T1 T2 integrated biochemistry & cell biology

steps of enzyme function

binding of substrate → conversion to product → release of product

cofactor

organic / inorganic (Mg2+, Ca2+)

coenzyme

organic

lactate dehydrogenase - function

reversible conversion of pyruvate & lactate

lactate dehydrogenase convert pyruvate to…

lactate + NAD+

NAD+ generated from lactate dehydrogenase

allow glycolysis to continue in low O2 state

LDH-1

heart

LDH-5

striated muscle

northern blot detects…

RNA

southern blot detects…

DNA

western blot detects…

protein

RFLP - restriction fragment length polymorphism

restriction enzyme, specific site cut, palindromic sequence

PCR - polymerase chain reaction

DNA template, dNTPs, DNA primers, Taq polymerase

standard technique of DNA sequencing

sanger sequence

recombinant DNA technology - human cell

obtain gene responsible for controlling insulin formation from sliced chromosome

recombinant DNA technology - bacteria

obtain isolated plasmid → cut with enzyme

recombinant DNA technology - transformation

desired gene inserted into plasmid → recombinant DNA

sanger sequencing - steps

reaction mixture → primer elongation & chain termination → capillary gel electrophoresis separation of DNA fragments → laser detection of fluorochromes

sanger sequencing - dNTP

chain elongation

sanger sequencing - ddNTP

chain termination bc no OH group

karyotyping

complete set of chromosomes of each individual

karyotyping - arrangement of chromosomes

size, banding pattern, centromere position

karyotyping - detect what

chromosome abnormalities → turner syndrome, klinefelter syndrome, down syndrome, cri du chat, prader-willi syndrome

DNA fingerprint - detect what

minisatellite DNA (10-60 bp)

DNA fingerprint - when

detect in crime scene

DNA profiling - detect what

microsatelllite DNA → short tandem repeats (2-3 bp)

DNA profiling - when

detect family → if child match w mom/dad

point mutation / substitution - types

silent, nonsense, missense

silent mutation

same fuction

nonsense mutation

stop codon → protein shorter

missense mutation - types

conservative & non-conservative

missense mutation - conservative

same function

missense mutation - non-conservative

function change

frameshift mutations

insertion & deletion

purine bases

adenine & guanine

pyrimidine bases

cytosine, thymine, uracil

cell cycle - components

interphase & mitotic

interphase - components

G1 → S → G2

mitotic - components

prophase, metaphase, anaphase, telophase, cytokinesis

G1 phase

metabolically active, growing cell

S phase

DNA replication

G2 phase

cell growth continues, preparation for mitosis

G0 stage

quiescent stage, metabolically active,

cells that remain in G0 normally

skin fibroblasts

skin fibroblast - function

produce collagen at epidermis

platelet-derived growth factor

stimulate fibroblast to S phase → proliferation → close wound

no telomerase leads to…

gene shortens every cycle → affect DNA stability

trinucleotide repeat expansion

expansion in DNA repeats

trinucleotide repeat expansion - location

5’UTR, exon, intron, 3’UTR

trinucleotide repeat expansion at intron

abnormal splicing → unstable mRNA

examples of trinucleotide repeat expansion

huntington’s & fragile x syndrome

RBC production - location

red bone marrow

heme production - location

mitochondria & cytoplasm of RBC

ROS production

H2O2 → fenton reaction → •OH

antioxidant production

O₂^- → H2O2 → H2O

superoxide dismutase

convert superoxide (O₂^-) → H2O2

catalase

convert H2O2 → H2O

ROS production - where

ETC, cytosolic enzyme, cosmic radiation, drugs

ROS production leads to…

disease, mutation, aging

antioxidant system

superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, NADPH, vitamins C, E

antioxidant production leads to…

growth promotion, signaling pathway, pathogen defense mechanism

pentose phosphate pathway

glucose → glucose-6-phosphate → … → ribose-5-phosphate

pentose phosphate pathway - useful products

NADPH & ribose-5-phosphate

pentose phosphate pathway - purpose of NADPH

antioxidant defense, regenerate GSH, reductive biosynthesis of fatty acids, steroid hormones, neurotransmitters

pentose phosphate pathway - GSH

protect cells from oxidative stress → detoxifies H2O2 & free radicals

pentose phosphate pathway - purpose of ribose-5-phosphate

nucleotide & nucleic acid synthesis

G6PD deficiency - distribution

resembles malaria

G6PD deficiency - genetics

x-linked recessive → missense mutation

G6PD deficiency - pathophysio

decrease NADPH → cannot reduce glutathione → less detoxification → ROS accumulates → oxidative damage → acute hemolytic anemia

G6PD deficiency - what leads to hemolysis

O₂^- , H2O2, •OH

G6PD deficiency - symptoms

asymptomatic (mostly), jaundice, dark urine

G6PD deficiency - triggers of oxidative stress

fava beans, infection, some drugs

primary lactase deficiency

genetic defect

secondary lactase deficiency

acquired / transient

lactose intolerant - symptoms

stomach ache, diarrhea, nausea/vomit

carbohydrate digestion - membrane transporters

SGLT1, GLUT5, GLUT2

SGLT1 - membrane & function

apical membrane, co-transport Na+ & glucose/galactose

GLUT5 - membrane & function

apical membrane, transport fructose

GLUT2 - membrane & function

apical membrane & basal membrane, transport glucose galactose fructose

GLUT2 at apical membrane when?

high luminal hexoses

GLUT1 - location & function

ubiquitous → basal glucose uptake (every cell)

GLUT2 - location & function

liver, pancreas, intestine → remove of excess glucose from blood

GLUT4 - location & function

muscle, fat, heart → activity increased by insulin

galactose metabolism - enzymes

galactokinase & galactose-1-phosphate uridylyl transferase

galactokinase

convert galactose → galactose-1-phosphate

galactose-1-phosphate uridylyl transferase

convert galactose-1-phosphate + UDP-glucose → glucose-1-phosphate + UDP-galactose

galactosemia

defect in galactose-1-phosphate uridylyl transferase deficiency

galactosemia - symptoms

failure to thrive, mental retardation, death from liver damage, cataract (accumulation of galactitol in lens)

SGLT inhibitors in kidney

inhibit SGLT2

inhibit SGLT2 in kidney leads to…

prevent renal glucose reabsorption → promote urinary glucose excretion (independent of insulin)

why don’t inhibit SGLT-1 in kidney

SGLT-1 also important in intestine → if inhibit: severe diarrhea & malasorption

ATP production from glucose

glycolysis → pyruvate → acetyl-CoA → TCA cycle → oxidative phosphorylation

ATP production from amino acids

deamination / transamination → C skeleton enters pyruvate, acetyl-CoA, TCA intermediates

ATP production from free fatty acids

beta oxidation → acetyl-CoA → TCA cycle → oxidative phosphorylation

ATP production from ketone bodies

convert to acetyl-CoA → TCA cycle → oxidative phoshphorylation

chylomicron transport…

TG from diet → exogenous pathway

VLDL transport…

TG synthesized from liver → endogenous pathway

after chylomicron in lymph…

thoracic duct → left subclavian vein → systemic circulation

after chylomicron in blood…

hydrolyze by LDL on capillary endothelium

lipoprotein lipase

hydrolyze TG → free fatty acid + glycerol