Salvage pathway of purine nucleotide synthesis
Q. 1 The purines salvage pathway is for:
| A | Hypoxanthine and Xanthine | |
| B | Hypoxanthine andAdenine | |
| C | Adenine and Guanine | |
| D | Xanthine and Guanine |
Q. 1 The purines salvage pathway is for:
| A | Hypoxanthine and Xanthine | |
| B | Hypoxanthine andAdenine | |
| C | Adenine and Guanine | |
| D | Xanthine and Guanine |
Ans.
B
B
B
B
B
Explanation:Hypoxanthine andAdenine
Q. 2
Which among the following are the substrates needed for purine salvage pathway?
| A |
Hypoxanthine and Xanthine |
|
| B |
Hypoxanthine and Adenine |
|
| C |
Adenine and Guanine |
|
| D |
Xanthine and Guanine |
Q. 2
Which among the following are the substrates needed for purine salvage pathway?
| A |
Hypoxanthine and Xanthine |
|
| B |
Hypoxanthine and Adenine |
|
| C |
Adenine and Guanine |
|
| D |
Xanthine and Guanine |
Ans.
Explanation:
Conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides involves “salvage reactions”.
The more important mechanism involves phosphoribosylation by PRPP of a free purine (Pu) to form a purine 5′-mononucleotide (Pu-RP). Phosphoryl transfer from ATP, catalyzed by adenosine- and hypoxanthine-phosphoribosyl transferases, converts adenine, hypoxanthine, and guanine to their mononucleotides.
A second salvage mechanism involves phosphoryl transfer from ATP to a purine ribonucleoside. Phosphorylation of the purine nucleotides, catalyzed by adenosine kinase, converts adenosine and deoxyadenosine to AMP and dAMP. Similarly, deoxycytidine kinase phosphorylates deoxycytidine and 2′-deoxyguanosine, forming dCMP and dGMP.
Ref: Rodwell V.W. (2011). Chapter 33. Metabolism of Purine & Pyrimidine Nucleotides. In D.A. Bender, K.M. Botham, P.A. Weil, P.J. Kennelly, R.K. Murray, V.W. Rodwell (Eds), Harper’s Illustrated Biochemistry, 29e.
Q. 3
Salvage pathway of purine biosynthesis is important for ‑
| A |
Liver |
|
| B |
RBCs |
|
| C |
Kidney |
|
| D |
Lung |
Q. 3
Salvage pathway of purine biosynthesis is important for ‑
| A |
Liver |
|
| B |
RBCs |
|
| C |
Kidney |
|
| D |
Lung |
Ans.
Explanation:
Purine nucleotide synthesis occurs by two pathways :
1.De novo synthesis
2.Salvage pathway
Liver is the major site of purine nucleotide biosynthesis (de novo).
Certain tissues cannot synthesize purine nucleotides by de novo patyway, e g. brain, erythrocytes and polymorphonuclear leukocytes.
These are dependent on salvage pathway for synthesis of purine nucleotides by using exogenous purines, which are formed by degradation of purine nucleotides synthesized in liver.
Q. 4 Salvage pathway of purine nucleotide synthesis are used by all except ‑
| A |
Brain |
|
| B |
Liver |
|
| C |
RBC |
|
| D |
Leukocytes |
Q. 4
Salvage pathway of purine nucleotide synthesis are used by all except ‑
| A |
Brain |
|
| B |
Liver |
|
| C |
RBC |
|
| D |
Leukocytes |
Ans.
Explanation:
Purine nucleotide synthesis occurs by two pathways :-
1.De novo synthesis
2.Salvage pathway
Liver is the major site of purine nucleotide biosynthesis (de novo).
Certain tissues cannot synthesize purine nucleotides by de novo patyway, e g. brain, erythrocytes and polymorphonuclear leukocytes.
These are dependent on salvage pathway for synthesis of purine nucleotides by using exogenous purines, which are formed by degradation of purine nucleotides synthesized in liver.
