Basics

Question 1

Biotechnology defintion

Answer 1

the use of tissue culture, living cells, or cellular enzymes to produce a defined product

Question 2

Approximtely how many biotechnologies are approved by the FDA each year?

Answer 2

12

Question 3

how many biotechnologies are in development whether it be by clinical trials or other means

Answer 3

approximetley 1000 being developed for over 100 diseases

Question 4

what kind of diseases are biotechnologies being developed for?

Answer 4

cancer, rare diseases, neurological, HIV/AIDS, heart and stroke disease, mental helath disorders, alzehimer’s disease, skin disease, asthma and allery

Question 5

cDNA define

Answer 5

only coding DNA - DNA transcribed from specific mRNA via enxyne reverese trasncription

Question 5

What therpauetic categories have the highest biotechnology development rate?

Answer 5

pediatric, neurological, etc

Question 6

Stages of the cell cycel

Answer 6

G1
S
G2
M

Question 7

What is circular DNA

Answer 7

Forms a closed loop and has no ends

Question 8

What is a cloning gene

Answer 8

a common practice in molecular biology labs that is used by researchers to create copies of a particular gene for downstream applications

Question 9

what is an endonuclease

Answer 9

an enzyme that breaks down a nucleotide chain into two or more shorter chains by cleaving internal covalent bonds

Question 10

What is DNA restriction?

Answer 10

restrcition enzymes bind to certain DNA sequences

they then cut the sugar-phosphate backbone of DNA strands leaving stticky ends

the sticky ends can be reattached via enzyme liagse, whihc is used to catalzye the chemical reaction that rejoins DNA

Question 11

What is DNA transformation

Answer 11

using restriction and liagse to combine various gene segemnst

Question 12

How are low temperatures and heat shock used in DNA transformatiom

Answer 12

the lipid membrane has a negative charge and so does the DNA making them un able to interact

by reduce the temperature we are able to stabilize the cell membrane and add Ca2+ to stabilize the membrane.

Then using a heat shock a temperature imbalnce is formed on either side of the membrane and this sets up a current. Since we have a ionic shield in place the DNA can be swept through the adhesion zone

Question 13

What is Gentic enigneering

Answer 13

we can add or remove segments of DNA plasmids via DNA restriction and DNA ligase

Question 14

What is gel electrophoresis

Answer 14

it separates DNA fragments based on size and charge

it does so by having a current run through gel containing the molecules of interest

Question 15

what is a model organism?

Answer 15

a non-human species that is studied to understand biological processes

Question 16

How does PCR work

Answer 16

Raise temeprature - denature
Annel
extend primers

Question 17

Descrieb how monclonal antibodies are produced

Answer 17

a mouse is immunized with the desired antigen

the mouse will then porduce antibodies to combat this antigen

via test bleeds we can determine whether the mouse has the desires antoboides and if it does then the spleen is removed and dissocated in culture mediums

in this culture, we also have myeloma cells and the B cells can fuse with these cells and produce hybridomas

When exposed to PEG all unfused b-cells die and we have hybridoma and myeloma cells left

Then using HAT medium we can eliminated all myelominoma cells

the surviving cells then are put in indiviual wells these are knonw as clonla cultures

after culturally for a week weeks we can screen the lqiuid for the desires antigen to see wehther they bond with the desires antigen

clones that produced the right antibody are then frozen or used for mass culture

Question 18

What are monoclonal antibodies used for?

Answer 18

diagnostic and anti cancer drugs

Question 19

what is molecular biology used for?

Answer 19

tracers, genetic engineering, and DNA technology

Question 20

What are the building blacks for DNA, Peptides/proteins, and oligosaccharides?

Answer 20

• nucelotides - nucleosides
• AA
• carbohydrate sugars

Question 21

What are the different nucleotides?

Answer 21

AGCT and U in RNA

Question 22

What are the fours groups of amino acids ?

Answer 22

acidic, basic, uncharges polar side chains, nonpolar side cahins

Question 23

What do we know about optical isomers?

