Biochemistry Flashcards
Properties of vectors (2C)
Capable of replication inside the host cell
Contains at least 1 restriction site for 1 restriction endonuclease.
Types of vectors
Plasmids: Double stranded circular/small. Clone small DNA fragments up to 10 kb.
Phage: Virus, clones large DNA fragments 20 kb
Cosmid: responsible for packing DNA, up to 50 kb
Process of cloning consists of
Plasmids from bacteria/ foreign DNA are cut by restriction endonuclease
The cohesive ends of foreign DNA recombine with plasmid and joined by ligase to form recombinant DNA
Recombinant DNA is introduced into bacterial host cell. Uptake of DNA is called transformation
The transformed bacterial cell grow and divide and so does the recombinant plasma DNA
Requirements for PCR: TD TD B
- Two DNA primers.
- Deoxyribonucleoside triphosphate (dATP)
- Thermostable DNA polymerase (taq polymerase).
- DNA to be amplified.
- Buffer solution.
Polymerase chain reaction (PCR)
In vitro method for DNA amplification
PCR steps
a. Denaturation: Heating to 95° C.
b. Primer annealing: Cooling to 50° C.
c. Elongation: Heating 72 C allowing taq to elongate the primers. After 10 cycles DNA amplified 1000 times, after 30 cycles DNA amplified 10^9
Apparatus in PCR heating cooling heating is called
automated thermocycler
Advantages of PCR
Sensitive
Faster
Technically less difficult
Applications of PCR: 2S 2P FG
- Synthesis of DNA for sequencing and cloning.
- Synthesis of proteins: insulin - GH -Vaccines - monoclonal Ab.
- Diagnosis of genetic diseases as sickle cell anemia.
- Detection of bacterial and viral infection i.e., HIV and hepatitis B.
- Forensic medicine: amplification of DNA from hair or sperm.
- Gene therapy.
Hemoglobinopathies from Production of abnormal hemoglobin:
* Sickle cell anemia (HbS disease).
* HbC disease.
* HbM disease (Methemoglobinemia).
Hemoglobinopathies from Decreased Production of normal hemoglobin:
* alpha Thalassemia: Defect in production of the alpha chain.
* Beta Thalassemia: Defect in production of the beta chain.
Sickle cell anemia (HbS disease)
a type of Point mutation;
There are 2 genes for the B chain so, Patient may be:
* Homozygous for sickle cell anemia: Contains Hb S only.
* Heterozygous for sickle cell anemia (sickle cell trait): Contains Hb A and Hb S.
Thalassemia
Hereditary hemolytic diseases due to gene mutation or deletions.
alpha Thalassemia:
There are 4 a genes (2 on each chromosome 16).
a. Defect in 1 gene -> carrier for a thalassemia.
Symptoms: Completely normal (silent carrier).
b. Defect in 2 genes -> a thalassemia trait.
Symptoms: Mild anemia.
c. Defect in 3 genes -> a thalassemia major.
Symptoms: Severe anemia.
d. Defect in 4 genes -> Homozygous for a thalassemia.
Effects: - Dies intrauterine (Hydrops fetalis).
Beta thalassemia
There are 2 beta globin genes
Defect in 1 gene –> Beta thalassemia minor (trait)
Symptoms: mild anemia
Defect in 2 gene—–> Beta thalassemia major
Symptoms: severe anemia later on after birth
Patient with p thalassemia major appears normal at birth …. Explain why?
Due to presence of HbF
High levels of HbF and HbA2 in patient with p thalassemia………….Explain why?
To compensate the absence of HbA.