Q. 5 Salvage pathway of purine nucleotide synthesis are used by all except ‑
| A |
Brain |
|
| B |
Liver |
|
| C |
RBC |
|
| D |
Leukocytes |
Q. 5
Salvage pathway of purine nucleotide synthesis are used by all except ‑
| A |
Brain |
|
| B |
Liver |
|
| C |
RBC |
|
| D |
Leukocytes |
Ans.
Explanation:
Ans. is ‘b’ i.e., Liver
- Purine nucleotide synthesis occurs by two pathways :
- De novo synthesis
- Salvage pathway
- Liver is the major site of purine nucleotide biosynthesis (de novo).
- Certain tissues cannot synthesize purine nucleotides by de novo patyway, e g. brain, erythrocytes and polymorphonuclear leukocytes.
- These are dependent on salvage pathway for synthesis of purine nucleotides by using exogenous purines, which are formed by degradation of purine nucleotides synthesized in liver.
Factors affecting enzyme activity
Factors affecting enzyme activity
Introduction:
- Enzyme activity can be affected by a variety of factors, such as temperature, pH, and concentration.
- Decrease in activation energy leads to an increase in enzyme activity.
- Enzymes work best within specific temperature and pH ranges, and sub-optimal conditions can cause an enzyme to lose its ability to bind to a substrate.
- One unit enzyme activity is defined as the amount causing transformation of 1.0 micro mole of substrate per minute(Micromole / minute) at 25o C
- Temperature: Raising temperature generally speeds up a reaction, and lowering temperature slows down a reaction. However, extreme high temperatures can cause an enzyme to lose its shape (denature) and stop working.
- Bell shaped curve is obtained by plotting temperature against velocity of reaction
- The optimum temperature for most human enzymes is between 35 and 400C.
- The temperature coefiicient (Q10) is the factor by which the rate of a biologic process increases for a 10’C increase in temperature.
- pH: Each enzyme has an optimum pH range. Changing the pH outside of this range will slow enzyme activity. Extreme pH values can cause enzymes to denature.
- Enzyme concentration: Increasing enzyme concentration will speed up the reaction, as long as there is substrate available to bind to. Once all of the substrate is bound, the reaction will no longer speed up, since there will be nothing for additional enzymes to bind to.
- Substrate concentration: Increasing substrate concentration also increases the rate of reaction to a certain point. Once all of the enzymes have bound, any substrate increase will have no effect on the rate of reaction, as the available enzymes will be saturated and working at their maximum rate
Exam Important
- One unit enzyme activity is defined as the amount causing transformation of 1.0 micro mole of substrate per minute at 25o C.\
- Decrease in activation energy leads to an increase in enzyme activity
Don’t Forget to Solve all the previous Year Question asked on Factors affecting enzyme activity
RNA Polymerase (RNAP)
RNA Polymerase (RNAP)
- RNA polymerase (ribonucleic acid polymerase), both abbreviated RNAP or RNApol, official name DNA-directed RNA polymerase, is a member of a family of enzymes that are essential to life.
- RNAP locally opens the double-stranded DNA (usually about four turns of the double helix) so that one strand of the exposed nucleotides can be used as a template for the synthesis of RNA, a process called transcription.
- They are DNA dependent RNA Polymerase.
- No primer is needed in RNAP
- No proofreading activity in RNAP.
- Prokaryotic RNA polymerase is of only one type.
- There are three types of Eukaryotic RNA Polymerase, RNA Polymerase I, II and III.
- Bacterial RNAP enzyme contains two alpha, two beta subunits, one omega subunit, and one sigma factor and two zinc molecules.
- RNAP type II or Bis the main enzyme synthesizing mRNAs. It is inhibited by alpha-amanitin.
- Phosphorylation activates RNAP II.
- RNAP type I or A is responsible for the synthesis of Rrna (ribosomal); it is not inhibited by amanitin.
- RNAP type III or C is responsible for the production of tRNA; it is moderately sensitive to amanitin.
- RNAP type III catalyzes the synthesis of flRNA, small nuclear RNA (sz-RNA) and miRNA
- Sigma factor enables the holoenzyme (RNA polymerase) to recognize and bind to The promoter sequence. Different sigma (σ) factors recognize the different group of genes
- Alpha-amanitin inhibits RNA polymerase II of eukaryotes.
