Flashcards in Frailty Deck (72):
What is RT-PCR used for?
Used to detect RNA expression in a sample, for example viral RNA or mRNA (to detect gene expression)
What are the steps of RT-PCR?
Purify the RNA
First strand synthesis: Use RT to convert RNA into double stranded cDNA
cDNA amplified by PCR
What is one step PCR? Also give one pro and one con
One step combines the steps of first strand synthesis and PCR amplification in a single tube
Minimises chance of contamination
Analysis limited to a few genes per sample
What is two step PCR? Also give one pro and one con
Reactions for first strand synthesis and PCR amplification take place in separate tubes
Allows for optimisation of reaction conditions
Increased chance of contamination
What is senescence?
Deterioration with age
What is cellular senescence?
Cells lose the ability to divide with age, so growth becomes halted
What causes cellular senescence?
Ageing causes telomere shortening
DNA damage detected by p53
p53 halts cell cycle at G1/S
Cells with arrested cell cycle will eventually apoptose
What is immunosenescence?
Deterioration of the immune system brought on by ageing
What is inflammaging?
Chronic, low grade inflammation
Chronically elevated levels of circulating proinflammatory cytokines
What causes inflammaging?
Ageing, cellular senescence and ROS trigger NFkB signalling
Products of NFkB signalling cause inflammation
How can inflammaging increase the risk of cancer?
Triggers genetic mutations
What is apoptosis?
Programmed cell death
What is ELISA and what is it used for?
Enzyme Linked Immunosorbent Assay
Used to detect levels of protein/antigen/antibodies/glycoprotein in a biological sample
Can be used for pregnancy tests and detecting HIV
What are the steps of ELISA?
1. Coat ELISA plate with capture antibody (raised to antigen of interest)
2. Add sample - antigens captured by antibody
3. Add enzyme-labelled detection antibody - binds to antigens bound to plate
4. Add substrate to initiate chromogenic reaction
5. Coloured product measured using plate reader
How can ELISA be used to detect apoptosis?
Apoptosis results in release of nucleosomes
Use ELISA to detect cytoplasmic nucleosomes in a sample
How is apoptosis initiated?
Cytokines activation JAK/STAT receptors, leads to activation of caspases
Bad protein binds to and forms a complex with Apoptosis Inhibitory Protein
What are caspases?
Protease enzymes that initiate apoptosis
Cysteine residue carries out protease activity
Where to tyrosine kinases phosphorylate?
Serine and Threonine residues
Steps of apoptosis
Small blebs form
Nucleus breaks down, DNA breaks down into smaller fragments
Organelles contained in blebs, still functioning
Breakdown into several apoptotic bodies
What is necrosis?
Non-programmed, premature cell death
Caused by infection, toxins, trauma
Steps of necrosis
Small blebs form
Structure of nucleus changes
Blebs fuse and become larger
Loss of cell membrane integrity; membrane ruptures (autolysis) and releases cell contents
What is autophagy?
Ordered degradation and recycling of cellular components
What is autophagy used for?
Maintain cellular energy levels during starvation
Degrade damaged organelles
Promote cell death
What are the 4 types of autophagy?
What is macroautophagy?
Phagophore engulfs organelles that are damaged or unused
Forms double membraned autophagosome
Autophagosome fuses with lysosome
What is microautophagy?
Direct engulfment of material into the lysosome, via invagination of the lysosomal membrane
What is chaperone-mediated autophagy?
Proteins with consensus motif (penta-peptide motif) recognised by Hsc70 chaperone protein
Substrate/chaperone complex taken to lysosomal membrane
Protein unfolds and translocated across lysosomal membrane
What is Hsc70?
Produced by cell in response to stressful conditions
What is mitophagy?
Degradation of defective mitochondria
What are ULK proteins?
Unc-51 Like Kinase
ULK1 and ULK2 activate downstream components of autophagy
How is autophagy inhibited?
mTOR and AMPK phosphorylate ULK1 and ULK2
Inhibits ULK1 and ULK2 from initiating autophagy
What are the genes called that regulate autophagy?
How is autophagy induced?
Dephosphorylation of ULK1 and ULK2
What is ubiquitination?
Ubiquitin ligase adds a ubiquitin tail onto a protein
This marks them to be degraded by a proteasome, or may alter their cellular location or activity
What is a proteasome?
Protein complex that degrades unused proteins via proteolysis
Cylinder-like structure made of alpha and beta subunits
Proteins degraded in the hollow core
What are the steps of the ubiquitin/proteasome pathway?
1. Activation - Using ATP, Enzyme E1 activates ubiquitin
2. Conjugation - Enzyme E2 transfers ubiquitin from E1 to E2
3. Ligation - Enzyme E3 transfers ubiquitin onto the substrate
4. Substrate degraded by proteasome
What is the ubiquitin/proteasome pathway used for?
