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BMSC 240 > Lectures 11, 12, 13 > Flashcards

Lectures 11, 12, 13 Flashcards

1
Q

What does bactericidal, bacteriolytic, and bacteriostatic mean?

A

Bactericidal- kills bacteria
Bacteriolytic- lysis/bursting of bacteria
Bacteriostatic- prevents growth/division
Therapeutic index- dose that kills humans over the dose that kills bacteria, less than 1 means the dose will kill human faster than bacteria
Slide 4 L11

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2
Q

How is does the closet an organism is to humans affect its ability to be distinguished?

A

Closer an organism is to humans, the harder it is to distinguish because it’s harder to damage the bacteria without damaging humans
Slide 6 L11

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3
Q

What are the 5 microbial targets?

A
  1. Inhibition of cell wall synthesis- penicillins, cephalosporins, bacitracin, vancomycin (inhibit peptidoglycan production or anything that damages cell well)
  2. Inhibition of protein synthesis- chloramphenicol, erythromycin, tetracyclines, streptomycin
  3. Inhibition of nuclei acid replication and transcription- quinolones, rifampin (could inhibit DNA replication since it’s circular in bacteria and linear in humans)
  4. Injury to plasma membrane- polymyxin B
  5. Inhibition of synthesis of essential metabolites- sulfanilamide, trimethoprin
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4
Q

What are cell wall inhibitors?

A

Natural and semi synthetic forms (more resistant to acid breakdown, more stable, broaden activity spectrum)
All contain β-lactam ring
Natural penicillin only works on gram +
Bacteriolytic

Slide 8 L11

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5
Q

What are protein synthesis inhibitors?

What are the 4 common drugs?

A

Many different classes interfere with ribosome/mRNA/tRNA complex

Chloramphenicol- can cause anemia, broad spec
Streptomycin- gram -, can cause deaf, targets mitochondrial ribosomes
Neomycin- topical streptomycin variant
Tetracycline- yeast over proliferation discolours developing teeth

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6
Q

What are the 2 plasma membrane damaging antibiotics?

A

Polymyxins- damage gram - membranes
Poorly absorbed and toxic to neurons and kidneys
Daptomycin- depolarizes gram + membranes

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7
Q

What are 2 nucleic acid synthesis inhibitors?

A

Rifamycin/rifampin- most useful against mycobacteria (TB, leprosy)
Inhibits mRNA synthesis
Highly permeable into cells and through cell walls
Quinilones- inhibit DNA gyrase->DNA replication

Slide 11 L11

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8
Q

What are metabolic inhibitors?

A

Sulfonamides (sulfadrugs)
Completely synthetic (originally used in dye making)
WWII soldiers got sulfa packets to spread on wounds to prevent gangrene

Completely inhibits an enzyme needed to generate folate (folic acid)

Folic acid is used to produce thymine and uracil
Slide 12-13 L11

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9
Q

What is sensitivity testing? (Disk diffusion and e tests)

A

Disk diffusion assays can determine what drugs are useful against bacteria (non quantitative, distance ranges for sensitivity and resistance)

Epsilometer tests (E tests) use a strip of antibiotic to determine a therapeutic dose
MIC= minimal inhibitory concentration
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10
Q

How does antibiotic resistance work?

A

Even with proper use, bacteria will eventually become resistant to any antibiotic
Misuse of antibiotic accelerates the development of resistance (discontinuing the antibiotic before the pathogen is eradicated, using poorly choosed antibiotic, use of antibiotics against viral infections, preventative use of antibiotics in animal feed)

Resistance can also be natural in some bacteria and can spread to other bacteria

Slide 7 L11

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11
Q

What are the 5 modes of resistance in antibiotics?

A
  1. Drug modification/destruction- some can create enzymes to break down antibiotic (β-lactamases)
  2. Pathway protection- synthesis of false targets for tetracycline
  3. Target alteration- single mutation in a ribosome to prevent binding
  4. Rapid efflux- actively pumping out the antibiotic (heavy metal pumps)
  5. Alternative pathways- folate scavenging against sulfa drugs
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12
Q

What is synergistic inhibition in antibody resistance?

A

Using a combination of drugs to greatly decrease the chance of developing drug resistance
Combinations of antibodies result in both being more effective than one

Slide 10 L11

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13
Q

What is the basic structure of proteins? (Hydrophobic and hydrophilic)

A

Charge of R group is pH dependant
Hydrophobic- non polar R groups fold internally
Hydrophilic- polar R groups fold externally

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14
Q

What is an enzyme?

A 
A protein (or RNA) that acts as a catalyst for a chemical reaction
Makes reaction more favourable (more fast or more possible)

Needs right shape to hold the substrate and a catalytic center to facilitate the reaction (enzyme specificity)

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15
Q

How do we measure reaction rate and velocity?