- Rifampicin inhibits b-subunit of RNA polymerase.
- RNA polymerase (holoenzyme)= core enzyme+ sigma subunit
- RNA polymerase holoenzyme requires:
- A template of ds/ss DNA
- Four ribonucleotide triphosphates GTP, UTP, ATP, CTP
- Mg” or Mn”
- RNA primers are required for DNA replication, not for transcription.
- RNA polymerase is The major enzyme involved in transcription.
Exam Important
- They are DNA dependent RNA Polymerase.
- No primer is needed in RNAP
- No proofreading activity in RNAP.
- Prokaryotic RNA polymerase is of only one type.
- There are three types of Eukaryotic RNA Polymerase, RNA Polymerase I, II and III.
- Bacterial RNAP enzyme contains two alpha, two beta subunits, one omega subunit, and one sigma factor and two zinc molecules.
- RNAP type II or Bis the main enzyme synthesizing mRNAs. It is inhibited by alpha-amanitin.
- Phosphorylation activates RNAP II.
- RNAP type I or A is responsible for the synthesis of Rrna (ribosomal); it is not inhibited by amanitin.
- RNAP type III or C is responsible for the production of tRNA; it is moderately sensitive to amanitin
- The sigma (σ) subunit of prokaryotic RNA polymerase Specifically recognizes the promoter site.
- Alpha-amanitin inhibits RNA polymerase II of eukaryotes.
- Rifampicin inhibits b-subunit of RNA polymerase.
- RNA polymerase (holoenzyme)= core enzyme+ sigma subunit
Don’t Forget to Solve all the previous Year Question asked on RNA Polymerase (RNAP)
POST TRANSCRIPTIONAL MODIFICATION OF RNA
- The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are post-transcriptionally modified by cleavage of the original transcripts by ribonucleases.
- In Prokaryotes mRNA are is not subjected to post-transcriptional processing.
- In eukaryotes, The collection of all the primary transcripts synthesized in the nucleus by RNA polymerase II is known as heterogeneous nuclear RNA (hnRNA).
- The primary transcripts are extensively modified in the nucleus after transcription.
These modifications usually include:
A) 5′ “Capping”:
- The first of the processing reactions for pre-mRNA, The cap is a 7-methylguanosine attached “backward” to the 5′-terminal end of the mRNA, forming an unusual 5’→5′ triphosphate linkage.
- Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase.
B) Addition of a poly-A tail:
- Poly A tail is added to the 3′ end of the hnRNA
- Polyadenylate Polymerase is the enzyme
- Takes place in the nucleus
- Poly (A) tail translates into Polylysine
- Length of Poly-A tail is up to 200 Adenine bases
- These tails help to stabilize the mRNA (by protecting from 3r-exonuclease), facilitate exit from the nucleus, and aid in translation.
- Some rRNAs do not have a poly-A tail, e.g. mRNAs of histones and some interferons.
C) Removal of introns (splicing):-
- Intron: an Intervening sequence that does not code for the amino acid
- Introns are exised by RNA splicing by Sn-RNAs/Snurp.
- Exon: Amino acid coding sequence Molecular machinery that carries out splicing is called Spliceosome
- The process by which introns are excised and exons are linked to form functional mRNA are called splicing, Thus mature mRNA does not contain introns.
- snRNA combines with proteins to form small nuclear ribonucleoprotein particles (snRNPs or snurps)
- It is the snRNA component of snurps that catalyzes splicing
- Snurps are U, U4, Us and Uu. Defective splicing (splicing mutation) is the most common cause of β-thalassemia.
- only about 1.5% of human DNA has coding sequence (exons), remaining is non-coding(introns).
- Two important gene-silencing RNAs are :- (i) Micro RNAs, and (ii) interfering RNAs (siRNAs).
D) Alternate splicing :-
- The hn-RNA molecules from some genes can be spliced in alternative way in different tissues.
- Thus two or more different mRNA (and therefore 2 or more proteins) can be synthesized from same hnRNA.
