydrogen bonds that stabilize the triple helix so our body temp does not degrade the protein 4) Starting in the ER and finishing in the Golgi selected hydroxylysines are glycosylated 5) Chains are aligned and a procollagen triple helix is formed by cysteine disulfide bonds 6) N-glycosylation of procollagen starts in ER and ends in Golgi 7) Procollagen keeps N/C propeptides so it is soluble. After golgi pack procollagen in secretory vessels and secrete it, the propeptides are removed. The propeptides are removed by endopeptidases N/C-propeptidase. The results is a COLLAGEN protein while non-triple helix telopeptide domains at the ends 8) Once collagen is formed the molecules sponateously polymerize outside the cell |
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Collagen Assembly
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-collagens assemble with a space between the head of one and tail of another for calcium deposition
-They are in a 1/4 staggered pattern giving them the 67nm cross striation pattern -collagen fibrils are crosslinked by lysyl oxidase which forms covalent bonds between Lys/HLysa in the telopeptide region. Crosslinking stabilizes collagen making them insoluble -Lysyl oxidase is Cu dependent and uses oxygen to form 2 aldehydes. These aldehydes with form a covalent bond by spontaneous aldol condensation -Collagen fibrils associated into collagen fibers which associated into collagen fiber bundles |
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Collagen Malfunctions
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1) Without a citrus source a ascorbic acid deficiency leads to a problem with Prolyl/Lysyl hydroxylase hydroxylation
2) A defect with crosslinking leads to CT defect. Accomplished by copper deficiency, lysyl oxidase mutation, or consumption of lots of peas which contain -aminopropionitrile which inhibits lysyl oxidase 3) Osteogenesis Imperfecta - due to mutations in collagen or collagen-modifying genes. Dominantly inherited and leads to bone fractures and dentinogenesis imperfecta 4) Ehlers-Danlos Syndrome - Due to mutation in Col I/III or collagen modifying enzymes. Leads to joint hypermobility, skin elasticity, and aortic aneurysms -In EDS Type 4 col 3 is deficient leading to thin collagen fibrils, thin fibers, and thin tissue. Skin, BV< and bowels are thin and easily rupture. No hypermobile joints except hands |
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Collagen Variations
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-collagen fibrils are usually heterotypic and contain more than 1 type of collagen
Ex: PDL has I, III, and XII (minor and appears during eruption). PDL collagen is in bundles Dentin has I and III -Cartilage made of major Col II and minor Col XI which lies on fibril surface using N-terminus domain. Determines fibril diameter -Col IX associated with other fibrils allowing them to interact with other matrix components |
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Type IV Collagen
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-the heart of the basil lamina
-Has many imperfections in the triple helix and keeps C/N terminal domain so procollagen -Instead of forming fibrils it forms a flat sheet-like lattice using 1) Monomers form dimers by C-terminal association 2) Dimers form sheets by lateral association via triple helix domains 3) Sheets form multilayers by N-terminus projecting above and below the sheet plane forming covalent bonds with other sheet N-terminals (lots of disulfide bonds) |
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Other Collagen Uses
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-propeptides of Col IV (tumstatin)) and Col XVIII (endostatin) have anti-angiogenic properties
-Can inhibit cancers -Works well in mice but results in humans mixed |
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Elastin Fibers (Elastin)
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-give tissues (skin, bladder, bv, lung) their elasticity
-made of elastin, fibrillin 1/2, and microfibril associated glycoproteins (MAPs) -microfibrillar proteins are deposited first and help organize amorphous elastin into elastic fibers by assisting in covalently crosslinking elastin -Elastin is secreted as a hydrophobic tropoelastin by fibroblast and SMC with G, V, A, and P (little hydroxyproline and no glycosylation) -Lysyl oxidase deaminates lysins forming desmosine and isodesmosine which form cross-links necessary to keep structure when stretched and recoil |
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Elastin Syndromes
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1) COPD - disruption of elastic fiber architecture leading to emphysema and chronic bronchitis
2) Mafan Syndrome - Problem with fibrillin-1 gene leading to tall individual with long appendages, affected aortas, and vision problems 3) Williams Syndrome - Deletion of elastin gene on chromosome 7 leads to tooth + cardio problem, elfin appearence |
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Proteoglycans
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Proteoglycan - protein with lots of glycosylation usually in the form of a GAG
Ex: aggrecan in cartilage ecm, decorin on col 1, syndecan on FGF, and serglycin on mast cell histamine GAG - polysaccharide with repeating disaccharides attached to a core protein -one sugar is a amino sugar in the form of n-acetylglucosamine or n-acetylgalactosamine -they are negatively charged due to a sulfate or carboxyl group -attach to core protein via Ser residue |
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Types of GAGs
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1) Chonrdoitin Sulfate - in cartilage
2) Heparan Sulfate - found in the basement membrane and binds growth factors 3) Heparin - Found in mast cells and basophils as a anticoagulant 4) Hyaluronic Acid - found in cartilage (part of aggrecan), synovial fluid, and loose conn. tissue. Used as shock absorbers |
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Hyaluronic Acid
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-the disaccharides (glucuronic acid and N-acetylglucosamine) are not sulfated
-super large -not attached to a protein -due to its size it is synthesized at the cell surface by hyaluronan synthases -it is a good shock absorber because it can expand when given water. Loses water to help resist compressive forces, then quickly expands when compressive removed -HA quantity and quality decrease with age -The drug Restylane is composed of HA and injected in skin to prevent wrinkles |
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Other Proteoglycans
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1) Aggrecan - secreted, major PG in cartilage helps cartilage form original shape after compression.