A

Reaction rate (v) is how fast substrate is converted to product or how fast product is accumulated
V= -Δ[S] / Δt
V= +Δ[P] / Δt

Units are concentration over time (μM/s from μmol/L/s or mg/mL/s)

Slide 20 L11

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16
Q

What is initial velocity of an enzyme reaction?

A

Quickly measured for short period of time
Want it to be as fast as possible so reactions fast
Want optimum temperature and pH as well (have to find these at different times)

17
Q

What are the two things rate is limited by?

A

Rate can be limited by the availability of the substrate
E + S -> ES
Rate can be limited by the production of the product
ES -> EP -> E + P

Slide 21 L11

18
Q

What is the limiting step under optimal conditions with an excess of substrate?

A

The limiting step is how fast the enzyme can catalyze ES->E + P

Slide 22 L11

19
Q

What is the Michaelis-Menten equation?

A

Vo = [S] Vmax / [S] + Km

Km= [S] at a Vo of 1/2 Vmax
Lower Km, more effective

Slide 3-5 L12

20
Q

What is ALDH1 and ALDH2?

A

ALDH1 is found in the cytoplasm and has a high Km 0.1 mM
ALDH2 is found in the mitochondria and has a low Km 0.01 mM

ALDH2 if more efficient since we want a lower Km to work at

Mutations can cause ALDH2 to be slow to ALDH1 would kick in

21
Q

What are the 5 steps to enzymatic studies?

A
  1. Come up with an assay that can be quantified
  2. Determine optimal conditions by plotting product accumulation vs time (determine fastest Vo)
  3. Determine the enzyme rate as we increase [S]
  4. Determine the Michaelis constant (Km)
    [S] when Vo=1/2Vmax
  5. Determine turnover number
    -Kcat = Vmax/[E]t

Slide 9 L12

22
Q

What is the lineweaver burk plot used for the michaelis mentis values?

A

Slide 10 L12
Uses double reciprocal of rate and concentration to place Vmax and Km on the X and Y intercepts

Used to visualize the mechanism of inhibition a compound may exert on an enzyme

23
Q

What is competitive inhibition, uncompetitive inhibition, and non-competitive inhibition?

A

Competitive inhibition- a compound binds the active site of the enzyme and excludes the reactant
Slide 13-14 L13
Uncompetitive inhibition- a compound binds the ES complex and slows release to E + P
Slide 16-17 L13
Non-competitive inhibition- a compound binds the enzyme and decreases the rate the enzyme can bind to or react with the substrate
Slide 18-19 L13

24
Q

What could happen with competitive inhibition if an enzyme has 2 or more possible substrates?

A

One substrate may act as a competitive inhibitor for the other

Someone consumes methanol (or even spills it on their skin)
Alcohol dehydrogenase converts

Ethanol treats methanol poisoning

25
Q

What is enzyme standardization?

A

1 unit (U) of an enzyme will catalyze 1μmol in 1 minute at an optimal temperature and pH and high substrate concentration

Once U is established, responsible experiments can be made

26
Q

What is enzyme activity and enzyme specific activity?

A

Activity= (Δc/min)(reaction volume) / (enzyme volume)
U/mL or U/μL

Specific activity= activity / [protein]
U/mg or U/μg

27
Q

What is biomolecular purification?

A

If you wanna know how a system works, disassemble it and look at the parts

Cells are best studied by breaking them apart and isolating organelles, proteins DNA, RNA and lipids for individual studies

28
Q

What are the 6 ways to break cells, tissues and organisms apart?

A

Sonication- sound waves/vibration
French press- high pressure -> rapid drop
Osmotic shock- low salt concentration
Digestion- enzyme breakdown- lysozyme digests
Detergents- dissolve cell membranes
Homogenization- force

Must go through a series of steps to remove cellular contaminants

29
Q

How are proteins stabilized after extraction?

A

Proteins removed from a cel may become degraded by the non physiological conditions

Should be careful with:
pH changes: extract/keep in buffer solutions
Heat denaturation: keep sample cold, heat only as needed
Oxidation: thiol group on cysteine (-SH) May react to form disulphides
Proteolysis: enzymes that break down proteins
Can lower temp or add protease inhibitors

30
Q

What are the 4 qualities of assays?

A

Sensitive
Specific- substrate only worked by the enzyme in question
Rapid
Quantitative

31
Q

What is yield and purity?

A

Yield- total activity before purification / total activity before purification

Purity: specific activity after / specific activity before

32
Q

What is ion exchange chromatography?

A

Like charges or neutral molecules will elite quickly

Greater the charge on a protein, the slower it will travel down the column

BMSC 240 (9 decks)

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