Exam Important
- Poly (A) tail translates into Polylysine
- Length of Poly-A tail is up to 200 Adenine bases
- These tails help to stabilize the mRNA (by protecting from 3r-exonuclease), facilitate exit from the nucleus, and aid in translation.
- Introns are exised by RNA splicing by Sn-RNAs/Snurp.
- Exon: Amino acid coding sequence Molecular machinery that carries out splicing is called Spliceosome
- The process by which introns are excised and exons are linked to form functional mRNA are called splicing, Thus mature mRNA does not contain introns.
- snRNA combines with proteins to form small nuclear ribonucleoprotein particles (snRNPs or snurps)
- It is the snRNA component of snurps that catalyzes splicing
- Snurps are U, U4, Us and Uu. Defective splicing (splicing mutation) is the most common cause of β-thalassemia.
- only about 1.5% of human DNA has coding sequence (exons), remaining is non-coding(introns).
- Two important gene-silencing RNAs are :- (i) Micro RNAs, and (ii) Small interfering RNAs (siRNAs
Don’t Forget to Solve all the previous Year Question asked on POST TRANSCRIPTIONAL MODIFICATION OF RNA
Disorders of heme biosynthesis
Disorders of heme biosynthesis
- porphyrias are a group of inborn errors (or occasionally acquired) defects of heme biosynthesis.
- This results in in the accumulation and increased excre-tion of porphyrins or porphyrin precursors.
- The mutations that cause the porphyrias are heterogenous.
- Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway.
- “Porphyria” refers to the purple color caused by pigment-like porphyrins in the urine of some patients with defects in heme synthesis.
Clinical manifestations:
- The porphyrias are classified as erythropoietic or hepatic, depending on the enzyme deficiency in bone marrow or in the liver.
- Most of the porphyrias are inherited autosomal dominant `EXCEPT
- ALAD enzyme Deficiency [ADP]
- Gongenital Erythropoeitic Porphyria [CEp]
- Erythropoetic Protoporphyria [EPP]
- X Linked Protoporphyria [XLp]
- Accumulation of ALA and PBG causes Neuropsychiatric and accumulation of porphyrin causes Photosensitivity.
Erythropoietic porphyrias
- usually present with cutaneous photosensitivity. Most common Porphyria in children is Erythropoietic protoporphyria (Epp).
- This disease is caused by to a deficiency in ferrochelatase. Protoporphyrin accumulates in erythrocytes, bone marrow, and plasma.•Patients are photosensitive.
PORPHYRIA CUTANEA TARDA:
- This disease is caused by a deficiency in uroporphyrinogen decarboxylase. Uroporphyrin accumulates in the urine.
- It is the most common porphyria. Patients are photosensitive.
- Uroporphyrinogen-I synthase (also called PBG dearninase or HMB synthase) causes Acute intermittent Porphyria
- Uroporphyrtnogen III Synthase causes Congenital erythropoietic.
- Ferrochelatase (Heme synthase) causes Protoporphyria.
VARIEGATE PORPHYRIA
- An acute disease caused by a deficiency in protoporphyrinogen oxidase.
- Protoporphyrinogen IX and other intermediates prior to the block accumulate in the urine.
- Patients are photosensitive.
HEREDITARY COPROPORPHYRIA:
- An acute disease caused by a deficiency in coproporphyrinogen oxidase.Coproporphyrinogen III and other intermediates prior to the block accumulate in the urine.
- Patients are photosensitive.
ACUTE INTERMITTENT PORPHYRIA:
- An acute disease caused by a deficiency in hydroxymethylbilane synthase1.Porphobilinogen and δ-amino-levulinic acid accumulate in the urine.
- Urine darkens on exposure to light and air. Patients are NOT photosensitive.
- Porphyrias leading to accumulation of ALA and porphobilinogen, such as acute intermittent por phyria, cause abdominal pain and neuro psychiatric disturbances.
- In the liver, heme normally functions as a repressor of the gene for ALAS1. Therefore, the absence of this end product results in an increase in the synthesis of ALA synthase1(derepression).
Exam Important
Erythropoietic porphyrias
- usually present with cutaneous photosensitivity. Most common Porphyria in children is Erythropoietic protoporphyria (Epp).
