lecture 11 Flashcards
what is pulse chase?
- add radioactive material to cell culture
- wash cells to remove radioactive material
- put new media in
- follow where radioactive material goes
how does HA exit cell?
- HA formed in plasma membrane
- can exit cell into extracellular matrix
- ABC transporter that forms and exports the HA
what do the chondrocytes contain lots of and how is it known?
- core protein
- inhibit protein synthesis, using cyclohexamide
- pool of core protein as glycoproteins keep appearing
what is glycoproteins synthesised as?
- single polypeptide chain
what is the signal for secretion?
- addition of glycosaminoglycans
- passes through intracellular pool to become glycosylated and rapidly secreted
what post-translational modifications take place?
- smaller cell free system, dont have enough machinery to do modifications after translation
what is the normal aggregation process?
- release of proteoglycan with link protein attached
-reelase of hyaluronic acid - stable aggregate formed in extracellular matrix
how is this process interfered with?
- by adding 10-20 sugars hylaronase
- binds proteogylcsn and slows ability
- isn’t long enough to bind proteoglycan and link protein
- ## equilibrium between attached and ability of proteoglycan to loose small chain and bind to stable aggregate
what does this tell us?
- more than 20 sugars needed to bind the link protein with the proteoglycan
what is the function of proteoglycans in cartilage?
- highly negatively charged
- retain large volumes of water in extracellular matrix
- immobilised in collagen matrix as aggregates
- restrained from swelling by collagen meshwork
what happens if collagen was removed?
- protoelgycans will expand and form a gel
what can the hydrated proteoglycans do?
- be reversibly compressed by displacement of water from their hydration shells
how does reversible compressibility occur?
- proteoglycan isn’t expanded as much as needed, Ks and Cs closer than should be
- force trying to push apart (intramolecular force)
- intermolecular force as proteoglycans too close and hydration spheres are overlapping
what happens if load is applied?
- squash gylcosaminogylcans closer
- hydration spheres overlap more
- some water lost
where does the fluid lost go?
- redistributed within tissue away from point of compression
- redistribution is slow
why is the redistribution slow?
- proteoglycans are entrapped in the collagen meshwork which impedes flow of fluid
how is a large frictional drag caused?
- by bottlebrushes structure sliding past each other
what provides the damping effect?
- cartilage onlu deforming gradually under a load
what occurred in sokoloffs experiments?
- showed cartilage is reversibly compressible
- set up rig that applies force including cartilage
what does the addition of lanthanum chloride cause?
- precipitation of proteoglycans
- reversible compressibility is lost
what occurred in the Thomas experiment?
- injected papain into rabbits ears
- papain destroys proteoglycans so cartilage support function is lost
what is the function of versican?
- important for reversible contractility
- found in intima of aorta
- lots found in head
what are some other functions of proteoglycans?
- cell adhesion
- DNA regulation
- nervous system
- lipid metabolism
- cell growth
- basement membrane permeability
- killer T lymphocytes
- platelet adhesion to endothelial surfaces
- HIV
- preventing tumour growth in brain
- amyloid plaque formation
- embryo implantation
what is the role of heparin sulphate proteoglycans in embryo implantation?
- non receptive phase = heparin sulphate protelgycans on surface of embryo but cant reach receptors, mucins expressed on surface (MUC1) so receptors hidden underneath so embryo cant implant
- receptive phase = MUC1 expression stopped so can implant
what is osteoarthritis?
- disease of synovial joints
- characterised by progressive deterioration and focal erosions
what is the appearance of the hyaline cartilage?
- white ish blue
- glossy in appearance
what occurs to hyaline cartilage appearance in osteoarthritis?
- loss of glossy appearance
- fibrillation
- erosion of cartilage
- exposure of subchondral bone
what are the first signs of osteoarthiris ?
- joint gets warm
- changes in collagen and glycosylation
- causes relax
what are the enzyme changes in osteoarthritis?
- shows degree of severity is proportional to level fo. degradative enzyme activity
- MMPs, ADAMTs and cysteine proteases increased
- TIMPs cant inhibit all degradative activity
- aryl sulphatases remove sulphate (levels raised)
what does the increased enzyme activity cause?
- faster matrix breakdown
-degradation fragments diffuse out tissue - increased porosity of extracellular matrix
- degradative enzymes penetrate further
- synovial fluid can diffuse in and leech out more proteoglycans
- unsupported collagen looses stiffness
what occurs in the acute inflammatory response in oestoarthirits ?
- can get lots of polymorphonuclear leucocytes (damage tissue)
- produce large quantities of lysosomal enzymes
- difference in diffusion rates due to size differences
- enzymes diffuse quick as small, inhibitors diffuse much slower so lots of damage occurs
what are the chondrocytes doing in this process?
- increased synthetic activity (increased DNA synthesis, cell division occurring)
what does 35S sulphate show?
- increased biosynthesis = increased levels in newly synthesised glycoproteins
what is a signal for an immature proteoglycan?
- newly synthesised richer in C4S than C6S
what occurs with newly synthesised proteoglycans?
- less associated with matrix
- more easily degraded
why cant degradation occur to all?
- repair fails as cant stop both newly synthesised and adult proteogylcasn
what does this lead to?
fall in synthetic activity in severe disease
- programmed cell death
- lacunae appear in tissue
what are the treatments for osteoarthritis?
- platelet rich plasma taken from patients
- centrifuged at low speed to bring down RBC, high speed to pellet the platelets
- platelets taken up in plasma
- injected into joint
what is another treatment method for osteoarthritis?
- intra-articular HA injections
why is more HA needed?
- increases concentration in synovial fluid
- so becomes more viscous
- increases viscous-elasticity properties
how is damaged cartilage repaired?
- open joint up and finding lesion, drilling through subchondral bone- bleeds into split (fibrous cartilage forms to repair)
- make small tubes of alginate (from seaweed) and fill with chondrocytes culture
how is the alginate used?
- culture for week
- strands of cartilage forned
- used as substrate for 3d printer
- able to compose the matrix which can be transplanted into the damaged area
how are drugs delivered to articular cartilage using nanoparticles?
- nanoparticles penetrate the cartilage matrix
- treated cultures with IL-1 to form proteoglycan breakdown
- tested with fluorescence to see if nanoparticles enter cartilage
what is shown in loss of joint space in OA?
- symptoms worsened
- left side of joint loose fine
- cartilage disappeared on right side
- bone articulating on bone causing friction and bone necrosis due to heat from friction
what occurs in synovial fluid in OA?
- increased fragments of aggrecan proteogylcan
what are the biomarkers of articular cartilage degradation?
- fragments of aggrecan
- fragments of cartilage