-It is composed of HA non-covalently attached to a linker protein whcih is connected to a PG (core protein and GAG) 2) Syndecan - Bound to surface by transmembrane domain and attaches to HS and CS. Low-affinity receptor for GF and ECM proteins -Binds to FGF controlling availability and can release when needed. Syndecan can also induce a conf-c in FGF allowing it to bind the FGF receptor 3) Glypican - Attaches to surface by GPI anchor and has HS -viruses can use PG as receptors to enter cells |
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ECM Interaction Overview
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-ECM provide information to cell so they express tissue specific genes and divide, differentiation, migrate, and alter metabolism
-Cell need to attach to a ECM to grow, proliferate, and survive -ECM helps endothelial cells undergo angiogenesis, OB for bone resorption, and blood clot to have cells migrate |
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ECM Main Players
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1) ECM Proteins
-collagen, proteoglycan, multi-adhesive proteins -multi-adhesive proteins serve as bridge between cell and ECM. Example is fibronectin, laminin (BM), and osteopontin (bone resorption) ECM Receptors: integrins and syndecan (PG) Intracellular Components - help transduce signal, maintain structure, and move cell. -cytoskeleton component and signaling molecules |
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Fibronectin
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-FN has several domains with different functions to act as a bridge to connect ECM components/cells together
-Insoluble FN made by fibroblast and soluble FN made by hepatocytes Structure: -FN has 2 elongated subunits connected by a C-terminus disulfide bond -domains include fibrin binding, collagen binding, heparin/heparin-sulfate PG binding, cell receptor binding -lots of alternative splicing at 3A, 3B, and 3CS produce fibroblast vs. hepatocyte (no 3A/B) version Function: -essential for embryonic development -soluble FN crosslinks w/fibrin at blood clot |
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Fibronectin Domains
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-domains are formed by repeating modules with a specific purpose
Module 3 9/10- Encodes B-sandwich structure that binds integrin. III(10) has R-G-D sequence loop and III (9) is a synergy site that enhancing RGD binding Module 3 (13/14) - Have positively charged basic amino acids that interact with negatively charged heparin sulfuate PG and heparan |
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Osteopontin
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-involved in bone resorption and the immune system
-high phosphorylated -has a acidic domain with ASP/GLU which binds hydroxyapatite -Has a RGD domain that binds integrin on osteoclast to stimulate resoprtion -Has a Ca domain to bind calcium -When OP aVb3 domain binds integrin on osteoclast it helps OC adhere to bone so it can be resorbed. W/O OP mice have too much bone |
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Laminin
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-the major cell adhesion protein in basement membranes it has globular domains to bind carbs and integrin (w/o RGD); a-chain, b-chain to bind lipids and Col IV, and g-chain to bind collagen and lipids
-Is a cross-shaped trimer of a,b,g chains stabilized by coiled-coil interaction |
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Basement Membrane
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-surround muscle fibers and nerves, and lines epithelial and endothelial cell sheets
Function: -structural support, tissue separation, kidney filter of macromolecules, injury scaffold to promote migration and guide rehabilition of NMJ Structure: 4 components of BM can bind each other -formed by a Col IV lattice with C-dimers, N-tetramers, and triple helix lateral associations -Has laminin, entactin, and HS-PG perlecan as well -Entactin (nidogen) is a sulfated glycoprotein which binds 1:1 laminin and also Col IV |
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Integrins
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-Heterodimeric transmembrane proteins in which alpha and beta subunits must come together to make the matrix binding domain
-for the domains to unit the alpha domain must bind divalent cation calcium or magnesium -Has a super low binding affinity (Kd) because ligand concentration in the ECM is high and it must be able to rapidly bind and dissociate -many combination of alpha and beta domain allow integrins to bind many different molecules -Some integrins bind many proteins while others (a5b1) only bind one (fibronectin) |
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Cell Binding and Focal Adhesion
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-A cell uses integrins to bind the ECM in many ways
1) Focal Adhesion - fibroblast use integrin to bind fibronectin. Integrin uses a adaptor protein to pass the signal to F-actin microfilaments 2) Hemidesmosomes - Epithelial cells use integrin to bind laminin in the basal lamina. The signal is passed by adaptor proteins to keratin intermediate filaments |
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Focal Adhesion Specifically
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1) Integrin binds fibronectin
2) Cytoskeletal and signaling proteins associate with cytoplasmic tail of the B-integrin 3) Other integrins cluster and actin microfilaments assemble 4) This leads to activation by autophosphorylation of focal adhesion kinase (FAK) 5) FAK relays a message to the rest of the cell to start organization of cytoskeleton for motility, cell divison, and apoptosis or survival -focal adhesions are heavily regulated and dynamic, always changing |
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Focal Adhesion SIgnaling
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1) Inside Out - Cell receptor binds ligand and this sends signal to integrin cytoplasmic tail to change affinity to increase binding to ECM leading to more FAK
2) Outside In - After the integrin changes affinity it will bind to the ECM transducing a signal to the inside 3) Response to Chemokine - Binding of cell to chemokines changes integrin inside-out signaling and cytoskeleton reorganization and movement. Direct and speed of movement determined by direction and amount of chemokine -When FAK activated in chemokine movement focal adhesions near gradient are increased while focal adhesion and attachment to back is decreased |
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Crosstalk Between Signals
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-Integrin signal acts in conjunction with other signals like GF pathways, cytokines, chemokines, and syndecan PG
1) Receptor Pathway Modulation - Activation of integrin enhances or inhibits another pathway (Erk activation) 2) Receptor Compartmentalization - Integrin activation helps organize signaling components in another pathway to facilitate their activation (cytoskeleton rearrangement) 3) Receptor Transactivation - Integrin activation can activate another receptor like EGF, PDGF, VEGF 4) Receptor Coordination - Integrin signal works with another pathway signal to trigger downstream event (works with PDGF, syndecans) 5) Receptor Expression Modulation - Integrin signal upregulations/downregulations the expression of another receptor like syndecan |
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ECM and Cell Proliferation
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-the more a cell can bind and spread of a surface the more likely it will enter S-phase of mitosis
-Integrins and other GF receptor work together to activate Ras pathway which activates cyclins and S-phase -If a cell cannot bind a substrate then lack of integrin signal can cause apoptosis (anoikis) -In cancer cells Src (proto-oncogene) mutation leads to constant FAK expression and increase survival, growth, and motility, and ECM destruction w/o a signal |
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Integrin Angiogenic Properties
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-Only in the presence of a ECM can VEGF induce angiogenesis
-Activated integrins like aVb3 bind ECM proteins like laminin and help VEGF bind to its receptor -Molecules that bind integrin can be used in cancer therapy to block RGD site and prevent integrins from inducing angiogenesis (Vitaxin, endostatin) |
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Proteoglycans as Cell Surface Receptors
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Syndecan - Membrane bound proteoglycan with heparan sulfate and chondroitin sulfate.
-low-affinity coreceptor for ECM proteins -Works with integrin to bind cell to ECM: Integrin binds RGD on FN while syndecan heparin sulfate binds the HS domain on FN -Syndecan-4 can work with a5b1 to bind ECM and activate GTPase for actin remodeling (movement) and endocytosis |
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Hemidesmosome
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-formed at dermal/epidermal junction when basal keratinocytes bind basal lamina
1) Intermediate keratin filaments in basal cell bind adaptor plaque protein attached to a6b4 integrin 2) basal cell a6b4 binds laminin which is connected to the rest of the basement membrane 3) The basement membrane binds to dermis anchoring fibrils Col VII ...if this connection is severed... 1) Epidermolysis Bullosa - mutation of gene in dermal-epidermal junction (keratin 5, integrin, laminin, col 7). Leads to separation of epidermis from dermis because no anchoring. Seen as skin fragility, nail and enamel problems, blistering and scarring all over with skin is (mouth, ass, esophagus) |