- This disease is caused by to a deficiency in ferrochelatase. Protoporphyrin accumulates in erythrocytes, bone marrow, and plasma.•Patients are photosensitive.
PORPHYRIA CUTANEA TARDA:
- This disease is caused by a deficiency in uroporphyrinogen decarboxylase. Uroporphyrin accumulates in the urine.common porphyria. Patients are photosensitive.
- Uroporphyrinogen-I synthase (also called PBG dearninase or HMB synthase) causes Acute intermittent Porphyria
- Uroporphyrtnogen III Synthase causes Congenital erythropoietic.
- Ferrochelatase (Heme synthase) causes Protoporphyria.
VARIEGATE PORPHYRIA:
- An acute disease caused by a deficiency in protoporphyrinogen oxidase.Protoporphyrinogen IX and other intermediates prior to the block accumulate in the urine.Patients are photosensitive.
ACUTE INTERMITTENT PORPHYRIA:
- An acute disease caused by a deficiency in hydroxymethylbilane synthase1.Porphobilinogen and δ-amino-levulinic acid accumulate in the urine. Urine darkens on exposure to light and air. Patients are NOT photosensitive.
Don’t Forget to Solve all the previous Year Question asked on Disorders of heme biosynthesis
xenobiotics
- A xenobiotic is a compound that is foreign to the body
- may be accidentally ingested or taken as drugs or compounds produced in the body by bacterial metabolism (Greek, xenos = strange).
- They are metabolized, a process called biotransformation(detoxification).
- Metabolism of xenobiotics in two phases (phase I and phase II).
- The cytochrome P450 enzymes are involved in the biotransformation reactions, the most important enzyme present on smooth endoplasmic reticullum.
- They are heme containing enzymes, localized in the endoplasmic reticulum of liver.
- so, Liver is the major site for metabolism of xenobiotics.
- Cytochrome P450 contains phosphatidyl choline.
- Phase one is the alteration of the foreign molecule, so as to add a functional group.
- The products of metabolic transformations are either excreted directly or undergo further meta-bolism by phase two reactions.
Phase | (non-synthetic) reactions
1. Oxidative Reactions:
- metabolism of compounds by oxidation (including hydroxylation), reduction, hydrolysis, cyclization and decyclization.
- The oxidation and detoxification of alcohol is also an important function of the liver.
- the alcohol dehydrogenase is an NAD linked enzyme, which is located in the cytosol. Aldehyde dehydrogenase is an NAD+dependent mitochondrial enzyme.
2. Reduction Reactions:
- compounds which are reduced and detoxified by the liver are nitro compounds, These are reduced to their amines, while aldehydes or ketones are reduced to alcohols.
3. Hydrolysis:
- addition of water splits the toxicant into two fragments or smaller molecules.,
- Esters, amines,hydrazines, amides, glycosidic bonds and carba-mates are generally biotransformed by hydrolysis.
PHASE TWO REACTIONS; CONJUGATIONS:
- a new metabolite from phase 1 contains a reactive chemical group, e.g. hydroxyl (-OH),amino (-NH2), and carboxyl (-COOH).
- Glucuronide conjugation is the most common Phase two reactions. Bilirubin is a good example for a compound conjugated and excreted as its glucuronide.
- Formation of bilirubin diglucuronide is a normal metabolic reaction for detoxification of bilirubin by phase 2 reactions.
- The glucuronic acid is added to xenobiotics by UDP-glucuronyl transferases, present in the endo-plasmic reticulum.
- sulfation decreases the toxicity of xenobiotics, for eg Phenolic and alcoholic compounds are conju-gated with sulfate.(steroids and indole compounds.)
- cysteine is derived from glutathione, which is the active conjugating agent,
- For example, catechol-O-methyl transferase converts epinephrine to metanephrine. Pyridine is excreted as N-methyl pyridine.
- Mercapto ethanol is excreted as 5-methyl mercapto ethanol.
Exam Important
- metabolism of compounds by oxidation (including hydroxylation), reduction, hydrolysis, cyclization and decyclization.
- The oxidation and detoxification of alcohol is also an important function of the liver.
