Biotech Exam 1

L1

molecular biology

study of bio on a molecular level inc structure, function, and makeup of biologically impt molecules such as DNA, RNA, and proteins

recombinant DNA tech

set of techniques for manipulating DNA including identification and cloning of genes; study of expression of cloned genes; production of large quantities of gene product

genetic enginering

process of transferring DNA from one organism into another that results in a genetic modification

biotechnology

production of goods and services using biological organisms, systems, and processes

biotech timeline

1. first antibiotic - moldy tofu used to treat boils

2. first insecticide - powdered chrysanthemums

3. variolation - dried scabs/fluid directly from ppl sick w virus for smallpox

4. first vaccination - Edward Jenner uses similar virus (cowpox) and inserts into a person to prevent smallpox

5. proteins are discovered

6. first enzyme is discovered and isolated

7. Louis Pasteur conducts experiments that support germ theory of disease - specific microscopic organisms cause infectious diseases

8. Gregor Mendel discovers laws of inheritance; starts science of genetics

9. Sir Alexander Fleming discovers antibiotic penicillin

10. Oswald Avery, Colin MacLeod, and Maclyn McCarty prove that DNA carries genetic info

11. James Watson and Francis Crick describe the double helical structure of DNA; shared Nobel Prize w Maurice Wilkins

12. Frederick Sanger discovers amino acid sequence of insulin

13. First biotech drug approved by FDA - Human insulin produced in genetically modified bacteria

14. genetic code for DNA is determined

15. Hamilton Smith isolates first site-specific restriction enzyme

16. Stanley Cohen and Herbert Boyer perfect genetic engineering techniques to cut and paste DNA using restriction enzymes

17. first expression of a human gene in bacteria is found

18. George Kohler and Cesar Milstein develop tech to produce monoclonal antiodies

19. Fred Sanger, Allan Maxam and Walter Gilbert developed rapid DNA sequencing methods

20. Joachim Messing developed shotgun DNA sequencing method

21. first transgenic animals are produced by transferring genes from other animals to mice

22. first patent for GMO is granted - bacteria that can break down crude oil

23. Kary Mullis discovers PCR

24. first recombinant vaccine is approved for human use - hepatitis B

25. first anti-cancer drug is produced through biotech - interferon

26. Human Genome Project launched - international effort to map all genes in human genome - and published

27. first GLP-1 agonist - exenatide (Byetta) approved by FDA for Type 2 diabetes

    1. then Liraglutide (Victoza), then Dulaglutide (Trulicity), then Semaglutide (Ozempic), then Tirzepatide (Mounjaro), then Semaglutide oral (Wegovy)

28. recombinant vaccine - HPV receives FDA approval

29. genome editing w CRISPR-Cas 9

30. FDA approves anti-PD-1 checkpoint inhibitors

31. first CAR-T cell therapy approved by FDA - Kymriah

32. SARS-Cov2 (COVID-19) mRNA vaccines

33. FDA approves engineered T-cell therapy for advanced synovial sarcoma

34. FDA approves first-in-class RAS inhibitor for metastatic pancreatic cancer

DNA genomics

• Replication

• Transcription - info coded in the sequence of base pairs in DNA is passed to molecules of RNA

• Translation - info in RNA is passed to proteins; never passes from proteins to nucleic acids

• Secretion - of complete protein after glycosylation folding and cross-linking

how to measure a genome

1 base = 1 nucleotide = 1 basepair (bp)

1000 bases = 1 kilobase (kb)

1000 kb = 1 megabase (Mb)

1000 mb = 1 gigabase (Gb)

polymorphism

an area of DNA sequence that varies from person to person

single nucleotide polymorphism (SNP)

polymorphism in which a single base in the DNA differs from the usual base at that position

copy number variant (CNV)

a polymorphism in which the number of repeats of a DNA sequence (controls production of proteins) at a location varies from person to person

indel

a polymorphism in which a DNA sequence is either present (insertion) or absent (deletion) at a location, varying from person to person

druggable genome

subset of genes whose protein products can bind small, drug-like molecules; what chemistry can reach NOT a fixed property of a gene

small molecule drugs

• molecular weight </= 1000 (preferably </= 500)

