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G Ghosh

Publications and source records attributed to G Ghosh.

53 records · Page 3Linked to original sources
PubMed1991

Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains the major recognition site required for aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase (MetRS) and have located part of the anticodon binding domain on the enzyme at a site close to Trp461 [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L.H. (1990) Biochemistry 29, 2220-2225]. In order to gain information about other possible sites of contact between MetRS and its tRNA substrates, we have examined the effects of mutations at a series of positively charged residues on the surface of the C-terminal domain of the enzyme. Conversion of Arg356, Arg366, Arg380, or Arg453 to Gln had little or no effect on enzyme activity. Similarly, conversion of Lys402 or Lys439 to Asn failed to significantly alter aminoacylation activity. Conversion of Arg380 to Ala or Arg442 to Gln produced a 5-fold reduction in kcat/Km for aminoacylation of tRNAfMet, with no effect on methionine activation, indicating a possible minor role for these residues in interaction of the enzyme with the tRNA substrate. In contrast, mutation of a phylogenetically conserved residue, Arg395, to Gln increased the Km for aminoacylation of tRNAfMet about 30-fold and reduced kcat/Km by 25,000-fold. The mutant enzyme was also shown to be highly defective by its inability to complement a strain of E. coli having an altered chromosomal MetRS gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1991

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.

Adenosine Triphosphate↗
PubMed1990

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1990

In-vitro studies with ramoplanin (MDL 62,198): a novel lipoglycopeptide antimicrobial.

Ramoplanin is a novel lipoglycopeptide antimicrobial complex, isolated from the fermentation products of a strain of Actinoplanes sp. (ATCC 33076), which comprises three closely related polypeptides, each containing chlorinated phenyl moieties and D-mannose. The in-vitro activity of ramoplanin was compared with those of vancomycin and teicoplanin. Ramoplanin was very active against Staphylococcus spp., irrespective of methicillin susceptibility, with all isolates inhibited by 1 mg/l or less. Concentrations of vancomycin and teicoplanin required to inhibit the same population of bacteria were 4 and 16 mg/l, respectively. Ramoplanin was also very active against Streptococcus spp. (alpha- and beta-haemolytic species, Str. pneumoniae and Enterococcus faecalis, Corynebacterium spp. (including Cory. jeikeium), Listeria monocytogenes, Gardnerella vaginalis, Propionibacterium acnes and Gram-positive anaerobic bacteria, with all isolates inhibited by 2 mg/l, or less. In general, the activity of ramoplanin against these species was either equal to or only slightly less than teicoplanin and equal to or somewhat greater than vancomycin. With the exception of Bacteroides melaninogenicus and Bact. bivius, ramoplanin was not active against Gram-negative bacteria.

Anti-Bacterial Agents↗
PubMed1990

Maternal and cord serum glycosylated protein in neonatal macrosomia and correlation with birth weight.

Maternal glycosylated hemoglobin and glycosylated protein and cord glycosylated protein were measured at delivery in 20 normal mothers of 20 macrosomic neonates over 4000 g (group I) and compared with values in two groups of mother/infant pairs: 20 normal/20 appropriate for gestational age (group II) and nine diabetic mothers/ten neonates (group III). Infants in group I, by design, weighed more (mean +/- SD 4403 +/- 337 g) than those in group II (2902 +/- 278 g) or group III (3365 +/- 898 g) (P less than .001). There was no significant difference in weight between group II and group III infants. Birth weight ratio was greater (P less than .001) in group I than in group II or group III (1.39 +/- 0.1, 0.9 +/- 0.08, and 1.08 +/- 0.25, respectively); group III infants had a higher birth weight ratio (P less than .05) than those in group II. Hematocrit (%) was higher (P less than .05) in group III (62 +/- 3) than in group I (59 +/- 5) or group II (57 +/- 6) infants. Glycosylated hemoglobin values were similar in all three groups. Mean serum glycosylated protein was higher (P less than .001) in group III (13.8 +/- 2%) than in group I (10 +/- 2%) or group II (9.8 +/- 2.5%) mothers. Cord glycosylated protein was also higher (P less than .001) in group III (12.3 +/- 1.9%) than in group I (9 +/- 1.3%) or group II (8.6 +/- 1.7%) neonates.(ABSTRACT TRUNCATED AT 250 WORDS)

Birth Weight↗
PubMed1988

Muscle fatigue and pain after eccentric contractions at long and short length.