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ECM Degradation
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-ECM degradation both a normal and pathogenic process
-Balance between degradation and synthesis important in adults -ECM degradation in pathologen in tumor metasisis, periodontitis, and arthritis -ECM normal in a process like embryonic development with the resopriton of the frog tail during metamorphosis using the enzyme collagenase-1 |
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ECM Degradation Proteins
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1) Collagenases - ONLY enzyme in vertebrates that can cleave triple helical collagen. Part of the MMP family
a) Fibroblast - produce it during immune respone b) Cancer - Produce to metasisize and migrate through tissue c) Bacteria use it to spread |
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Matrix Metalloproteinases
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-MAJOR enzyme to carry out ECM degradation
-Zinc dependent and can be secreted or membrane bound -Many different types organized by what they cleave, all together can degrade everything in the ECM -synthesized as inactivate zymogen and propeptide cleaves at right time and right place Ex: development, angiogenesis, mammory gland at puberty, bone in remodeling during inflammation |
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MMP and Dentistry
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-MMP-20 is enamelysin and expressed by ameloblast to remove organic matrix to allow for mineralization
-MMP-20 mutation leads to amelogenesis imperfecta -MMP also causes periodontal CT and bone loss. Bacteria in periodontitis leads to inflammation and upregulation of MMP around gums as a result MMP Drugs -Therapy include MMP inhibition by chelating zinc (Periostat) -Santyl is a collagenase that can be used to digest collagen from necrotic tissue in ulcers of burns -Can also inject collagenase to treat cellulite |
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ADAMs Family
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-A distintegrin and metalloproteinase
-Cell surface protease that do no degrade ECM but degrade other cell surface proteins -Col 17 at hemidesmosomes of dermal-epidermal junction is cleaved by ADAM 9,10,17 |
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Random Taste
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-chlorhexidine kill taste receptors salt and bitter and replace with pain
-if block 3/4 of receptors then brain pays attention to pain -Pain associated with the tongue is due to CN V while taste is due to CN VII and CN IX -sweet, sour, salty, bitter (toxins, alkaloids, peptides), umami (GLU, ASP, nucleotides), and olfactory receptor too (taste + olfactory = chemical irritation) -umami or savory is the taste of glutamate or meat |
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Feline Taste
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-cannot taste sweet because receptor T1R2 is defective
-it has lots of stop codons and exon 3 deleted, so now it is a pseudogene -Cannot prep body for sucrose digestion |
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Taste 1
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-taste helps trigger cephalic phase of digestion and metabolism
-taste receptors in the alimentary tract to signal brain what food is incoming -orbital frontal cortex takes in taste from tongue through CN 7, 9, 10 -you need to trigger both seratonin and gustducin on 2 diff. cell for taste to happen |
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Taste Receptors
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-taste cells have tight junction btw them
-Type I is a dark cell, support -Type II is a receptor cell -Type III has synaptic machinery -Type IV is a basal cell - Type V is a perigemmal cell |
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Taste Receptor II
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T1R Family - sweet and umami heterodimer. 1 sweet and 4 umami but always heterodimer
-related to Ca receptor and glutamate T2R: bitter -similar to olfactory receptor -broad specificity -stimulating the receptors trigger ion channels -taste bud is a multicell organelle of 50-75 cells of which 7-10 function as receptors and cell turnover is high. |
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Mineral Biology
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-Calcium and phosphate are essential macronutrient
Calcium: -Calcium is important for bone formation and in the EC fluid and cytoplasm Ca concentration maintains several processes like muscle contraction/nerve conduction -Most Calcium is in skeleton as hydroxyapatite, very little is in the cytosol Phosphate: -Also in hydroxyapatite but also in cell intermediates and in the EC it is important as a buffer -Most phosphate is in skeleton but a high percentage is not in skeleton and in cytosol or EC than calcium -Not as tightly regulated as calcium |
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Calcium and Phosphate Regulation
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1) Built and maintain a healthy skeleton
2) Maintain blood Ca 3) Maintain cellular and extracellular fluid phosphate 4) Prevent ectopic calcification. Calcium and phosphate form a salt so if they were close by in a high conc this would happen -calcium demands increase with age until adulthood -calcium supplements w/o extra vitamin-D leads to risk of myocardial infarction -phosphate requirements increase with age and then dip to baseline in adulthood |
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Mineral Homeostasis
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-blood calcium balance is achieved by hormones that regulate Ca/P levels in bone, intestine, and the kidney
1) 1,25 Vitamin D - targets kidney, intestine, and bone 2) Parathyroid Hormone - targets kidney and bone 3) Calcitonin - targets bone and a bit on the kidney |
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Mineral Hormones Reactions
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-Low Ca trigger PTH to release Ca from bone and excess is phosphate is execreted by FGF22 stimulating kidneys to be less permeable to phosphate reabsorption
-FGF22 is made by OB/OC and regulated by Fex/DMP1 resoption - bone breakdown absorption - uptake mineral from intestine to blood reabsorption - kidney function, take in molecules and spit back to blood as needed |
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Calcium and Phosphate Ingestion Tract
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1) Calcium is taken in through oral cavity and either absorbed in the intestine or execreted in feces
2) Calcitonin increase and PTH decrease to trigger Ca for bone deposit or urine (calcitonin lowers blood Ca). Between meals these trends are reversed 3) When needed PTH increase to release mineral from bone and kidney 4) Calcitonin and PTH are rapid and short term. For long term effects Vitamin-D is used as a steroid hormone to change gene levels. PTH triggers Vitamin-D activation from 24,25 to 1,25 |
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Hormone Effects on Bone
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-can be counterintuitive
-PTH and Vitamin-D can increase bone resoprtion and formation. It seems that long term PTH decrease bone formation and short intermittant PTH icnreases bone formation -Glucocorticoids Glucocoritoid makes sense and it increase bone resorption and decrease bone formation -Gonadal steroids decrease bone resorption and increase bone formation -Calcitonin decrease bone resoprtion -Thyroid hormones increase bone resoption -Growth Hormone increase bone formation |
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The Calcium Receptor
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-found on parathyroid gland, thyroid gland C-cells, kidney tubules, chondrocytes, and osteoblast
-GPCR that detects small chances in calcium conc and leads to signal transduction PTG: When calcium level is low the PTG CaR sense it and release PTH and the PTG cells proliferate. Opposite happens when levels high TG: When blood Ca concentration high the C-cells detect it and release calcitonin |
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Parathyroid Hormone
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-released by PTG cells when serum Ca drops below a certain number
-monomeric hormone -PTH increases blood calcium and decrease blood phosphorus -In the bone PTH increase bone resoprtion and decreases formation -In the kidney PTH decrease urine calcium and increase urine phosphate and conversion of 1,25 to Vit-D -Binds a GPCR called PTH1R whichis coupled to adencyclate cyclase (more cAMP) and IP3/DAG -PTH1R that binds PTH is found in kidney and bone -In tissues other than kidney and bone like growth plate chondrocytes PTH1R also binds PTHrP -This is more a autocrine/paracrine mechanism for growth and differentiation -Increase PTHrP is released to hypercalcemia of malignacy and bone metasisis of breast cancer -Both PTH and PTHrP bind PTH1R, different responses. PTH continue to send signal when internalized whereas PTHrP stops |
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Parathyroid Hormone Cont...