• structure and purity determined

• active pharmaceutical ingredient (API) is usually a solid

• focused on metabolism

• often formulated in a solid dosage form (tablet, capsule)

• often dosed orally

advantages

• oral bioavailability

• wide distribution in vivo - tissue penetration; ability to reach intracellular and CNS targets

• ease of use and manufacturing better than for biologics

  • identity, purity, stability, multiple formulations

  • multiple dosage forms - oral, IV, transdermal, fixed dose combos

biologics

• high molecular weight » 1000

• primary structure defined; post-translational modifications not so well

• cannot be delivered orally

• generally will not penetrate inside cells

• formulation and solution stability may be a challenge; can get inside brain based on barrier leakiness

• formulated in parenteral dosage form (SQ, IV)

• difficult to genericize

advantages

• top selling

• reduced attrition in clinical development

• broader use

• toxicity is generally mechanism-based

Drug development facts

• over 90% of drugs entering clinical trials fail to make it to market; esp for cancer

• avg cost to bring a new drug to market is > $1 billion

imatinib (Gleevec) example

• philadelphia chromosome - translocation of chromosome 9 and chromosome 22 creates BCR-ABL oncogene

• imatinib blocks ATP from binding to BCR-ABL kinase which inhibits CML - but it is not perfectly selective

• more research and resistance drove the finding of asciminib binds at a different site on the kinase, making it more targeted to the specific kinase

L2

cellular problems that lead to diseases or injuries

• too much cell division

  • ex: cancer

• too little cell proliferation → interfere w repair

  • ex: damaged neuron, weakened heart muscle cells

• abnormal ell function

  • ex: hemophilia (impaired ability of hepatocytes to make clotting factors)

living organism

• capable of growth and movement

• carries out metabolism and uses and generates energy

• can respond to stimuli

• can carry out reproduction

cell theory

1. every living thing on earth is made of one or more cells

2. the cell is the basic unit of structure and organization in living organisms

3. cells arise from pre-existing cells

Robert Hooke

• improved the microscope - key to discovery of cell theory

• observed individual compartments in cork plants - cells

Anton Van Leeuwenhoek

• also made a microscope

• observed bacteria and protozoa

Theodore Schwann

• observed animal tissue under microscope

• began to formulate cell theory w Schleiden

• early realization that cells were ubiquitous

abiogenesis

origin of life from nonlife

• presence of small organic molecules

• to spontaneous formation of monomeric biomolecules

• to polymerization of monomers into macromolecules

• to phospholipid membranes formed and some molecules became encapsulated in phospholipid bilayer

• to first protocells followed by first cells

Miller-Urey experiment

• earth conditions were mimicked in an apparatus made in lab

• conditions included water, ammonia, methane, hydrogen, heat, and electric sparks

• results

  • amino acids formed after about a week

• provided support for abiogenesis theory but faced criticism as experiment may not have perfectly replicated the environment

first protocell

• phospholipid bilayer spontaneously formed the ring structure

• RNA → first genetic material

• transition from RNA to DNA led to development of more complex cells

• led to the diversity of life that now exists on earth

• first prokaryotic cells were formed abt 3.5 billion years ago

prokaryotic cells

usually unicellular cells

ex: bacteria and archaea

• capsule

  • some bacteria have

  • thick structure for protection, structural support, and adhesion

• cell wall

  • sometimes thinner than capsule

  • provides structural support and protection

• nucleoid

  • region containing chromosome

• flagella

  • used for movement

• no nucleus, no membrane bound organelles, but yes ribosomes!

eukaryotic cell

• nucleus

• membrane-bound organelles

• cytoskeleton

• first cells abt 1.5 billion years ago

evolution of eukaryotic cells

• ancestral prokaryotic cell developed internal membrane infoldings (inc SA) that gave rise to endomembrane system