Respiratory (Volatile) acids
as carbonic acid H2CO3, excreted by lungs
Metabolic (Fixed) acids:
Not excreted by the lungs as:
Sulfuric acid/ phosphoric acid
Uric acid and nucleic acid
lactic acid
Mechanisms of regulation of pH:
- Buffers:
- 1st line of defense
- Acts in a fraction of a second (immediate response) - Respiration (Ventilation):
- By increase/decrease CO2 excretion so regulates the amount of H2CO3 in the body - Kidneys:
- 2nd line of defense
- Slow process (takes several days to reach maximum capacity)
- By excretion of excess acids (H+) or bases (HCO3)
Buffers can be
A weak acid and its salt with strong base
H2CO3/ Na bicarbonate (NAHCO3)
Acetic acid/ Na acetate (CH3COOH/CH3COO)
A weak base and its salt with strong acid
Ammonium hydroxide/ ammonium chloride
Physiological Buffers types
- Bicarbonate buffer system: mainly in Extracellular fluid
- Phosphate buffer system: in all types of cells
- Protein buffer system: in cells and plasma
- Hemoglobin buffer system: in RBCs and Specific for buffering CO2 Produced by oxidation in tissues
Bicarbonate buffer system:
BHCO3/H2CO3, ratio 20:1, main buffer system in lungs
Advantages of Bicarbonate buffer system:
- Present in higher concentrations than other buffers
- Easily formed at the tissues from CO2 by Carbonic Anhydrase enzyme
- Easily corrected by respiration; CO2 is excreted with expired air at Lungs
Phosphate buffer system:
Structure: B2HPO4/ BH2PO4 (Alkaline phosphate/ Acid phosphate)
Ratio: 4 : 1
Characters: Strictly related to the kidneys (healthy kidneys are needed for proper
function)
. Protein buffer system:
Structure: H-proteinate / B-proteinate (Acid protein / proteinate salt)
Characters: include hemoglobin in RBCs and plasma proteins of the blood
Normal levels of blood
■ Bicarbonate: 22- 26 mEq/L
■ PCO2: 35-45 mmHg
■ pH: 7.35-7.45
■ PO2: 80-100%
Acidosis
- 4, BHCO3 / H2CO3 Less than 20/1
- PH Less than 7. 4
Alkalosis
- BHCO3 / H2CO3 More than 20/1
- PH More than 7. 4
Causes of respiratory acidosis
- Failure of Lungs to excrete CO2 -> ‘b H2CO3
• Extensive Lung disease : A BEP
► Asphyxia
► Bronchia Asthma
► Emphysema
► Pneumonia - Morphine or barbiturate Poisoning -> 4, of respiratory center
Causes of respiratory alkalosis
- Hyperventilation -increase Loss of CO2 decrease H2CO3
HFHAM
• High altitudes
• Fever
• Hysterical
• Aspirin overdose
• Meningitis & encephalitis
Compensation of Respiratory Acidosis :
- ↑ excretion of acids ( H ),
- ↑ reabsorption of (HCO3-) by the kidneys.
Compensation of Respiratory Alkalosis
- ↑ Excretion of HCO3.
- ↓ Excretion of H+ by the kidneys.
Causes of metabolic acidosis :
( decrease of HCO3- )
- High protein diet & severe muscular exercise.
- Renal failure & Diarrhea.
- Ketosis (severe uncontrolled diabetes mellitus , starvation and carbohydrate deficiency).
- vomiting
Compensation of metabolic Alkalosis :
- Depression of the respiratory center →↓ rate
of respiration. - ↓ Excretion of co2 by the lungs.
- Renal compensation .
ELECTROPHORESIS
Definition:
Migration of a charged molecule in an electric field.
Used for separating amino acids , proteins , peptides and nucleic acids .
Unit of electrophoresis consist of
1-Electrodes :
Cathode (-ve).
anode (+ ve) .
Best types of electrodes are made of platinum
2-Buffer reservoirs (tanks or chambers ) .
3-A support for the electrophoretic medium.
4-A transparent insulating cover to minimize evaporation of buffer.
Importance of buffer in electrophoresis
1- Transmit electric current.
2-Adjust the pH: determine the electric charge on the solute to be separated .
3- Facilitate migration of the substance to be separated .
High ionic strength buffers
give sharp bands,↓ rate of migration.
↑ Current & heat production → denature
proteins
Low ionic strength buffers
Bands become more diffuse and wider,
↑ rate of migration
↓current & hence heat production
Types of supporting media in electrophesis
1 Paper (old fashioned)
2 Cellulose acetate membrane
3 Agar
4 Agarose gel (common in use)
5 Polyacrylamide gel (common use)
Paper Electrophoresis :
Not in common use as it is not inert (charges on paper may interfere with
electrophoresis).
- It is time consuming (16-18 hr).
Agarose Gel Electrophoresis: advantages
- Inert→ no adsorption & very little interference with migration of charged samples.