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Bone: PTH acts on osteoblast to increase RANK-L which OC precursor RANK to stimulate OC activation and bone resoprtion
-A bit of evidence that in certain doses PTH can increase bone formation in osteoporatic patients, drug is Teriparatide |
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Vitamin-D
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-Ingested in one of two inactive forms
1) V-D2 from plant steroid 2) V-D3 from cholesterol -Can be synthesized in UV light by B-ring photolysis but we don't get enough exposure Activation: Done by 2 hydroxylations, one in liver and one in kidney 1) In liver blood circulating vitamin D3 (cholecaliferol) is acted on by Vitamin D-25 hydroxylase to make 25-0H-Vit-D3 2) In kidney PTH trigger cytochrome P-450 (CYP) known as vitamin D-1 hydroxylase to make 1,25Vit-D3 which is active -Rate of this conversion controlled by serum levels of PTH and phosphate -Major role of vitamin D3 is Ca/P absorption in the small intestine, Ca reabsorption by the kidney, and resoprtion of bone |
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Vitamin-D Deficiency
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Skeletal Disease
1) Rickets - soft bones leads to fractures -Osteomalacia is a adult form of rickets 2) Vitamin D Resistant Rickets -hereditary defect in enzyme to make active vitamin-D. a) Hypophosphatemic Rickets - problem in phosphate metabolism so lots of bone resoprtion and low vit-D production b) Pseudo Deficiency Rickets - Problem in Vit-D metabolism that can be corrected 2) Non-Skeletal DIsease -Cardiovascular, multiple sclerosis, arthritis, diabetes, crohn's |
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Vitamin D Mechanism of Action
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-increase 1,25 leads to osteoblast stimulating OC formation and upregulating BM proteins
-Increase 1,25 leads to increase of calcium binding protein calbindin -Calbindins (EF hand) are vitamin-D dependent Ca binding protein that increase Ca transport -Vitamin D works by steroid hormone mechanism (genomic) leading to gene expression and a cell membrane mechanism (non-genomic) where transcription not the final product |
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Vitamin-D Mechanism of Action
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-The vitamin-D receptor is a ligand-inducible nuclear transcription factor
-N-terminal binds DNA and C-terminal binds ligand -They form a heterodimer with RXR and bind vitamin D response elements VDRE using 2 hexameric sites space by 3 nucleotides Nongenomic Effect - change membrane fluidity, fatty acid acylation of proteins, AA and PGE2 production, activation of PKC, PLC, MAPK, and openeing of L-type Calcium channels |
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Calcitonin
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-works mostly on bone
-leads to decrease in blood calcium and phosphate -binds receptos that increase intracellular cAMP and Ca -Acts on OC to inhibit mineral release from bone -Act on kidney to increase excretion of the minerals -Act on vitamin-D to increase another CYP, vitamin-D-24 hydroxylase to inactivate 1,25D3 |
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Phosphate Homeostatis
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-phosphate is needed to make nucleic acids and form energy metabolism as ATP
-Cells get phosphate from blood using sodium/phosphate active transporter -1,25 increase phosphate absorption while PTH decreases phosphate reabsorption Cycle 1: Low calcium and increase phosphate increase PTH while high calcium, 1,25 and FGF23 decrease PTH. PTH acts on OB and kidneys to increase bone resoprtion and decrease renal phosphate reabsorption and stimulate 1,25 (cyclic). Net effect of this cycle is increase serum Ca and decrease serum phosphate Cycle 2: FGF23 is up-regulated by serum phosphate and 1,25 and acts to inhibit phosphate reabsoprtion and decrease 1,25 and PTH. Inhibited by DMP and PHEX Cycle 3: Klotho is expressed in the kidneys and it helps FGF23 bind to FGFR1 by bringing them all together. Makes kidney FGF23 responsive when 1,25 and phosphate high |
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What's in Bone?
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-osteoblast precursors become osteoblast and not osteoid which is a uncalcified bone matrix. Eventually that will become a calcified bone matrix entrapping the osteoblast as osteocysts which communicate via canaliculi
-In osteoid there is no HA, mostly collagen and proteoglycans like decorin, fibromodulin. In calcified bone matirx there is HA within the collagen and only a few noncollagenous proteins like OCN, BSP -cementum and bone are very similar with mineral %, Col I content, and non-collagenous OP/BSP |
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Biological Mineral
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-the vertebrate mineral is hydroxyapatite (HA)
1) Clusters of ions come together in extracellular space to form a critical nucleus 2) Cell secrete protein which help sequester (chelate) ions and stabilize the critical nuclei. Proteins like collagen act as a scaffold for crystals to grow on -Collagen scaffold accelerates mineralization by allowing crystal to begin growing between fibrils. Mineralization of bone is initiated at the hole zones of type I collagen fibrils |
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Mineral Regulators
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1) Osteocalcin - Regulates crystal size
2) Bone Sialoprotein - Nucleates HA 3) Alkaline Phosphatase (ALP) - Converts mineralization inhibitor PPi (pyrophosphate) into organic phosphate -Intracellular nucleotides are pumped out by ankylosis protein (ANK) and cleaved by pyrophosphate hydrolase to yield PPi. Membrane bound ALP acts on this PPi -In cranial metaphysical dysplasia a mutated ANK leads to too much PPi in extracellular space |
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Bone Cell Origin
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1) Osteoblast - synthesize and secrete matrix protein to make osteoid and then mineralize it
-Becomes a bone lining cell, osteocyte, or dies by apoptosis 2) Osteocyte - Maintain bone quality and transmit mechanical signal. Regulate redmoeling and regulate calcium 3) Osteoclast - Resorb bone |
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Osteoclast Action
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1) Degrade osteoid matrix by secreting proteolytic enzymes
2) Degrade mineral by secreting acid -Osteoblast make OPN which binds the osteoclast aVb3 integrin allowing osteoblast to stick down on bone matrix -By creating a sealed border osteoclast can increase local concentration of acid and protease without causing non-specific damage -osteoblast create a ruffled border resoprtion pit and secrete proteases Cathepsin K and MMP and pump out protons. They will remove calcium and protein products from the pit and recycle in themselves or excrete to ECM and blood |
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Trabecular Bone Remodeling
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-Most bone remodeling occurs at trabecular bone, not coriticol
-osteoclast are activated to bone and resorb it quickly while osteoblast lay down new bone slowly -Osteoblast and osteoclast live for a short time and are constantly repopulated by adult stem cells -Osteoblast precursors MSC are present in bone marrow stromal cell region, which lines the trabecular bone |
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Stem Cell Differentiation
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-precursor activation stimulated by mechanical force, GF, and hormones
-Commitment requires expression of 1 or more key transcription factors 1) MSC to pre-osteoblast requires GF BMP and TF RUNX2 - The GF TGF/BMP are released from bone matrix during osteoclast resorption 2) Pre-osteoblast to osteoblast requires TF OSX and wnt/b-catenin signaling molecule |
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Hormones and GF Involved in Differentiation
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1) TGFb - Promotes pre-osteoblast proliferation and osteoid production
2) BMP - Affect MSC to osteoblast differentiation and survival 3) Hormones PTH/Insulin/VIt-D - In small doses can promote the final differentiation step -VItamin-D and its receptor bind to a hormone responsive element to increase proteins involved in osteogenesis and bone formation -The combination of RUNX2 and Vitamin-D help express OCN |
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Osteoclast Differentiation
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-come from hematopoetic stem cells
-multinucleated -involve osteoblast-osteoclast communication 1) PTH binds PTHR1 on osteoblast 2) Osteoblast upregulate membrane bound RANK-L 3) RANK-L binds RANK on osteoclast to trigger osteoclast differentiation 4) To inhibit osteoclast osteoblast can produce OPG which is a soluble RANK-L decoy receptor. Looks like RANK and binds RANK-L |
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Leptin Regulation and Bone Mass
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-When you gain or loss weight you gain or loss bone
-Accomplished by adipocyte hormone leptin which acts via the hypothalamus 1) Leptin regulates appetite 2) Leptin can trigger hypothalamus to act on osteoblast to increase RANK-L production stimulating osteoclast for bone resorption -leptin KO leads to increase bone mass -leptin overexpression reduces bone mass |