• cells engulfed aerobic bacteria, which evolved into mitochondria

  • mitochondria and chloroplasts have their own DNA

endosymbiotic theory

theory that a larger host cell engulfed aerobic bacteria that evolved into mitochondria and chloroplasts

plasma membrane

• function:

  • semi-permeable membrane that helps determine what can go in or out of a cell; has transporter proteins

• structure:

  • phospholipid bilayer with proteins and cholesterol within it

  • polar hydrophilic head

  • nonpolar hydrophobic tail

cystic fibrosis

involves abnormality in a chloride ion transporter in the membrane

hypercholesterolemia

can involve a defect in the LDL (low density lipoprotein) receptor in the membrane

• LDL → carries cholesterol in bloodstream

• causes abnormalities in cholesterol levels

cytoplasm

space inside the cell with organelles and cytoskeleton

• contains cytosol: jelly-like fluid

endomembrane system

set of membrane bound regions consisting of:

• nuclear envelope

• rough and smooth ER

• golgi apparatus

• lysosomes

• endosomes

• plasma membrane

• NOT mitochondria

membranes are interconnected either by direct contact or vesicle transport; can pinch off one organelle and add to another

nucleus

• function:

  • stores genetic info

• structure:

  • contains chromatin, nucleolus, nucleoplasm, nuclear envelope

chromatin

the DNA and associated proteins

nucleolus

contains ribosomal RNA (rRNA) and ribosomal proteins; key role in making ribosomes

nucleoplasm

fluid area in nucleus other than chromatin and nucleolus

nuclear envelope

double membrane contains pores to let things in and out

ribosomes

• function

  • process of translation while making proteins

• structure

  • NOT membrane bound

  • some are free ribosomes (free in cytoplasm)

  • some are ER-bound (attached to ER membrane)

  • made of ribosomal RNA (rRNA) and protein

rough endoplasmic reticulum

• functions

  • protein synthesis

    • ER-bound ribosomes synthesize proteins destined for secretory pathway

    • polypeptides enter ER lumen during protein synthesis process

  • protein processing

    • proteins are folded and modified

    • some proteins are glycosylated (carbohydrates added to form glycoproteins)

  • protein trafficking - secretory pathway

    • proteins are either

      • secreted

      • inserted into plasma membrane

      • delivered to endomembrane organelles

• structure

  • studded w ribosomes

  • ER lumen inside ER

    • have cisternae - flattened vesicles inside lumen

smooth endoplasmic reticulum

• functions

  • production of phospholipids which are delivered to various membranes in cell

  • production of steroids including cholesterol

  • site of detoxification

    • ex: hydroxylation (addition of hydroxyl groups) to toxins to enhance water solubility

  • metabolism of lipids

  • synthesis, metabolism, and storage of calcium ions

• structure

  • connected to rough ER

  • NOT covered w ribosomes

  • ER lumen inside ER

    • have cisternae - flattened vesicles inside lumen

HMG-CoA-Reductase

enzyme important for cholesterol synthesis; located in membrane of ER (both rough and smooth)

Statins

drugs that target HMG-CoA-Reductase to lower blood cholesterol levels

golgi apparatus

• function

  • modify proteins from ER

  • modify lipids

  • sort proteins so they are sent to correct destinations

  • cis face

    • receives vesicles carrying newly made proteins and lipids from ER

  • medial region

    • adds modifications or tags which are impt for sorting

  • trans face

    • tags are recognized and items are packaged into new vesicles and sent to correct destinations

• structure

  • made up of cisternae - series of membrane bound sacs

  • different cisternae contain diff enzymes that modify incoming proteins

  • cis face - closest to ER

  • trans face - furthest from ER

  • medial region - in between cis and trans faces

  • associated vehicles - vesicles that transport

  • most animal cells have one Golgi

Golgi fragmentation

• seen in cells undergoing apoptosis

• associated w some types of cancer

• common in neurodegenerative diseases; ex: Parkinson’s

• not always known what causes it

lysosome

• function

  • digest cellular waste

  • digest molecules no longer needed as well as damaged organelles - autophagy

• structure

  • membrane-bound sac

  • acidic environment

  • enzymes that digest large molecules (hydrolytic enzymes)