2 Small amount of sample applied (0.6-3 / µ) .
3 Short electrophoretic time (30-90 minutes).
- clarity which permits excellent densitometric scanning .
Atherosclerosis is promoted by high level of______ (bad cholesterol) without
adequate removal of cholesterol by functional _________(good or protective
cholesterol) .
LDL, HDL
Mechanism of LDL:HDL ratio
- Hyperglycemia (as in diabetes mellitus) →↑ Plasma LDL level → modified
into oxidized & glycated LDL by oxidants (ROS). - LDL is taken up by macrophage and arterial smooth muscle by:
- Scavenger receptors. - or non - receptor pinocytosis . - Macrophages become overloaded with cholesterol → foam cells .
- Accumulated foam cell in arterial wall stimulate :
o Release of growth factors.
o Proliferation of smooth muscle.
o Formation of plaque (atheroma) .
o Narrowing of blood vessels → predispose to thrombosis .
Total plasma cholesterol normal level is ______, high risk is ______
< 200 mg/dL , >240 mg/dL
is an excellent indicator for early
acute myocardial infarction .
An isoform ratio of 1.5 or greater
______Are released into blood stream with myocardial injury. _______ accurate than CK-MB but _______diagnose reinfarction
Troponin I and T. More accurate. cannot
Myoglobin is a sensitive indicator to _________. It is not specific to _______muscles
muscle injury, cardiac
Because the ______(level) the myoglobin, the larger the size of infraction, a negative myoglobin rise can rule out _________
higher, myocardial infarction
Plasma AST
returns to normal about 5th day
Plasmas LDH
returns to normal after a week
Risk factors of myocardial infarction
Hypertension. hypercholesterolemia, obesity, diabetes, smoking, stress, sedentary life style and family history
Symptoms of Myocardial infarction
Anxiety, nausea, chest pain, shortness of breath, rapid irregular heart beat
Jaundice
↑↑ serum bilirubin above 2 mg/dL → yellowish discoloration of the skin, sclera and mucous membranes.
Unconjugated hyperbilirubinemia types
Hemolytic jaundice
Physiological neonatal jaundice
Crigler-Najjar Syndrome type 1
Crigler-Najjar Syndrome type 2
Gilbert syndrome
Conjugated Hyperbilirubinemia types
Obstructive Jaundice
Dubin-Johnson And Rotor Syndrome
Difference between Conjugated and unconjugated hyperbilirubinemia
Liver to can’t conjugate bilirubin → unconjugated hyperbilirubinemia.
Liver cells swell → blocking the biliary canaliculi → conjugated hyperbilirubinemia.
In both conjugated and unconjugated hyperbilirubinemia
↓ Stereobilin in the feces (faint stool).
Both urobilinogen and bilirubin appear in the urine → dark brown in color.
Serum levels of the enzymes ALT and AST and γ-GT are elevated Due to liver cell damage.
Hemolytic jaundice
↑ Stercobilin in feces → dark brown stool.
↑ Urobilinogen in urine.
Causes of hemolytic jaundice
Abnormal hemoglobin
Abnormal cell membrane
Red cell enzyme deficiency (G6PD and pyruvate kinase)
Red cell antibodies
Infections; malaria
Physiologic neonatal jaundice
↑ hemolysis & immature UDP-glucuronyltransferase Enzyme.
Physiologic neonatal jaundice treatment
Exposure to blue fluorescent light (phototherapy)
Phenobarbital
Crigler-Najjar Syndrome, type I
Total deficiency of UDP-glucuronyltransferase
Serum unconjugated bilirubin exceeds 20 mg/dL → kernicterus.
fatal within one year as phenobarbital therapy doesn’t work
Crigler-Najjar Syndrome, type II
Partial deficiency of UDP-glucuronyltransferase.
Serum unconjugated bilirubin does not exceed 20 mg/dL.
Patients respond to large doses of phenobarbital therapy
Gilbert Syndrome:
Benign condition caused by a defect in the uptake of unconjugated bilirubin by the liver.