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Osteoporosis General
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DEXA Scan: Gives a T-score that indicates level of osteoporsis if 2.5 STD below mean for healthy adults of same gender (if T is 1 then at risk)
-Osteoporosis can strike at any age and osteopenia can lead to it -Affects more women than men above 50 for a while -Osteoporosis leads to loss in bone architecture and strength leading to increase risk of fractures. End up with lower bone density (less trabeculae) and lower bone quality (disjointed/thin trabeculae) and more fat infiltration |
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Risk Factors
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Uncontrollable: gender, age, size, ethnicity, heredity
Controllable: sex hormone level, eating habits, medications (glucocoritoicds increase resorption and decrease bone volume), inactivity, smoking, boozing -as we lose bone the chance of fracture and the risk of dying from fracture increase -Juvenille osteoporosis can be causeed by heredity, bad eating habits, certain medications, and inacitivty, smoking, alcohol Osteoporosis is preventable and treatable by watching mineral intake, exercising, avoid alcohol and smoking, watching meds, and avoid falling |
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Pathyphysiology of Osteoporosis
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Bone has 3 major functions
1) Mechanical support and muscular attachment 2) Protective for vitals and marrow 3) Metabolic source of ions -in osteoporosis more osteocytes are active so inbalance favor bone loss |
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Diagnosing Osteoporosis
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1) Meausre bone mineral density with DEXA to get T-score
2) Biochemical markers in serum and urine measure bone resoprtion and formation markers: pyridinoline, deoxypyridinoline, OC, ALP, N/C terminal collagen 3) MicroUT to see bone porosity/density 4) Dental Radiographs |
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Osteoporosis Management
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-osteonecrosis can occur spontaneously, through injury/surgery, and by increased bisphosphonate
-Bisphosphonate jaw osteonecrosis BJON seen in dentistry thr |
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Trabecular Bone Remodeling
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-since trabecular bone does 80% of the turnover it is heavily involved in bone disease
-In age related osteoporosis a drop in estrogen causes accelerated bone loss |
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Drug Trials
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-most drugs for osteoporosis inhibit osteoclast
-Teriparatide is the only that stimulates osteoblast proliferation through PTH action. -anabolic, builds bone Conjugated Equine Estrogen - Reduced fractures but caused stroke/cancer |
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SERMS
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-selective estrogen response mediators
-These drugs tamoxifene and raloxifene mimic estrogen and bind to the receptor selectively (breast/reproductive tissue respectively) -These protect bone from loss |
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Osteoclast Formation
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-use this description to understand drug goal
1) Requires RANK binding to osteoblast RANK-L for differentiation 2) Needs aVb3 integrin to bind OPN and bind bone surface 3) Need a proton pump to create acidic condition - H+-ATPASE and CIC-7 channel 4) Need to release Cathepsin K and MMP to degrade proteins 5) Binds calcitonin which inhibits bone resorption 6) Uses membrane bound GTPase to have ruffle border |
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Bisphosphonates
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-most widely used for osteoporosis
-inhibit osteoclasts -similar to pyrophosphate but not hydrolyzable and have nitrogern at 2 R-groups -BPs inhibit a step in cholesterol synthesis needed for isoprenylation. WIthout this there is no GTpase binding so O.C. lose ruffled border, resoprtion, and die Ex: 1) Alendronate (Fosamax) - first approved. Clinical trial showed increase bone density |
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Other Osteoclast Targets
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1) Denosumab - Binds RANK-L to block osteoclastogenesis
2) Integrin inhibitor L-845704 prevents osteoclast attachment and migration 3) Cathepsin K blocker - Cathepsin is a thiol protease with a catalytic triad C-H-N to chop collagen. Odanacatib is a cathepsin K inhibitor and increases both trabecular and corticol bone density. Attractive because unlike other drugs it inhibits bone resorption without inhibiting bone formation 4) Sclerostin Ab - Blocks SOST which is needed by osteoclast. In diseases with SOST mutation (sclerosteosis and van buchem disease) there is massive bone formation. Sclerostin Ab increases bone thickness |
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Genetics Overview
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-genetic disease come from single-gene, chromosomal abnormality, and multifactorial disorder (multiple gene/protein involved)
-monogenic disorder based one 1 gene, mendelian inheritance, complex disorder revolves around many genes, odd inheritence pattern Autosomal Recessive - sickle-cell anemia Autosomal Dominant - osteogenesis imperfecta X-Linked - hemophilia, amelogensis imperfecta -always basal mutation during DNA replication but UV and carcinogens make it worse -Nondisjunction during mitosis cause chromosomal abnormality -one mutation every DNA replication cycle, but usually not noticed because 95% of DNA does not encode a gene, and some mutation do not chance codon or substitute 2 similar AA |
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Polymorphisms
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-region of DNA (protein or nonprotein sequence) that exist in 2 or more forms in humans
ex: 1) Restriction Fragment Length Polymorphism - cleaved by MST II differently 2) Single Nucleotide Polymorphism - one nucleotide different. Example is sickle cell where b-globlin AA goes from an E to V. Also consider a RFLP because cleavage site disappear. Because you replace a hydrophilic protein at exterior with a hydrophobic one. Under low oxygen condition the affected globin will aggregate and precipitate on the RBC causing a shape change so it cannot fit through capillaries 3) Variable Number of Tandem Repeats - Short, identical DNA segments aligned head-to-toe and location and number of repeats different between people. Used for paternity test, same for identical twins |
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Type of Mutations
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1) Point Mutations - Can be
a)missense 1) Promoter - Normal protein but transcription and protein levels decrease 2) Poly (A) Site - mRNA is less stable to get less protein 3) Splice Site - aberrant splicing because 5' looks for another splice site, can cleave exon, so less mRNA and protein b) nonsense (closer to N-terminal it occurs the worse) 2) Frameshirt - Due to deletion/insertion of 1-2 NT that usually makes protein unstable and nonfunction. If frameshift by 3 then it can be OK depending on the site 3) Large insertion/deletion |
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Pattern of Inheritance
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1) Autosomal Recessive - 1 gene mutated and disease requires 2 mutant genes for phenotype. Men/women affect equally. Both parents must at least be carriers and siblings usually carrier/affected
-These diseases usually affect enzymes and carrier proteins 2) Autosomal Dominant - Only takes 1 mutated gene for the phenotype to be present. Men/women affected equally, no carriers, and child has 50% chance to acquire trait. Every affected person has a affected parents -Dominant instead of recessive because more than 50% of normal gene needed for normal function (hypercholesterolemia) or mutant gene interferes with wild-type (osteogenesis imperfecta). Usually if 2 bad genes then disease even worse. Dominant disorders usually affected structual protein or receptor |
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Autosomal Dominant Disease Detail
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1) Familial Hypercholesterolemia - mutation affects LDL receptor and 50% of LDL receptor won't cut it
-Heterozygotes have bad cholesterol and heart disease -Homozygotes have very high cholesterol and early heart attack 2) Osteogenesis Imperfecta - Example of dominant negative because mutant hinders function of WR. Affects one part of heterotrimer (2 a1 chains, 1 a2 chain) -Usually glycine is substituted and substitute too large so steric hinderance and interferes with Col conformation so Col is weaker and more susceptible to proteolysis -So dangerous because Col 1 is a trimer. Even if only 1 a1 gene mutated, 3/4 collagen molecules will have it incorporated and will be mutated |
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Epidermolysis Bullosa
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-autosomal dominant disease cause by mutation in any protein involved in basal lamina: keratin intermediate filaments, adapter plaque protein, integrin a6b4, laminin, col VII anchoring fibrils