Gaucher disease

• abnormalities in gene for a lysosomal enzyme that breaks down certain lipids

• causes accumulation of lipids in lysosomes

• some types of disease are treatable but others esp for infants are fatal

• differences lie in diff mutations in the gene for the enzyme

mitochondria

• function

  • sites of later stages of cellular respiration where ATP is generated

  • houses citric acid cycle (Kreb’s cycle) and oxidative phosphorylation

• structure

  • NOT part of endomembrane system

  • has 2 membranes

    • outer membrane → smooth

    • inner membrane → has folds called cristae; also has enzymes

  • intermembrane space → space between two membranes

  • mitochondrial matrix → innermost part; has mtDNA, ribosomes, and enzymes

  • has its own DNA (mtDNA) - makes some of its own proteins

Leigh syndrome

• rare genetic disorder caused by mutations that impair oxidative phosphorylation

• mutations may occur in nuclear DNA or mtDNA

• symptoms: overall weakness, tremors, and neurological symptoms

• usually fatal

peroxisome

• function

  • lipid metabolism: oxidation (breakdown) of very-long-chain fatty acids

  • detoxification of harmful substances (ex: alcohol)

  • catalase: enzyme that breaks down hydrogen peroxide (a byproduct of many reactions) to prevent toxicity

Zellweger syndrome

• disorder in which peroxisome function is severely impaired

• caused by mutations in genes that encode proteins necessary for the proper assembly of peroxisomes

• symptoms: impaired development, neuronal symptoms

• patients do not usually survive beyond a year of age

cytoskeleton

• NOT an organelle

• function

  • maintenance of cell shape and integrity

  • cell motility

  • provide tracks for motor proteins that can transport vesicles

• structure

  • network of fibers throughout the cell

  • components

    • microtubules

    • microfilaments (actin)

    • intermediate filaments

centrosome

• function

  • major microtubule organizing center (MTOC) in animal cells

  • centrioles organize microtubules and are critical for cell division

  • microtubules extend from centrosomes and play a role in chromosome alignment and separation

• structure

  • pair of centrioles - 13 triplets of microtubules surrounding a hollow ring

  • pericentriolar material (PCM) - contains tubulin

single cellular eukaryotes

ex: yeast, algae

developed billions of years ago

multicellular eukaryotes

• ex: animals, plants

• developed abt 600 M years ago

specialized cells within multicellular organisms

• diff cells have specialized functions

• can be organized into cells that make up 4 broad categories of tissues

  • cells within a tissue share a common embryonic organ

  • epithelial

  • connective

  • muscle

  • nervous

epithelial tissue

sheets of cells that line organs, glands, and exterior surfaces

connective tissue

found between other tissues (ex: tendons, ligaments, cartilage, bone)

muscle tissue

impt for movement

nervous tissue

communication and signaling; have neurons

L3

plasma membrane components

all components move around within their layer

• phospholipids - the major component

• glycolipids - lipids attached to a sugar

• glycoproteins - proteins attached to a sugar

• sterols - such a cholesterol

• lipids

• proteins

phospholipids structure

• polar head group

• phosphate group

• glycerol backbone

• 2 fatty acid side-chains; nonpolar tail

  • one saturated

  • one unsaturated; has double bond - kink in chain

4 major phospholipids in membranes

differ in phosphate group

• phosphatidyl-ethanolamine

  • ethanolamine phosphate head

• phosphatidyl serine

  • serine phosphate head

• phosphatidyl choline

  • choline phosphate head

• sphingo-myelin

  • choline phosphate head

  • no glyerol backbone

  • sphingosine backbone instead

Acid Sphingomyelinase (ASM)

enzyme that breaks down sphingomyelin in lysosome

ASMD (ASM deficiency) or Niemann-Pick disease

• rare and severe lysosomal storage disease

• mutation in gene encoding Acid Sphingomyelinase (ASM)

• disrupts balance between sphingomyelin and its breakdown products

• causes accumulation of sphingomyelin in lysosomes of macrophages

  • these macrophages travel throughout body and cause tissue damage and chronic inflammation