Obstructive Jaundice causes
obstruction of the biliary passages: gallstones, pancreatic cancer or inflammation of pancreas
↑ Serum bilirubin, mainly conjugated bilirubin.
feces clay colored
Dark brown urine with bile salts
Dubin-Johnson and Rotor Syndrome
Cause: defect in the ability of hepatocytes to secrete conjugated bilirubin into bile.
Hepatic lobules contain dark pigment
Normal fasting glucose blood levels are:
2 h post prandial are
70-100 mg/dL
less than 140 mg/dL
Sources of blood glucose:
During fasting: glycogenolysis and gluconeogenesis
During feeding: carboydrates
GIT (Gastrointestinal tract): prevents sudden ↑ in blood glucose through
Slow rate of evacuation: Allows good time for gradual absorption
Secretion of gastric inhibitory peptide
Liver: Main glucose homoeostat during hyperglycemia and hypoglycemia?
During hyperglycemia “↑insulin”: Liver ↓ blood glucose by:
↑ Uptake of glucose (GLUT2)
↑ Utilization of glucose (oxidation and storage)
During hyperglycemia “↓insulin” (fasting):
Liver ↑ blood glucose by:
↑ glycogenolysis & gluconeogenesis
Kidney regulation of insulin
During hyperglycemia: Glycogenesis & excretion of glucose (if exceeds 180mg/dl) .
During fasting: ↑ gluconeogenesis
Skeletal muscles regulation of glucose
During hyperglycemia: ↑ Uptake by Glut 4 ↑ storage (Glycogen)
During hypoglycemia:↓ Uptake & ↑ catabolism of muscle & release of amino acids
Adipose tissues regulation of blood sugar
During hyperglycemia:-↑ Uptake by Glut 4 (insulin dependent)& ↑storage (Lipogenesis)
During hypoglycemia: ↓ Uptake& ↓catabolism of adipose tissue & release of FA → FA oxidation
Insulin, where its from and what it does?
Secreted by the β - cells of pancreatic islets in response to hyperglycemia.
↑ Glucose uptake by GLUT- 4, by (heart, skeletal muscles & adipose tissues)
↑ utilization of glucose: Storage and oxidation
↓ glycogenolysis & gluconeogenesis
Anti-insulins and what they do
Glucagon: ↑ Glycogenolysis & gluconeogenesis & ↓ Glycolysis, glycogenesis
Adrenaline: ↑ Glycogenolysis, gluconeogenesis, lipolysis & ↓Glycolysis, glycogenesis
Glucocorticoids: ↑Gluconeogenesis, lipolysis&↓ uptake, glycolysis
GH: ↑ Gluconeogenesis, lipolysis&↓ uptake, glycolysis
Hypoglycemia defined as
↓ Blood glucose less than 70 mg/dl
Blood glucose below 45 is fatal
Symptoms of hypoglycemia
Early signs and symptoms of mild hypoglycemia usually include:
Hunger , tremors , sweating , accelerated heart rate and tingling lips
Late signs and symptoms when more severe hypoglycemia:
Concentration problems , confusion and loss of consciousness .
Causes of fasting hypoglycemia (more than 6 hours)
Insulin overdose
Decrease anti-insulins
Renal failure
Alcohol abuse
anorexia nervosa
genetic diseases
. Hyperglycemic glucosuria: causes
Diabetes mellitus: The commonest cause
adrenaline glucosuria
Normoglycemic glucosuria causes
renal glucosuria
Renal failure
Pregnancy
Importance of ketone bodies
Main source of energy during starvation
KB can be oxidized easier than FA during fasting
Ketogenesis represents a preparatory step performed by liver,
Causes of ketosis
Starvation, low carbohydrates and high fat in diet
Severe uncontrolled diabetes mellitus
Prolonged use of anti-insulin
Sever muscular exercise
Effects of ketosis
↑ Production of acetoacetate & β-hydroxybutyrate → ketoacidosis → loss of buffer cations → electrolyte imbalance
Ketogenic substances
FA, ketogenic AA, Anti-insulin
Anti-ketogenic substances
Carbohydrates, glycerol, insulin and glucogenic AA.
Management of ketosis
Hospitalization to Management of the cause
IV glucose and insulin
Bicarbonate to correct acidosis
IV K+
Fluids/electrolytes
Where is insulin made and store?