-leads to blister, skin fragile, nail fragile, and enamel hypoplasia -can't maintain oral hygiene and require LOTS of calories since body constantly repairing itself |
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X-linked Inheritance
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-affects many more men than women cause men are hemizygous for it and cannot be carriers
-Affected male pass it to ALL his daughters and none of his sons -example is clotting disorder hemophilia |
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X-Linked Inheritance and Barr Bodies
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-Women carries of X-linked disorder can show some symptons does to lyonization/X-inactivation, dosage compensation
-In somatic cells one X is inactivated as a barr body -Early on stem cells decide which X-is active so some regions express mutant and some have WT creating a mosaic -Example is Duchenne Muscular Dystrophy where some muscle cells have mutation in dystrophin right next to normal ones |
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Amelogenesis Imperfecta
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-many causes each having own phenotype and treatment
-due to mutated amelogenin, enamelin, kalikrein-4, MMP-20, and distal-less homeobox 3 -can be autosomal dominant/recessive and x-linked recessive One Type: Gene on X/Y chromosome, X mutation gives X-linked RECESSIVE AI -Men predominantly have the disease, however women carriers have striped teeth. SInce men cannot be carriers they do not have striped teeth -Can lead to hypoplastic (less enamel), hypomaturation (less maturation of final enamel), and hypocalicified (defect in initial enamel) -Hypoplastic can be caused by ENAM mutation (enamelin) leading to abnormal tooth eruption and coronal resoprtion |
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Genetic Testing
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1) Newborn Screening - early intervention helps. There is a easy, reliable, and rapid testing method. Example is PKU
-Enzyme Phe Hydroxylase mutated so cannot convert F to Y. F builds up and causes neuro damage. Easily detected by F levels in blood and treated by avoiding F-rich foods. Autosomal recessive 2) Heterozygote Screening - If you are in a ethnic population susceptible to a certain disease you have a screening before conceiving. Helps to identify if children could be at risk for X-link or autosomal recessive disease. -Tay-Sachs in Jews, sickle-cell in blacks, B-thalassemia in Cyprus/Sardinia 3) Prenatal Diagnosis - This is for people who are conceiving late in life or have a family history of a genetic disorder. Include cystic fibrosis, PKU, sickle cell, and tay-sachs. Due to increase in nondisjunction with age 4) Disease Susceptibility Screen - Screen for genes that if mutated increase cancer risk. Rb, NF1, BRCA (cause 85% of familial dominant BC and even heterozygrous have a large increase because 1 good one already down) |
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Dental Pellicle
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-organic film that coats tooth after eruption
-contains salivary proteins that absorb to the the enamel surface by ionic charge: statherin (prevents Ca precipitation), mucin (texture), cystatin, PRP, amylase -functions to regulate ion flux, prevent calcium/flouride escape -also a anchoring surface for bacteria and nutrient source for them |
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Dental Plaque
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-first primary organizers bind to the salivary proteins and then secondary colonizers (s. mutans) bind to the primary substrates
-These bacteria make a insoluble matrix (biofilm/plaque, same) |
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Cariogenic Bacteria
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-bacteria like S. mutans are cariogenic because they are acidophiles that produce organic acids as a result of the carbohydrate metabolism
-S. mutans are gram positive, faculative anerobes, and love acid, and eat sucrose. The plaque matrix is a glucose polymer called glucan -plaque matrix is a combination of bacteria, bacteria proteins, and carbs -S. mutans allow other bacteria to bind, create a acidic environment, and dissolve teeth |
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Sucrose Metabolism
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1. Bacteria use sucrose to synthesize their plaque matrix and then stick to that matrix
-Have a enzyme class called glucosyltransferase that converts sucrose to glucan and fructose 1) Dextransucrase converts sucrose to dextran (a kind of soluble glucan) and fructose. Can add glucose to a growing dextran polymer, needs fructose to do so. Dextran help attach bacteria to each other and also a food storage source 3) Mutansucrase converts sucrose to mutan (a kind of insoluble glucan) and fructose. Mutan help attach bacteria to hard surface -also have fructosyltransferase that convert sucrose to fructan (insoluble) and glucose. Short term food storage 2. Bacteria use sucrose (or glucose) to fuel their metabolism in the production of lactic acid |
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Retrieving Glucans/Fructans for food
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-when needed bacteria secrete enzymes extracellular to digest carbohydrates into simple sugars. When glucose concentration high enough these carbohydrate degraded are catabolically repressed. This is a transcriptional regulation by bacterial opersons that shut down sugar-metabolizing and transport enzymes
1. Dextranase converts dextran to sucrose 2. Fructanase converts fructan to fructose |
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Sucrose Metabolism (1)
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-in a anerobic environment catabolic repression drives sucrose metabolism to fermentation and lactate. This generates ATP for the bacteria. If oxygen present oxidative phosphorylation will occur
1) When needed sucrose is hydrolyzed by sucrase/invertase to glucose and fructose 2) Even though sucrose, glucose, and fructose can be brought in the cell and taken through glycolysis, only the glucose transporter is usually on -Bringing a sugar into the bacteria requires the phosphotransferase system (PTS).Enzymes in the cell membrane not only bring in the sugars, they also use PEP to phosphorylate them so they cannot leave the cell. PEP Is regenerated with glycolysis -PTS converts glucose to G6P |
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Sucrose Metabolism (2)
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Fermentation:
1) Pyruvate is acted on by lactate dehydrogenase and reduced to produce lactate 2) This reaction regenerates NAD so further glycolysis can occur -NAD needed as cofactor of GapDH to oxidize and phosphorylate glyceraldehyde-3-phosphate -when glucose/sucrose present catabolite repression converts glucose to lactate (homofermentation). When glucose not plentiful repression is turned off and other sugars are used less efficiently and yield a variety of products (heterogermentation) -Since lactic acid (lactate) is a strong organic acid it decreases the local pH and increases the HA solubility -enolase conversion of 2-P-glycerate to PEP is blocked by flouride |
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Factors Protecting Against Caries
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1) Saliva - acts as a bicarbonate buffer, uses flow to remove bacteria, has lots of anti-microbial enzymes (lysozyme, lactoferrin, IgA, aggregation, defensin, cystatin, histatin)
2) Xylitol - in fruits and a artificial sweetner. S. mutans cannot metabolize and it actually reduces bacterial growth, lactate/dextran production, and decreases plaque formation |
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Saliva
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1) Lysozyme - Degrades bacteria peptidoglycan cell wall by cleaving the B1,4 link between N-acetylmuramic acid and N-acetylglucosamine
2) Lactoferrin - Deprives bacteria of iron they need for their metabolism 3) IgA - Has specific activity to bind bacteria surface inhibiting bacterial adhesion to plaque 4) gp340 -Part of innate immune system, aggregates bacteria and clears from oral cavity 5) a-defensins - cysteine-rich peptide produced by neutrophils and ductal cells of submandibular gland. Resistant to degradation and destabilizes the bacterial membrane |
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Saliva 2
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Buffering Capacity
-Enamel demineralization occurs below pH 5.5 while flourapatite occurs below pH 4.5 -Sucrose lactic acid decreases pH -saliva contains constant level of carbonic acid that can be converted into bicarbonate buffer when saliva flow high. -If salivary flow is low then there is less bicarbonate and the oral pH will decrease -The way the buffer works is that bicarbonate incorporates acidic proton to make carbonic acid. Carbonic anhydrase IV converts the carbonic acid to water and Co2, thereby removing the H+ |
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Saliva 3
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-Salivary flow is important to act as a shear force and remove bacteria (why brushing so important)
-Tooth morphology comes into play because a sheltering tooth site can protect/harbor bacteria from shear flow -caries tetrad is the host (tooth/saliva), microbes (plaque biofilm), environment (diet), and time (oral hygiene) |