- Bilirubin is a good example for a compound conjugated and excreted as its glucuronide.
- Formation of bilirubin diglucuronide is a normal metabolic reaction for detoxification of bilirubin by phase 2 reactions.
- The glucuronic acid is added to xenobiotics by UDP-glucuronyl transferases, present in the endo-plasmic reticulum.
- catechol-O-methyl transferase converts epinephrine to metanephrine. Pyridine is excreted as N-methyl pyridine.
Don’t Forget to Solve all the previous Year Question asked on xenobiotics
RNA editing
RNA editing
- RNA editing is a process through which the nucleotide sequence specified in the genomic template is modified to produce a different nucleotide sequence in the transcript.
- Even after mRNA has been fully processed, it may undergo additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing.
- There are two generic classes of RNA editing in nuclei, involving enzymatic deamination of either C-to-U or A-to-T nucleotides.
- Current estimate suggest that 0.01% of mRNA is edited in this fashion
- The best characterized example of C-to-U RNA editing is that of apolipoprotein B (apoB), which is mediated by a holoenzyme that contains a minimal core composed of an RNA-specific cytidine deaminase apobec-1, and its cofactor apobec-1 complementation factor (ACF).
- (apo) B—an essential protein component of chylomi-crons and very low density lipoproteins (VLDL).
- the C residue in the codon (CAA) for glutamine is deaminated to U, changing the sense codon to a nonsense or stop codon (UAA).
- This results in a shorter protein (apo B-48, representing 48% of the message) being made in the intestine (and incorporated into chylomicrons) than is made in the liver (apo B-100, full-length, incorporated into VLDL)
- the linear relationship between the coding sequence in DNA, the mRNA sequence, and the protein sequence is altered.
- It is an exception to Central Dogma of molecular genetics.
- The single apoB gene is transcribed into an mRNA that directs the synthesis of a 512kDa proteiry apo 8100 with 4536 amino acid residues.
- A cytidine deaminase converts a CAA codon in the mRNA to UAA at a single specific site
- Other examples of RNA editing o Glutamate Receptor (Glutamine changed toArginine) Trypanosome mitochondrial DNA.
Exam Important
- Even after mRNA has been fully processed, it may undergo additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing.
- There are two generic classes of RNA editing in nuclei, involving enzymatic deamination of either C-to-U or A-to-T nucleotides.
- Current estimate suggest that 0.01% of mRNA is edited in this fashion
- The best characterized example of C-to-U RNA editing is that of apolipoprotein B (apoB), which is mediated by a holoenzyme that contains a minimal core composed of an RNA-specific cytidine deaminase apobec-1, and its cofactor apobec-1 complementation factor (ACF)
- It is an exception to Central Dogma of molecular genetics.
- The single apoB gene is transcribed into an mRNA that directs the synthesis of a 512kDa proteiry apo 8100 with 4536 amino acid residues.
- A cytidine deaminase converts a CAA codon in the mRNA to UAA at a single specific site
- Other examples of RNA editing o Glutamate Receptor (Glutamine changed toArginine) Trypanosome mitochondrial DNA.
Don’t Forget to Solve all the previous Year Question asked on RNA editing
Transport of Bilirubin
Transport of Bilirubin
- Unconjugated bilirubin (UCB), the principal mammalian bile pigment, is the end intravascular product of heme catabolism.
- Bilirubin is toxic to tissues; therefore, it is transported in the blood bound to albumin.
- Bilirubin in the bloodstream is usually in a free, or unconjugated, state.
- Bilirubin leaves the site of production in the reticuloendothelial system and is transported in plasma bound to albumin .
- The capacity of serum albumin to bind bilirubin is known as the binding capacity, and the strength of the bilirubin-albumin bond is referred to as the binding affinity.
- In 100 ml of plasma, approximately 25 mg of bilirubin can be tightly bound to albumin at its high-affinity site.
- The conjugation of bilirubin is catalyzed by a specific enzyme called glucuronyltransferase.
