16 Flashcards
Immunoelectron Microscopy Purpose?
combines antibody-based detection with resolution of electron microscopy
visualize and localize antigens within cells at an ultrastructural (fine) level
STEPS of immunoelectron microscopy
Fix tissue with a cross-linker (aldehyde), then apply an antibody to a cell/virus/bacterium
Apply a second Ab labeled with gold (dense) so electron beam cannot penetrate it
Use electron dense agents (Uranyl Acetate/Pb Citrate/Osmium Tetroxide) to stain cell
View in electron microscope
ROS Regulates:
a) Inflammasomes regulate Caspase-1 because of infectious microbes
b) NETs
What is NETosis induced by?
TPA (12-Tetradecanoylphorbol-13-acetate) &
PMA (12-myristate 13-acetate )
Role of Neutrophils (WBCs)
-first immune cells to attack microbes that get into body
-uses antimicrobials
-professional phagocytes
-forms NETs to trap microbes & kills them with antimicrobials
-prepares tissue for new growth
What does TPA/PMA induces ?
NETosis and ROS in cells
What is Netosis (simple terms)
Another type of cell death
What does PAD4 do to Histones?
causes arginine to form into citrulline in histones, = decondenses chromatin and promotes NETosis
Neutrophils contain?
AP-1
What activates AP-1 in neurophils?
MAPK
** AP-1 binds to specific enhancer sequences called TPA/PMA- response elements
what are podoctyes?
Podocytes are specialized cells in the kidneys that wrap around the glomerular capillaries (sit along GBM)
what is MAML-1 responsible for?
In the Notch signaling pathway, MAML-1 has been shown to influence the expression of certain MMPs (migratory proteins)
What is first secretase to cleave Notch?
ADAM-10 (alpha-secretase)
sources of ROS
- Mitochondria
- ER
- NADPH oxidases (NOX)
Drugs : Doxycycline, Azidothymidine (AZT), Cisplatin, Metformin
Heavy Metals
High fat diets
Indoor pollutants:
particulate matters,
metals from vacuums,
blenders,
laser printers
underproduction of oxygen byproducts can be signs that —— are affected
Mitos
What are normal byproducts of metabolixing oxygen?
peroxides, superoxides, hydroxyl radicals are VERY damaging
- can contribute to Oxidative stress
How are ROS beneficial?
-serve to activate/inhibit signaling pathways
- professional phagocytes use NOX to produce ROS
What is AZT
AZT is an antiretroviral drug used primarily in the treatment of HIV/AIDS.
How would Metformin prevent ROS formation?
Activates AMPK: Helps regulate mitochondrial function and reduces ROS production.
Inhibits mitochondrial complex I: Reduces ROS generated by mitochondria.
Improves insulin sensitivity: Reduces the need for excess insulin, lowering ROS formation.
what is Doxycycline?
Doxycycline is used to treat bacterial infections.
It works by inhibiting bacterial protein synthesis, preventing bacteria from growing and multiplying
What is end results of OXPHOS?
production of ATP
(ROS) are produced in Mitochondria where?
ROS are produced during electron transfer at Complex I and Complex III in the electron transport chain.
Enzymes that work to convert ROS into less harmful molecules and maintain cellular redox balance.
**Thioredoxin,
**Cytochrome P450s
Catalase,
Superoxide Dismutase,
Peroxiredoxin,
Glutathione Reductase,
Glutathione S-Transferase
Metabolites that help Neutralize ROS ?
Vitamins C & E,
Coenzyme Q,
Glutathione,
Cytochrome C
What organelle is linked to b-TrCP proteins?
The organelle linked to β-TrCP (E3-ligase) proteins is the proteasome.
Keap1 is a —–?
redox sensor
- linked to Ub-ligase
- WD40 Protein
STEPS in the Nrf2 Pathway ?
Unstressed Conditions:
- Nrf2 is inactive in the cytoplasm, bound to Keap1.
Stressed Conditions:
- Oxidative stress or signals stabilize Nrf2, preventing degradation.
- Nrf2 accumulates and translocates to the nucleus.
- Nrf2 activates genes involved in antioxidant defense by binding to AREs in DNA.
- GSK-3 can target Nrf2 for degradation, unless it’s sufficiently activated.
what is glutathione ?
Glutathione is a major antioxidant in the body that helps neutralize reactive oxygen species (ROS).
Vit E significance?
Vitamin E works alongside glutathione to protect cells from oxidative stress by scavenging free radicals and reducing damage.
How is N-Acetylcysteine (NAC) a precursor to glutathione?
NAC is converted into cysteine, which is a key building block for glutathione synthesis.