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Oral Fluid Overview
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Functions:
1) Digestion/Taste 2) Protect oral cavity from bacteria and viruses 3) Buffering System 4) Protect mucosal and hard tissue surface -each salivary protein can have many functions to serve as backups |
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Saliva in Digestion
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1) Taste - saliva needs to dissolve food in solution for taste
2) Mastication - without lubricated bolus food will injure the mucosa 3) Lubrication 4) Digestive enzymes - amylase, nuclease, protease, lipase -Amy-1 gene encodes alpha-amylase which cleaves starch at alpha 1-4 bond. alpha 1-6 branch cleave by a 1-6 glucosidase (debranching enymes) |
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Mucins
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-a highly o-linked glycosylated protein can be used for
1) tissue coating 2) lubrication 3) antibacterial/antiviral 4) heterotypic complexes - form complex with many other proteins to serve 1 ultimate function 2 types: MG1 - apomucin is large with many subunits and a few 0-linked sugar residues MG2- apomucin is small, single subunit, with many o-linked sugar residues |
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Maintainence of Hard Tissue
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-saliva has Calcium and Phosphate which help keep enamel in tact
-Use statherin and PRP to keep the calcium phosphate in solution, control ion regulation, and make sure tooth doesn't dissolve -Statherin - negatively charged AA so it binds Ca2+ tightly. Also binds and stabilizes hydroxyapatite. When it binds there is a conf-c and it can now bind bacteria |
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Saliva Antibacterial Enzymes
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-antibacterial/virus enzymes are similar and have many backups
1) Lysozyme - cleaves bacteria peptidoglycan at b1-4 bond between acetylmuramic acid and acetylglucosamine 2) Defensin - Because it is amphoteric with hydrophobic and cation AA it can bind bacteria membrane with charge and insert hydrophobic part. Creates leaky bacteria and destabilizes the membrane 3) Anti-Viral - Some saliva proteins like SAG and MG2 can interact with virus. Also, hypotonic saliva can lyse viral envelope 4) Salivary Agglutin - Known as GP340 and DMBT1. Can aggregate oral bacteria for clearence and bind HIV to block infectivity |
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Salivary Proteomics
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-Use 2D Page and then isolate, digest, and use mass spectrometry
-Use MALDI-TOF mass spectrometry to get the protein sequence -Between the parotid and the submandibular/sublingual glands there were tons of proteins, most overlapped, but still about 250 unique ones -found that protein in saliva are indicitive of other disease. Use saliva as test for other diseases since so easy to get and plentiful -EMT can use test to see if patient suffering a acute myocardial infarction -Pouch Cassette and Actuator moves saliva around, has a immunoassay to bind certain proteins, then uses flourescence to tell the amount |
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Proteomics Summery
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-Molecules in blood indicitive of disease also in saliva at low levels so use ELISA/PCR to amplify
-Existing tests monitor ABs, steroid hormones, drugs, biomarkers -Current test include caries, periodontal disease, cancer, and cardiovascular disease |
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Caries Overview
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-cargiogenic bacteria acid dissolves subsurface inorganic compounds and brings to surface. That is why even if surface in tact, can have lots of demineralization under
-Dr. McKay discovered the Colorado Brown Stain teeth has no cavities. Linked excess flouride to this phenomenom. Dr. Dean implemented the first studies of flourinated water and reduced caries |
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Flouride Overview
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-decrese apatite solubility
-prevents carbohydrate metabolism in bacteria -Helps remineralize carious legions -our hydroxyapatite is Ca10, PO46, OH-2. While flourapatite has F-2 instead of OH-2, our apatitite can have 10% of the OH replaced with F -Flouride uptake in enamel decreases from outer enamel to dentin |
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Flouride Solubility
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-measured by amount of calcium released at different pH
-Or, add sucrose, saliva, and bacteria and measure pH and Calcium released. pH tells you the level of acid produced while calcium tells you solubility -Solubility decreases from brushite (CaHPo4 soluble), to enamel (hydroxyapatite with carbonate and calcium deficient), real hydroxyapatite, and flourapatite |
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The Magic of Flouride
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-the structure of enamel mineral is 2 calcium hexagons. The hexagons are surrounded by phosphate. In the center of the hexagon is a calcium star of david. Above and below the hexagon is OH sticking out
-OH is usually outside the calcium plane, Flouride replaces -OH and makes a more compact crystal and pulls rings together. Also binds OH making a strong hydrogen bond |
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Magic of Flouride (2
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Flouride decreases the solubility of the outer enamel
-When lactic acid enters enamel it is undissociated by water and the H+ attacks HAP to dissolve into Ca, PO4, Pi -This makes the enamel weakened. When the enamel remineralizes it becomes a apatite veneer that is not compact and has lots of soluble HPO4 and carbonate incorporated. This is more like brushite than enamel. -When flouride incorporated into enamel you don't get a apatite veneer, instead of get regular flour-apatitite. This slow rate of mineralization is the main function of flouride Flouride and Bacteria -flouride blocks enolase for PEP isn't made. Without this step lactic acid isn't made. Without PEP phosphotransferase system cannot function so no glucose in. Without glucose and without NAD no ATP made, bacteria cannot function. |
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Inflammation Overview in Periodontitis
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-when bacteria infect the mucosa the body mounts a inflammatory response using white blood cells that secrete cytokines and prostaglandins (lipid)
-Cytokines are intercellular peptides that activate or inhibit immune response. There are proinflammatory, anti-inflammatory, and those that affect osteoclast (import. for perio, RANK-L and TNF) -A little inflammation is helpful but too much inflammation can lead to tissue destruction -divided to innate immunity quick is immediate and not-specific, and adaptive immunity which is delayed but specific |
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Innate and Adaptive Immunity
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Innate:
-recognize a pathogen surface characteristic not in humans, called a pattern recognition receptor -The goal is to recruit more cells of innate and adaptive immunity and to destroy bacteria or remove dead waste Adaptive Immunity: specific, found only in vertebrates, and has memory to make response quicker the second time -Cells are B-cells which secrete anybody and Tc which kill viruses and Th which help in killing |
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Periodontal Disease
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-Gingivitis is gum inflammation and this is reversible
-Periodontitis is not reversible and there is a loss of bone and the connective tissue holding the teeth (periodontal ligament, sharpy fibers) |
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Periodontitis
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1) Bad bacteria colonize the tooth
2) Bacteria invade the tooth connective tissue 3) Inflammatory response begins against bacteria. Both innate and adaptive immune 4) Inflammation causes alveolar bone loss (osteoclast) and connective tissue between tooth and gingiva loss (lytic enzymes released by immune cells) -two bacteria to know are P. gingivalis and A. A -periodontitis can occur a slow continuous episode or short burst -when bacteria cause initial inflammation it is gingivitis. Once inflammation reaches bone it is periodontitis. When invasion gets close to bone leukocytes stimulate osteoclastogenesis |
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What role does the host immune response play
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-study periodontitis by the ligature model (tie silk around molar to facilitate invasion) and the gavage model (inject bacteria into site)
-Results contradict, some show host defense cause periodontitis, some say it doesn't. May come does to the sptation location and duration of the host response |
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Cytokines in Periodontitis
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1) Cause Disease - IL-1, TNF, IL-6