Beta cells of the islets of Langerhans
Beta cells release insulin in _____phases
two
First phase of release is in response to
high blood glucose
Second phase is a
sustained slow release of insulin
Substances that stimulate release of insulin
Arginine and leucine, acetylcholine
Measurement of which of the following is a good index of insulin secretion
C-peptide
Insulin half life is
3-5 minutes
Insulin catabolized through two systems
Hepatic glutathione
Insulin specific protease
Action of insulin is terminated by
de-phosphorylation of the receptor.
followed by internalization of insulin and its receptor.
Degradation in lysosomes
Reason of Diabetes type 2 mellitus
resistance of insulin
Tissues affected in insulin resistance
In fat cells : ↑ lipolysis → release of free fatty acids in the blood .
In muscle cells : ↓ glucose uptake →↓local glycogen.
In liver cells : ↓ glycogen synthesis & ↑ glucose production by gluconeogenesis .
Causes of insulin resistance
Obesity: main cause
Anti-insulin antibodies
Hereditary mutations to receptors
Acquired causes: diet, aging, physical inactivity
IF fasting insulinto glucose ratio greater than 4.5, you have
insulin resistance
Type 1 diabetes
10% incidence, usually for under 20 years of age, some blood insulin left
Type 2 diabetes
90% incidence, Above 40, absent insulin (due to insulin resistance), treatment is diet control, exercise oral hypoglycemic drugs.
Roles of vitamin D
Increases pancreatic insulin release which reduces insulin resistance
Gestational Diabetes mellitus
Placenta causes low sensitivity to insulin, can go away after 4 weeks but usually turns into diabetes 2
Specific types of diabetes mellitus
Over production of anti-insulin
Surgical excision of pancreas
Hemochromatosis
Drug induced diabetes
Metabolic changes of carbs in diabetes mellitus
decrease glucose uptakes
causes polydepsia
Polydepsia
extreme thirst
lipid metabolism changes due to diabetes mellitus
decrease lipogenesis which causes weight loss
Increase uptake of FA
Changes in protein metabolism due to diabetes
decrease protein synthesis
Increase sensitivity to infection and slow healing time
Changes in mineral metabolism diabetes mellitus
Polyuria
Polyuria
pissing more than usual
Macro vascular and microvascular diseases form Diabetes mellitus complications
Macro: stroke and coronary artery disease
Micro: Damage to retina
Hyperglycemic coma types
Diabetic ketoacidosis
Hyperosmaolar hyperglycemic non-ketotic coma
Diabetic ketoacidosis
more common in type 1 diabetes, cause of coma is ketoacidosis
Manifestation of Diabetic ketoacidosis
Acetone smell on breath, deep rapid respirations, rapid pulse and low blood pressure
Treatment of Diabetic ketone acidosis
IV infusion of K and HCOs
IV infusion of glucose and insulin
Hyperosmolar Hyperglycemic Non - Ketotic Coma:
Occurs in elderly patients with uncontrolled type two diabetes, can be caused from kidney failure
Symptoms of Hyperosmolar Hyperglycemic Non - Ketotic Coma:
Severe dehydration
Severe hyperglycemia
Decreased concuisness and seizures
Treatment of Hyperosmolar Hyperglycemic Non - Ketotic Coma:
IV infusion of K, fluids, glucose and insulin
Hypoglycemia coma
occurs in any type of diabetes, caused by overtreatment of diabetes. Coma from low blood glucose
Hypoglycemia coma symptoms and treatment
Symptoms: headache, poor concntration, tremors of hand, sweating, strong rapid pulse
Treatment; give glucose
Diabetes mellitus symptoms
Polyuria , polydipsia , polyphagia , prolonged time of wound healing , and weight loss .
Diabetes mellitus diagnostic tests
Fasting and 2 hour post glucose plasma glucose levels
Oral glucose tolerance test
Measurement of Glycated-Hb (HbA1c)
Measurement of plasma fructosamine
Test for microalbuminuria
Normal, impaired glucose tolerance and diabetes level
FPG (mg/dL) normal<100 IGT 100-125 Diabetes > 126
2hour PG Normal <140, IGT 140-200 Diabetes >200
OGIT test steps
After 8 hours of fasting blood and urine samples taken
Patient takes oral 75g of glucose in water
4 samples are obtained at each half hour)
