Biomedical subjects

S Unnithan

Publications and source records attributed to S Unnithan.

6 recordsLinked to original sources

PubMed2000

Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3'-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3'-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3'-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

3' Untranslated Regions↗

PubMed1993

Poppy tea dependence.

A patient presenting with dependence on opium poppy tea infusion is reported. Poppy tea drinking, although previously described in certain parts of the UK, rarely presents in the form of a dependence syndrome. Issues relating to the management of poppy tea dependence are discussed, including the results of existing laboratory urinalysis for drugs of abuse, the calculation of opiate dose equivalence, and the likelihood of transition (or not) from use of poppy tea to use of other opiates.

Adult↗

PubMed1992

Factors associated with relapse among opiate addicts in an out-patient detoxification programme.

Relapse is a central problem in the treatment of addictive behaviour, and a specific problem in the out-patient treatment of the opiate withdrawal syndrome. This study investigated factors associated with relapse among 42 opiate addicts receiving out-patient detoxification treatment at a London drug-dependence clinic. All subjects completed a questionnaire about their social, psychological, and environmental circumstances in the week before interview, and were interviewed within the first two weeks of the programme. Forty per cent had lapsed to illicit heroin abuse within the previous week. Interpersonal factors and drug-related cues were associated with lapse to opiate use. Most subjects encountered a range of high-risk situations, such as regularly meeting other drug users and being offered drugs, and persistent negative mood states.

Adult↗

PubMed1990

Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.

Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.

Base Sequence↗

PubMed1989

Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant.

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.

Amino Acid Sequence↗

PubMed1984

A high-performance liquid chromatography assay for asparagine synthetase.

A highly sensitive method for assaying asparagine synthetase and its glutaminase activity is presented. The amino acids L-asparagine, L-aspartate, L-glutamate, and L-glutamine, are separated by derivatization with o-phthaldialdehyde followed by reversed-phase high-performance liquid chromatography on an Altex ultrasphere-ODS C18 column. The elution is isocratic and the mobile phase used is 50 mM sodium acetate buffer (pH 5.9) with 30% methanol. This assay can easily detect picomoles of asparagine, which may be difficult to do with the other assays that have been described.

Amino Acids↗