Degrade regulators of the cell cycle, regulators of apoptosis, transcription factors
What is IGF-1 and what does it do?
Insulin-like growth factor 1
Binds to tyrosine kinase receptors
Initiates intracellular signalling pathways such as MAPK and PI3K
Causes cell growth, inhibits apoptosis and autophagy
How is IGF-1 produced?
Produced by many cell types including hepatocytes
In response to growth hormone (GH)
What is the somatotrophic axis?
Nutrients/amino acids are sensed
Hypothalamus release GHRH (growth hormone releasing hormone)
Anterior pituitary releases GH
Liver produces IGF-1
What is the PI3K pathway?
1. IGF-1 binds tyrosine kinase receptor
2. Trans-auto phosphorylation
3. Attracts SH2 domain of PI3K
4. PI3K phosphorylates PIP2 --> PIP3
5. PIP3 attracts PDK
6. PDK phosphorylates Akt, activating it
What is PIP2?
A membrane phospholipid
Why can PIP3 attract PDK?
PIP3 can recruit proteins with a PH homology domain
What are the 3 effects of Akt, once activated by phosphorylation?
1. Akt inhibits 'Bad' protein, preventing apoptosis
2. Activates mTORC1, leading to growth
3. Inhibits transcription factor FOXO
What is mTOR?
Mechanistic Target Of Rapamycin
Forms complexes mTORC1 and mTORC2
Involved in regulation of many cellular processes
What is the function of mTORC1?
Upregulates protein synthesis, cell growth ribosome production
What is the function of mTORC2?
(Which then indirectly activates mTORC1)
What 2 things activate mTORC1?
Presence of calories/lipids/nutrients
What is FOXO?
Activates genes involved in autophagy/ubiquitin proteosomal degradation/lysis/muscle atrophy
What is Rheb?
Protein involved in how Akt activates mTORC1
What is sarcopenia?
Skeletal muscle atrophy associated with ageing
What is IRS-1 and what does it do?
Insulin Receptor Substrate-1
Transmits signals from insulin receptor to the PI3K and MAPK pathways
How does IRS-1 work in the PI3K pathway?
1. IGF-1 binds receptor
2. Trans-auto phosphorylation
3. Receptors phosphorylate IRS-1
4. IRS-1 recruits SH2 domain of PI3K
5. PI3K phosphorylates PIP2 --> PIP3
What is myostatin?
Type of myokine - which is a cytokine released by muscle cells
Regulates (inhibits) muscle growth
What is myostatin also known as?
Growth differentiation factor 8
What receptors does myostatin bind to? (and what type of receptors are these)
Autocrine Activin type I and type II receptors
which are Tyrosine kinase receptors
What happens if you lack myostatin?
You have hypertrophy and more muscle mass than usual
What 2 things does myostatin do?
1. Inhibits Akt, therefore reducing mTORC1 activity and reducing protein synthesis
2. Leads to activation of genes that inhibit myogenesis, by halting the cell cycle at G1/S phase
How does myostatin inhibit cell cycle progression?
1. Released by myocytes
2. Binds to activin type I and II receptors
3. Receptors dimerise and trans auto phosphorylate
4. Phosphorylation/Activation of SMAD2/3 transcription factors
5. SMAD2/3 form complex with SMAD4
5. Promotes expression of genes involved in atrophy, autophagy, proteasomal degradation
What family is myostatin a member of?
TGF beta (transforming growth factor-beta) superfamily
What is anabolism and catabolism?
Anabolism = creating molecules
Catabolism = Breaking down molecules
What is the damage/error theory of ageing?
Cumulative damage from environment
Greater rate of oxygen basal metabolism = shorter lifespan
Free radical exposure causes oxidation of macromolecules
What is the programmed theory of ageing?
Ageing follows biological timetable
Depends on expression of genes for maintenance, repair, defence systems
IGF-1 has key role
What is a quiescent cell?
Cells in 'G0' state
They are not participating in the cell cycle but can do in the presence of mitogens
What is thought to be the cause of Progeria conditions?
Faulty telomerase enzyme
Telomerase allows replication of telomere ends
Without this, telomeres will shorten with every cell division
In which cells is telomerase present?
Stem cells, cancer cells
Not normal somatic cells
What is the primary structure of a protein?
Sequence of amino acids
What is the secondary structure of a protein?
Hydrogen bonds form between amino acids
Can form alpha helix, beta pleated sheet, U turn
What is the tertiary structure of a protein?
Further folding, caused by formation of bond types
What type of proteins assist with protein folding?
They are heat shock proteins, as stressful conditions can cause proteins to fold incorrectly
What is the quaternary structure of a protein?
Arrangement of many peptides, forming a multi-subunit protein
Subunits held together by non-covalent bonds