1. The effect of muscle length on the development of muscle pain and fatigue has been studied. 2. Eight normal young adults performed maximal eccentric contractions of the elbow flexors. The muscles of one arm were exercised at short length, and the contralateral muscle at long length. Each contraction lasted approximately 1 s, and was repeated once every 10 s for 30 min. 3. Muscle strength and frequency-force characteristics were measured from isometric contractions before, immediately after and at 24 h intervals for the next 4 days. Muscle tenderness was assessed daily. 4. The muscle strength was reduced by approximately 10% by exercise at short length, and by 30% by exercise at long length. 5. The 20:100 ratio (force generated by stimulation at 20 Hz/force generated at 100 Hz) fell by 30% after exercise at short length and had recovered after 24 h. Exercise at long length reduced this ratio by 65% and the muscles had not fully recovered 4 days later. 6. Muscle pain developed after both exercise regimens, but was slightly worse after that at long length. 7. It is concluded that there is a length-dependent component in the development of pain and fatigue after eccentric exercise, which had previously been thought to be caused solely by high force generation.

Adolescent↗
PubMed1988

Resistance studies with ofloxacin.

The selection of ofloxacin-resistant mutants from susceptible wild-type bacterial populations was investigated by three methods. Resistant mutants selected from populations of Escherichia coli (NCTC 10418) and Staphylococcus aureus (NCTC 6571) by single-step passage at either four or eight times the MIC occurred at a frequency of less than 1 x 10(-10). Ofloxacin-resistant mutants of Pseudomonas aeruginosa (NCTC 10662), selected at four times the MIC, occurred with a 100-fold greater frequency at 3.3 X 10(-8), however, mutants of this species selected at eight times the MIC, occurred at a frequency of less than 1 X 10(-10). Sequential selection of ofloxacin-resistant mutants during multiple passages at 1/2 MIC, resulted in a 16- to 32-fold increase in MICs for isolates of P. aeruginosa and Klebsiella aerogenes, a four- to eight-fold increase in MICs for some strains of E. coli and a two- to four-fold increase in MICs for Staph. aureus and Streptococcus spp. During prolonged exposure of populations of E. coli (NCTC 10418) and Staph. aureus (NCTC 6571) to concentrations of ofloxacin at 1, 10 and 100 times the MIC no resistant-mutants were selected after seven days incubation. In similar experiments with P. aeruginosa (NCTC 10662) ofloxacin-resistant mutants were selected from populations exposed to the MIC which required 64 mg/l ofloxacin for inhibition. No ofloxacin-resistant mutants were selected from populations of P. aeruginosa (NCTC 10662) exposed to either ten or 100 times the MIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Resistance, Microbial↗
PubMed1985

Conference on insulin pump therapy in diabetes. Multicenter study of effect on microvascular disease. Assessment of fluorescein angiograms.

Quantitation of the earliest changes in abnormal retinal morphology using fluorescein angiography is potentially superior to retinal photography. However, the critical importance of flawless technique, limitations in the size of the field available for detailed study, and observer variability constitute major disadvantages. A protocol describing standards of photography and of injection was developed. Methodology for counting microaneurysms (Ma) was developed at a central laboratory and applied in suitable photographs obtained at 0 (baseline), 4, and 8 mo in 68 patients. Counts of "definite" or "possible" Ma were made on films projected under standard conditions by two observers known to achieve consistently reproducible results. Semiquantitative assessment of diffusibility of fluorescein reflecting capillary leakage was performed in 61 patients. Leakage was graded according to three degrees of severity permitting study of observer variation, concordance of change in pairs of eyes, and treatment effects. The average number of Ma at baseline was slightly higher in the continuous subcutaneous insulin infusion (CSII) group than in the conventional insulin treatment (CIT) group, but the difference was not statistically significant at the 0.05 level. At both 4 and 8 mo, definite Ma were more prevalent in the CSII group, and the difference was statistically significant at the 0.05 level using both parametric and nonparametric tests. In 27 CSII and 23 CIT patients having complete sets of 0, 4, and 8-mo photographs, Ma counts increased during the 0-4-mo interval. During the 4-8 mo interval, a further increase occurred in the CSII group but contrasted with a decrement observed in the CIT group, which showed no net change from baseline.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneurysm↗
PubMed1983

Severe diabetic retinopathy in adolescents.

This paper reports on 9 patients aged 13-18 years (mean 15.4) with severe diabetic retinopathy. Two patients were prepubertal and 4 were going through puberty. Hypertension was present in 2 patients, while 4 had proteinuria. Severe preproliferative disease was present in 3 patients initially and proliferative retinopathy in the remainder. In 5 this retinopathy was considered to be florid. Two patients seen prior to 1975 had pituitary ablation, while those seen after 1975 were treated by extensive argon and xenon arc photocoagulation. Proliferative lesions regressed in both groups. At the latest follow-up 7 of the 9 patients achieved a final visual acuity of 6/9 or better in at least one eye. One patient became blind. Proliferative retinopathy advances rapidly in adolescents, but photocoagulation, as used now, can maintain vision in most.

Adolescent↗