• symptoms

  • enlarged liver

  • spleen

  • difficulty eating

  • loss of muscle tone

  • lung disease

  • eye problems

  • low platelet counts

  • delayed growth

xenpozyme

enzyme replacement therapy for ASMD

• first FDA-approved treatment for ASMD

• recombinant human acid sphingomyelinase

• received breakthrough therapy designation

glycocalyx

• layer of carbohydrates that coats the membranes of many cells

• made up of glycoproteins and glycolipids

• functions

  • facilitate interactions w extracellular matrix

  • protect cells from damage

  • keep cells apart from each other

transporters (carriers or permeases)

• usually specific for their solute

• bind solute and change conformation to transport it across the membrane

• uniport

  • transports one molecule across membrane

  • ex: GLUT1

• symport

  • transports two molecules across membrane in same direction

• antiport

  • transports two molecules across membrane in opposite directions

channels

• may also be specific

• form pores through the membrane

• faster transport vs transporters

transporter kinetics

• vmax

  • max transport rate when all available transporters are fully engaged in transport

• Km

  • solute conc at which the transport rate is ½ Vmax

competitive inhibitor

• compete for same binding site as the solute

• inhibitor may or may not be transported

non-competitive inhibitor

• bind elsewhere in the transporter

• may alter transporter function, reducing its ability to transport to its solute

passive transport

• does not req energy

• solutes flow down their conc gradients (downhill)

• more common mechanism for transport of drugs

• diffusion

  • small gases, small lipids, and small uncharged molecules

• facilitated diffusion

active transport

• req energy

• solutes flow against conc gradients (uphill)

drugs that can pass through the membrane via diffusion

• ibuprofen

• aleve (naproxen)

• aspirin

• retrovir (azt)

• epivir (lamivudine)

• ethanol

• caffeine

• rheumatrex (methotrexate)

facilitated diffusion

• solutes travel down their conc gradient via a channel or transporter

• does not req energy

• used by many ions and polar molecules

• ex: aquaporins

  • allow transport of small amounts of water molecules

  • abundant in epithelial cells of kidney

• ex: ion channels

  • transport ions such as Na, K, Ca, Cl

  • some have gate which is only open under certain conditions

• ex: gated ion channels

  • open briefly usually in response to a stimulus

calcium channels

• muscle cells

  • skeletal + cardiac muscle cells → calcium flow regulates muscle contraction and relaxation

• neurons

  • calcium is essential for neurotransmitter release

• endocrine cells

  • ca influx triggers release of hormones into bloodstream

• immune cells

  • ca signaling regulates immune responses

calcium channel blockers (CCBs)

block flow of calcium ions by binding to L-type calcium channels and blocking them

• amlodipine (norvasc) → treats high blood pressure and angina (chest pain)

• cardizem (diltiazem)

• calan (verapamil)

• procardia (nifedipine)

• cardene (nicardipine)

ion channels and …

action potentials

• Na/K move through ion channels

• Na entry causes membrane depolarization - inside of the cell becomes less negative relative to the outside

• depolarization opens voltage-gated Ca channels at synapse

• Ca entry triggers release of neurotransmitters via vesicle fusion

• Ach can open ligand-gated ion channels(nAChR) on postsynaptic cells which allow influx of ions

• AChR on postsynaptic neurons are opened transiently in response to ACh

• leads to passage of ions through the channel

• flow of ions through postsynaptic neuron leads to various outcomes - excitatory effects

acetylcholine receptors

• AChR on muscle cells are opened transiently in response to ACh, resulting in passage of ions such as Na and K

• leads to contraction of muscle cells

vessel fusion

• as Ca enters neuron, it causes vesicle fusion and ACh release

• at synapse after action potential

  • ACh is in vessels in presynaptic cell

  • influx of Ca through Ca channels leads to vesicle fusion w membrane

  • SNAP25 has an impt role

  • ACh is released into synaptic cleft and left to act on nAChRs

Daxxify

• neurotoxin that blocks ACh signaling

• used to reduce wrinkles

• cleaves SNAP25 - protein necessary for synaptic vesicle fusion

• ACh release from presynaptic neuron is blocked so it leads to dec in muscle contraction, which is effective at reducing wrinkles