IL-1: both gain of function and loss of function role in periodontitis. May expedite migration to inflammatory front TNF: Has both a gain of function and loss of function role in periodontitis. Also seems to play a role osteoclastogenesis IL-6: Pro-inflammatory and stimulater osteoclastogenesis 2) Prevent Disease - IL-11 - anti-inflammatory reduces IL-1 and TNF |
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Adaptive Immunity in Periodontal Disease
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Cause: IFN--y (GOF and LOF proof) and RANK-L (GOF and LOF bone loss)
-studies have shown that b and t-cell cause more bone loss and periodontitis Prevent: IL-17 -studies have shown that B-cells and T-cells correlate with less periodontitis and more bone loss |
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Tooth Molecular Development Overview
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-caused by reciprocal signal between epithelial cells and mesenchyme
1) Tooth Position - Epithelial express FGF8 and mesenchyme express Pax9 there is a tooth. If mesenchyme receive FGF8 and BMP2/4 then there is not a tooth 2) Later Development (most epi/mesy interaction) - BMP4 from enamel epithelium induce TF MSXI, MSXII, and LEF in mesenchymal Basic Epithelial/Mesenchyme Interaction |
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Gene Interactions
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-Tooth development is due to the timing and location of proteins
-KO of MSX I, MSX II, and LEF stop development at bud stage for all teeth -some mutations affect only certain teeth, MSX1/2, DLX1/2, and Goosecoid important - DLX1/2 (individual KO = WT, combined KO = no maxillary molars). Opposite is Noggin where you lose mandibular molars. Activin lose mandible teeth. Gli2 lose maxillary incisor |
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Apoptosis and Teeth
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-apoptotic teeth found in bud stage invaginating dental epithelium
-found in cap/bell stage enamel knot -found in bell stage dental lamina breakdown to separate dental organ from surface -found in the crown stage as ameloblast die during maturation/secretion and stratum intermedium and stellate reticulum die when reduced dental epithelium made |
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Ameloblast Products
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1) Amelogenin secreted during maturation to regulate crystal grwoth and thickness
2) Enamelin there during secretion to control enamel thickness 3) Tuftelin at DEJ for crystal nucleation 4) MMP20 (enamelysin) is secreted to cleave amelogenin C-terminus 5) EMSP-1/Kallikrein-4 - During maturation they cleave amelogenin TRAP so it can be absorbed 6-8 Affter cell/matrix interaction 6) Shealthin during secretion in enamel rod 7) Ameloblastin during maturation at sheath 8) Amelin during root formation with HERS 9) FAM83H - intracellular enamel protein -if any of these mutated you get AI (except tuftelin) |
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Amelogenesis Imperfecta
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-Involved in several patterns of inheritance like autosomal dominant/recessive and X-linked
-Amelogenin genes leads X-linked hypoplatic |
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AI (2)
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1) X-linked amelogenin - in women there are vertical stripes due to X-linked inactivation by barr body. Causes by missense mutation of proline to threonine
2) Brown Hypomaturation - X-linked - discoloration and broken enamel due to softness 3) AMELX in C-terminal - Male hypoplastic, female with vertical grooves 4) AMELX in N-terminal - X-linked hypomaturation with brown teeth cause too much protein left |
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Other Enamel Mutations
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1) Autosomal dominant causing local hypoplastic
2) MMP20 missense lead to hypomaturation since too much protein left and enamel not high density. Can't cleave amelogenin to remove for degradation. Autosomal recessive 3) KLK4 mutation is autosomal recessive. Too much protein left over and teeth brown/soft 4) FAM83H deletion, C-terminus mutation causes diff. problems |
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Dentin Mutations
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-problem with DSPP gene leads to dentinogenesis imperfecta
-mice are a good model to study mutations to genes in the tooth |
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Tooth Engineering
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-scaffold polymer shaped like tooth coated with collagen and seeded with pig tooth bud cells. Forned dentin, enamel, and cementum
-mesenchyme and epithelium from different species under certain conditions form teeth. Mesenchyme directs epithelial tooth development -Tooth problem in birds may be due to loss of BMP4 |
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Lipid Mediators Intro
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-lipid mediator can inhibit/activate inflammation
-they are synthesized from lineolic acid or a precursor -The main precursors are arachidonic acid, eicosapentanoic acid, and docoshexanoic acid -lipid mediators are short range messengers for pararine or autocrine action |
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Arachidonic Acid
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-Phospholipase A2 in cell membrane cleaves lipid to make A.A. (rate limiting) (products of AA are eicosanoids made by O2 addition to acyl chain)
-A.A. can be converted into lipoxins, leukotrienes, or cyclooxygenase which is a precursor of prostaglandins -In turn prostaglandins make prostacyclin and thromboxane which is regulated by tissue -leads to pro-inflammatory prostaglandins and leukotrienes Mediate acute inflammatory process in response to injury/infection -makes anti-inflammatory lipoxins |
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A.A Continued
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-Vascular endothelium example of prostacyclin thromboxane balance
Normally vascular endothelium don't want random platlet aggregation so they produce prostacyclin which is a anti-aggregatory compound (paracine). Prostacyclin synthase and prostacyclin much higher than thromboxane -if vascular wall injured then you need platlets to aggregate. Platlets have thromboxane synthesis and make thromboxane which is a autocrine signal to allow platlet to aggregate. Prostacyclin is low |
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Docosahexanoic Acid
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-makes docosanoids which form resolvin D and protectins (neuroprotectins) which decrease inflammatory response and protect against oxidative stress
-first step in resolvin synthesis requires acylated Cox-2 enzyme |
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Eicosapentanoic Acid
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-makes resolvins E which are anti-inflammatory
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Cycloxygenase
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COX-1: Always active and in charge of vascular homeostatis, renal and GI blood flow. Binds to aspirin acetylsalicylate irreversibly to be inhibited. Without Cox prostaglandins and thromboxane not made
COX-2: Induced as needed in response to inflammation by macrophages (IL-1/LPS), neurons, and smooth muscle endothelial cells -When inflammation needs to be turned off we take aspirin. Aspirin will inhibit Cox1 and acylated Cox-2 will stop using A.A as a substrate and instead use EPA and DHA to make resolvins/protectins which are anti-inflammatory |
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Periodontal Disease and Resolvins
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-during periodontitis some of the inflammatory response is mediated by A.A. metabolites
-To stop inflammation EPA and DHA resolvins are created. If you apple resolvin to damaged tissue it can be restored in the oral cavity |
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Regulation of Skeletogenesis
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-FOXo1 is a TF that switches MSC to osteoblast differentiation and skeletal development
-FOXo1 regulates RUNX2 expression -Foxo1 works with RUNX2 to regulate expression of other osteogenic markers |
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Engineering Growing Bone
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-require cells, scaffold, and growth factors
-limited by access to vascular system -If they create a cartilage scaffold it will carry all the signals to make new bone when implanted in vivo -use chitosan as their scaffold to grow chondrocytes because it is similar to HA and GAGs |
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High Frequency Forces and Bone FOrmation
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-vibration have osteogenic potential on bones and are able to stimulate bone formation and increase in bone mass
-Add high frequency forces to jae bones to help osteoporotic patients and those who need implants but have poor bone quality |
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Inflammation Enhanced Tooth Movement
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-inflammation causes bone movement and anti-inflammation decreases bone movement
-Put small holes in bone to create inflammation allowing for quicker bone remodeling. After remodeling stop inflammation -perforations doubled the tooth movement -decided to put holes not in tooth (weaken) but in area around |