- proteins Ligandin (a member of the family of glutathione S-transferases) and Protein Y help in intracellular binding
- In the liver the bilirubin is removed from albumin, Taken up at the sinusoidal surface of the hepatocytes by a carrier-mediated saturable system (facilitated transport system). It is then concentrated to about 1,000 times the strength found in blood plasma.
- Much bilirubin leaves the liver and passes to the gallbladder, where it is further concentrated and mixed with the other constituents of bile.
- conjugated bilirubin passes from the gallbladder or liver into the intestine. There, it is reduced by bacteria to mesobilirubinogen and urobilinogen.
- Some urobilinogen is reabsorbed back into the blood; the rest goes back to the liver or is excreted from the body in urine and fecal matter. In humans, bilirubin is believed to be unconjugated until it reaches the liver.
- 80-90% of the urobilinogen stercobilinogen and stercobilin and excreted through feces.
- 70-20 % enterohepatic circulation reaches the liver, This is called enterohepatic urobilinogen cycle.
- A small fraction < 3 mg/dl escape hepatic uptake, filters across renal glomerulus and is excreted through urine.
Don’t Forget to Solve all the previous Year Question asked on Transport of Bilirubin
HYPERBILIRUBINEMIAS
HYPERBILIRUBINEMIAS
Depending on the nature of the bilirubin elevated, the condition may be grouped into:-
→ conjugated or Unconjugated hyperbilirubinemia.
A → Congenital Hyperbilirubinemias
They result from abnormal uptake, conjugation or excretion of bilirubin due to inherited defects such as
Crigler-Najjar Syndrome
- The defect is due to conjugation, there is severe deficiency of UDP glucuronyl transferase. The disease is often fatal and the children die before the age of 2.
- Unconjugated bilirubin level increases to more than 20mg/dl, and hence kernicterus results.
- Bilirubin level in blood exceeds 20 mg/dl in Crigler-Najjar syndrome Type 1 and does not exceed 20 mg/dl in Crigler-Najjar syndrome Type 2.
Gilbert’s Disease:
- It is inherited as an autosomal dominant trait.
- The defect is in the uptake of bilirubin by the liver.
- Bilirubin level is usually around 3 mg/dl, and patient is asymptomatic, except for the presence of mild jaundice.
Dubin-Johnson Syndrome:
- It is an autosomal recessive trait leading to defective excretion of conjugated bilirubin.
- The disease results from the defective ATP-dependent organic anion transportin bile canaliculi.
- There is a mutation in the MRP-2 protein which is responsible for transport of conjugated bilirubin into bile.
- The bilirubin gets deposited in the liver and the liver appears black. The condition is referred to as Black liver jaundice.
Rotor Syndrome
- exact defect is not identified. Bilirubin excretion is defective, but there is no staining of the liver. It is an autosomal recessive condition.
B→Acquired Hyperbilirubinemias:
Physiological Jaundice:
- Called as neonatal hyperbilirubinemia.
- Transient hyperbilirubinemia is due to an accelerated rate of destruction of RBCs and also because of the immature hepatic system of conjugation of bilirubin.
Breast milk jaundice
- In some breast-fed infants, prolongation of the jaundice has been attributed to high level of an estrogen derivative in maternal blood, which is excreted through the milk.
Conjugated Hyperbilirubinemias
- Dubin Johnson’s syndrome
- Rotor syndrome
- Benign Recurrent intrahepatic Cholestatsis (BRIC)
- Progressive Familial intrahepatic Cholestatsis (FIC)
Exam Important
- Unconjugated Hyperbilirubinemia is associated with > 85% indirect bilrubin or less than 15% of direct bilirubin.
- Hemolytic disorders and increased hemoglobin destruction cause unconjugated or indirect hyperbilirubinemia.
- Biliary atresia and neonatal hepatitis lead to conjugated hyperbilirubinemia.
- Uncojugated hyperbilirubinemias is-Gilbert’s disease, Crigler-Najjarsyndrome.