• over time, there is a gradual recovery of SNAP25

• daxxify inc stability of original botox so it lasts longer

drugs that affect AChR receptors

• nicotine

  • activates nAChRs

• varenicline (chantix0

  • partial agonist of nAChRs

• anesthetics

  • activates nAChRs on skeletal muscle cells

• curare (D-tubocurrarine)

  • antagonist of nAChRs on skeletal muscle cells

GABA

• inhibitory neurotransmitter

• binds to GABA_A receptors which are ligand-gated Cl- channels

• helps regulate anxiety, sleep, and muscle tone

drugs that affect GABA receptors

• benzodiazepines

  • enhance inhibitory effects

  • ex: diazepam (valium), lorazepam (ativan)

• barbiturates

  • enhances inhibitory effects

  • ex: phenobarbital (luminal)

Ztalmy (ganaxolone)

• anticonvulsant

• used to treat seizures associated w CDD

• acts on extra-synaptic GABA receptors - outside direct connection - making it a stronger effect

primary active transport

occurs directly via ATP hydrolysis

secondary active transport

occurs indirectly; often via coupled transport

• happens when conc gradient is set up via a pump that req ATP

• gradient used as a source of E to transport another molecule up its conc gradient

concentration gradient

for uncharged molecules; the diff in conc of the molecule on two sides of the membrane

electrochemical gradient

for charged molecules; the conc gradient and electrical gradient (net charge diff across membrane) combine

P type - ATP driven pump

phosphorylate themselves during pumping cycle;

ex: Na/K pumps, Ca pumps

Na/K pump

• P type pump

• pumps 3 Na ions out and 2 K ions in

• establishes electrochemical gradient

  • inside cell: low Na and high K

  • outside cell: high Na and low K

  • used for electrical signaling and maintenance of cell V and membrane potential

digoxin

drug used to treat heart conditions

• inhibits Na/K pump, which reduces Na gradient

• also reduces activity of Na/Ca exchanger, which uses Na gradient to remove Ca from cell

• intracellular Ca inc, which inc cardiac contraction

Ca2+ transporters

• Ca levels

  • in cytosol → low

  • outside cell → high

  • in ER → high

  • in sarcoplasmic reticulum (ER in muscle cells) → esp high

• impt for establishing gradient

SERCA (sarcoplasmic/endoplasmic reticulum calcium ATPase)

• P type calcium pump

• found in high levels in membrane of SR of skeletal muscle cells

• pumps Ca into SR, resulting in high Ca levels in SR and low CA in cytoplasm

• conformational changes

  • E1

    • SERCA binds ATP and 2 Ca from cytosol

  • E1 → E1-P

    • ATP phosphorylates pump; ADP is released

  • E1-P → E2-P

    • conformational change exposes Ca to SR lumen

    • 2 Ca are released and 2 H bind

  • E2 → E1

    • pump returns to E1 as 2 H are released into cytosol

    • H counter-transport helps balance charge

ABC transporters (ATP Binding Cassette Transporters)

have a diff structure than P-type and mostly pump small organic molecules

• transport solutes out of cytoplasm

• contain 2 transmembrane domains and 2 cytoplasmic ATP-binding cassettes

ex: MDR pump

MDR (multidrug resistance) protein

• p glycoprotein and transmembrane drug efflux pump

• export drugs and toxins from inside cell; inc resistance

• impt in cancer drugs as it makes cells resistant to many drugs

• strategies to overcome MDR

  • combination therapies

  • drug sequencing

  • MDR transporter inhibitors

  • nanotechnology

serotonin transporter (SERT)

• reuptake inhibitor

• uses Na conc gradient to transport serotonin back into the cell

• located on presynaptic neurons in CNS

• responsible for reuptake of serotonn from synaptic cleft back into presynaptic neuron