Answer 23

all L-form (mammalian cells) except Gly

Question 24

what are the acidic aa

Answer 24

glu, asp

Question 25

what are the basic aa

Answer 25

lys, arg, his

Question 26

what are the uncharged polar sidechains aa

Answer 26

asn, gln, ser, thr, tyr

Question 27

what are the nonpolar side chains aa

Answer 27

Ala, Val, Leu, Ile, Pro, Phe, Met, Trp, Gly, Cys

Question 28

What do stability and degradation in pharamcy mean?

Answer 28

to define how much of the active drug is available for use

Question 29

What kind of drugs can retain 100% oof the chemcial composition of the biologically active protein yet be biologically inactive

Answer 29

protein drugs

Question 30

How are traditional drugs degraded?

Answer 30

chemical degradation - oxidation, hydrolysis, racemization, etc

Question 31

How are protein drugs degraded?

Answer 31

chemical degradtaion physical degradtion

Question 32

when talking about chemical insatbility whta bonds are we focusing on?

Answer 32

covalent bond modification of proteins via bond formation or cleavage

Question 33

What is deamination and what drugs are suscpetible to it?

Answer 33

the removal of NH2 and replcing it with OH can only happen to aa with amide such as: Asn and Gln

Question 34

whats another word for hydrolysis?

Answer 34

Proteolysis

Question 35

What hydrolysis reaction is of special note?

Answer 35

Pro and Asp this creates a hot spot

Question 36

What does oxidatio affect? proteins in solution or lypholized form?

Answer 36

both

Question 37

How can we redue oxidization?

Answer 37

by packaging lympholized pretin under a nitorgen atmosphere

Question 38

What are common targets of oxidation?

Answer 38

Sulfur containing aa such as cysteine and methionine

Question 39

what does the oxidation of cysteine to sulfonic acid look like?

Answer 39

RSH - RSOH - RSO2H- RSO3H thiol from cysteine - sulfenic acid - sulfinic acid - fulfonic acid

Question 40

what can happen to unpaired cysteins?

Answer 40

they can be oxdized to form cystein di sulfide RSH + R'SH -> RS-SR'

Question 41

what does the oxidation of methionine look like?

Answer 41

Refer to the diagram on slide 36

Question 42

What does Disulfide exchange look like?

Answer 42

RSH + R'S-SR" - RS-SR' + R"SH

Question 43

How can disulfide exchange or oxidation of an unpaired cystein create a problem for proteins?

Answer 43

these will create new protein structure

Question 44

Racemization does what?

Answer 44

Conversion of L-amino acids to D

Question 45

Undertsadn beta-elimination

Answer 45

slide 42

Question 46

Whats a labile aa

Answer 46

unstable aa

Question 47

Primary reaction of Asn and Gln

Answer 47

deamidation, racemization

Question 48

Primary reaction of Asp

Answer 48

hydrolysis, racemization

Question 49

Primary reaction of Met, Cys, His, Trp, Tyr

Answer 49

Oxidation

Question 50

primary reaction of ser, thr, cys

Answer 50

beta elimination and racemization

Question 51

primary reaction for cys

Answer 51

disulfide excahnge and oxidation

Question 52

What does physcial instability do to structure?

Answer 52

it disrupts secondary, tertiary, and quaternary structures

Question 53

if proteins are more then 20 aa how will they degrade?

Answer 53

physical

Question 54

if proteins are less than 20 aa how will they degrade?

Answer 54

chemical

Question 55

Where are the polar and non-polar regions in a folded protein?

Answer 55

hydrophobic on the inside hydrophilic on the outside

Question 56

What are the 4 types of physical degradation?

Answer 56

denaturation absorption to surfaces/interfaces aggregation precipitation

Question 57

What does proetin denaturation do to bonds?

Answer 57

disruption of secondary and tertiary bonds

Question 58

is physcial degradation reversible?

Answer 58

may or may not be reversible

Question 58

what can cause physcial degradation?

Answer 58

heat, extreme pH, chemcials (organic solvents)

Question 59

why are denatured portain usualy insoluble?