- Conjugated Hyperbilirubinemias-A. Dubin Johnson’s syndrome, B. Rotor syndrome, c.Benign Recurrent intrahepatic Cholestatsis (BRIC), d.Progressive Familial intrahepatic Cholestatsis (FIC)
Don’t Forget to Solve all the previous Year Question asked on HYPERBILIRUBINEMIAS
POLYMERASE CHAIN REACTION (PCR)
POLYMERASE CHAIN REACTION (PCR)
- Karry Mullis invented this ingenious method in 1989, & awarded Nobel prize in 1993.
- millions of copies of a particular sequence of DNA can be produced within a few hours.
- PCR is a method of enzymatic amplification of a target sequence of DNA.
- It is sensitive, selective (specific) and extremely rapidmeans of amplifiing any desired sequence of double stranded DNA.
- DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq) because it is thermostable.
- Primers are amplified to produce desired sequence of DNA.
- Two DNA primers of about 20-30 nucleotides with complementary sequence of the flanking region can be synthesized.
Steps in PCR:
- PCR uses DNA polyme rase.
- Each cycle doubles the amout of DNA in the sample, leading to exponential increase.
- Thus amplification after’n’number of cycle in (2)n, twenty cycles provide an amplification of 106 (million) and 30 cycles of 10, (billion).
Step 1: Separation (Denaturation):DNA strands are separated (melted) by heating at 95°C for 15 seconds to 2 minutes
Step 2: Priming (Annealing): The primers are annealed by cooling to 50°C for 0.5 to 2 minutes. The primers hybridise with their complementary single stranded DNA produced in the first step.
Step 3: Polymerization: New DNA strands are synthesized by Taq polymerase.This enzyme is derived from bacteria Thermus acquaticus that are found in hot springs. Therefore the enzyme is not denatured at high temperature. The polymerase reaction is allowed to take place at 72°C for 30 seconds in presence of dNTPs (all four deoxy ribonucleotide triphosphates) and DNA polymerase.
- As Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.
- Both strands of DNA are now duplicated
- The steps of 1,2 and 3 are repeated, Thus, 20 cycles provide for 1 million times amplifications. These cycles are generally repeated by auto-mated instrument, called Tempcycler.
Thus following is required in PCR:-
- target double standard DNA,
- two specific primrers
- a thermostable DNA polymerase
- Taq polymerase,
- dNTP
APPLICATION OF PCR:
Detection of infectious diseases:
- AIDS, Tuberculosis, CMV, H1N1, etc.
- Lyme Disease-joint inflammation from tick bites.
- Detect 3 sexually transmitted diseases in one swab-herpes, papillomarvirus, chlamydia.
- PCR can diagnosis even one bacteria or virus present in the specimen.
- Latent viruses can also be diagnosed.
Detection of Variations and Mutations in Genes
- Detects people with inherited disorders and carriers
- Track presence or absence of DNA abnormalities, characteristic to cancer.
- Prenatal diagnosis of genetic disorders.
- PCR combined with RE and Southern blotting is used for mutation detection.
PCR and the Law
- DNA fingerprinting can multiply amounts of DNA
- found in blood samples, hair, semen, and other
- body fluids “contain only very few tubercle bacilli ,cytomegalo virus and HIV
Various types of PCR:
- Multipler PCR
- Reverse Tanscriptase PCR: DNA polymerase derived from thermus thermophillus organism has got additional reverse transcriptase activity and hence is preferred for reverse transcriptase type of PCR.
- Realtime PCR: a fluorescent dye known as “SYBR green is used to tag the primer, this helps in quantitative detection of PCR material.
- Invert PCR
- Nested PCR.
Exam Important
- PCR is a method of enzymatic amplification of a target sequence of DNA.
- It is sensitive, selective (specific) and extremely rapidmeans of amplifiing any desired sequence of double stranded DNA.
- the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq). Taq polyrnerase is used because it is thermostable.
- Each cycle doubles the amout of DNA in the sample, leading to exponential increase.
- In Chain extension:- DNA polymerase and deoxyribonucleotides are added. to the mixture.
- Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.
- DNA polymerase derived from thermus thermophillus organism has got additional reverse transcriptase activity and hence is preferred for rsverse transcriptase tnte of PCR.
Don’t Forget to Solve all the previous Year Question asked on POLYMERASE CHAIN REACTION (PCR)