Answer 59

because hydrophobic regions folded inside are now exposed to the outside environemnt

Question 60

Why do molecules localize to interfaces?

Answer 60

molecules are amphiphilic so they have both hydrophobic and hydrophilic components so at the interface both parts can be happy

Question 61

Since moleculesAbsorption to surfaces/interfaces implies what? where do proteins adhere?

Answer 61

Proteins adhere to the walls of the doage form containers or the delivery device

Question 62

larger or smaller proteins adhere more?

Answer 62

larger proteins adher more

Question 63

What kind of proteins are less likely to unfold?

Answer 63

disulfide linked proteins

Question 64

what happens when absorption at an interface happens?

Answer 64

proetin denaturation

Question 65

what is the removal of a protein from the surface and return to the solution called?

Answer 65

protein desorption

Question 66

what happens when a protein desorbs?

Answer 66

it's hydrophobic regions are exposed to water and aggregation or precipitation occurs

Question 67

Explain protein aggregation

Answer 67

interaction between hydrophobic regions of DIFFERENT denatured protein molecules

Question 68

What steps preceed precipitaion formation?

Answer 68

protein in native conformation - aggregates - precipitation

Question 69

why can protein aggregation be dangerous for drugs?

Answer 69

it has the potential to trigger an immune response which could lead to decreased drug effectiveness or even harmful side effects

Question 70

what can we do to prevent protein absorption?

Answer 70

surfactants other proteins albumin phospholipids - these all are amphilic or have surface activity

Question 71

how do surfactants work to prevent protein aggregation?

Answer 71

refer to pictures on slides 61-63

Question 72

Why is albumin used to prevent portein absorption?

Answer 72

- water soluble, highly charges - conatins serveerla disulfide bonds ( very stable ) - a competitor for inter-surface phenomenon ( decreased chances of intermolecular interactions of the protein interested)

Question 73

should we shake protein solutions? why?

Answer 73

no - precipitation provides interfaces for proteins to adhere to

Question 74

is precipitation accelerated in large or small air-water interfaces?

Answer 74

large

Question 75

what are some agents that can prevent protein aggregation?

Answer 75

- sugars and salts - polyols, PEG and other polymers - free AA (arginine, glutamate, glycine, etc)

Question 76

What is the effect of aggregation in insulin pumps?

Answer 76

precipates may blcok/obstruct the intake of the drug and pateint may not recive enough insulin

Question 77

Aggregation in insulin pumps may be due to:

Answer 77

- body tempertaure changes - agitation of insulin in reservoir with air - adsorption of insulin to hydrophobic surfaces of air and PVC

Question 78

rate constant describes what?

Answer 78

the kinetics of degradation active drug -k-> inactive drug

Question 79

as a drug decays what does it lose?

Answer 79

biological actvity

Question 80

Rate constant is association with what order of kinetics? a unit of whta?

Answer 80

first order rate constant a unit of time 1/time

Question 81

as the magnitude of k increase what happens to degration?

Answer 81

degradation of the active drug gets faster

Question 82

what can assays be used to measure?

Answer 82

the function, presence or amount of a substance

Question 83

how do we assay chemiacl degrdation? and what are we meauring?

Answer 83

the amount of intact prptain via electrophoresis

Question 84

how do we assay physcial degrdation? and what are we meauring?

Answer 84

physical degradation is harder to measure however we can meausr ethe biological activity a proetin such as thru clot lysis assay for t-PA

Question 85

roles of fibrin and plasminogen in actue M.I or pulmonary embolism

Answer 85

fibrin - clot plasminogen - cardiac repaire

Question 86

What is activase/altepase?

Answer 86

indicated for acute M.I and pulmonary embolism

Question 87

How does activase work?

Answer 87

t-PA (tissue plasmonogen activator) is an endogenous proetin that works by binding to fibrin an d activating plasminogen this results in fibrinolysis or the dissolution of the fibrin clot

Question 88

are different forms of degrdation additive?

Answer 88

yes