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Biomedical subjects

M Akhtar

Publications and source records attributed to M Akhtar.

At least 253 records · Page 14Linked to original sources
PubMed1991

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays.

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.

Animals↗
PubMed1991

Ramadan fasting and thyroid hormone profile.

A study comprising 41 males, 5 females of the age ranging from 28 to 56 years was conducted during Ramadan of 1989 to compare T3, T4 and TSH levels in fasting with the levels of non-fasting conditions. Each individual gave 6 blood samples: One sample was taken 20 days before the onset of Ramadan, 3 samples at different fasting days and last two samples were drawn 23 days and five months after the end of Ramadan, respectively. The results showed a significant gradual rise in TSH throughout the fasting month, although the mean levels remained within normal limits. Pre- Ramadan levels were re-attained well after the end of Ramadan. There was no significant change in T3 and T4 levels.

Adult↗
PubMed1991

Local preparation, standardization and quality control of technetium labelled macroaggregated albumin for lung perfusion studies.

Lung perfusion study is an important investigation in various pulmonary diseases. The radiopharmaceutical commonly used now-a- days is imported macroaggregated human albumin (in kit form), which is labelled with technetium (Tc99m-MAA). Due to its high cost the technique could not be fully exploited. We have tried to locally prepare freeze dried MAA particles. Various parameters like concentration of protein, pH value, temperature, quality and quantity of reducing agents were studied to find out the optimum conditions for radiolabelling and the desired particle size. More than 98% of the added radioactivity was found tagged to the MAA particles in the final preparation (confirmed by paper chromatography). Labelled agent was found to be radiochemically stable for up to 6 hours. Initial animal and later human studies showed an ideal spectrum of particle size.

Chemistry, Pharmaceutical↗
PubMed1991

Qualitative evaluation of in-house immunoassays of T3, T4 and TSH based on bulk reagents.

Increase in the cost of RIA kit assays has led to our attempts to seek cheaper alternates. Assays based on bulk reagents (supplied free of cost by INMOL in collaboration with IAEA) were started in 1988. Statistical and Quality control data on 50,51 and 52 assay batches of T3, T4 and TSH respectively has been collected from the beginning. Cumulative assay parameters show that T3 and T4 assays are almost equally precise. TSH assay is most imprecise in the group especially at low concentration levels. The working ranges of T3 and T4 assays defined at 10% error limit are quite wide and cover low, medium and high levels of hormones. In TSH the assay working range does not cover levels below 10mu IU/ml. The variability of curve parameters is similar in this group of assays. Quality control results are most reproducible in T4 assays with a between batch variability of 11.9%. T3 and TSH assay results are equally reproducible (20.50% variability). Overall within assay drift is low in all assays. IQC charts of these assays show occasional significant positive or negative shift of results from mean which might be related to methodological variations of quality among various distributions of reagents. The reproducibility and precision of results could be further improved by harmonizing the future distributions of reagents.

Data Interpretation, Statistical↗
PubMed1990

L-methionine decarboxylase from Dryopteris filix-mas: purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination.

L-Methionine decarboxylase from the male fern Dryopteris filix-mas has been purified 256-fold from acetone powder extracts to very near homogeneity. The enzyme is membrane-associated and requires detergent for solubilization during the initial extraction. The enzyme is a homodimer of subunit Mr 57,000 and shows a pH optimum at approximately 5.0 with 20 mM (2S)-methionine as substrate. The specific activity, kcat, for methionine is approximately 50 mol s(-1) (mol of active site)(-1) at pH 4.5 and below. A wide range of straight- and branched-chain (2S)-alkylamino acids are substrates for the enzyme. The values for the rate of decarboxylation, Vmax, and for the apparent Michaelis constant, Km, however, vary with structure and with the chirality at C-3. The pH dependence of V and V/K has been examined for three substrates: (2S)-methionine, valine, and leucine. Pyridoxal 5'-phosphate (PLP) is required for activity, and in the absence of excess PLP, the activity of the enzyme in incubations reduced with respect to time. The addition of PLP fully restores the activity, indicating that an abortive decarboxylation-transamination accompanies the normal decarboxylation reaction. The occurrence of the abortive reaction was confirmed by showing that [35S]methionine is converted to labeled 3-(methylthio)propionaldehyde while [4'-3H]PLP is converted to labeled pyridoxamine 5'-phosphate (PMP). The decarboxylation of (2S)-methionine gave 3-(methylthio)-1-aminopropane. Preparation of the N-camphanamide derivative of the amine allowed the C-1 methylene protons to be distinguished by 1H NMR spectroscopy. Synthetic samples of the camphanamide were prepared in which each of the C-1 methylene protons was replaced by deuterium. When (2S)-methionine and the C-2 deuteriated isotopomer were incubated with the enzyme in deuterium oxide and protium oxide, respectively, and the products were converted to their camphanamide derivatives and analyzed by 1H NMR spectroscopy, it was evident that decarboxylation occurred with retention of configuration at C-2. When the decarboxylation of six other substrates was studied, examination of the N-camphanamide derivatives of the amines indicated that decarboxylation occurred stereospecifically and, by analogy, with retention of configuration at C-2. When tritiated pyridoxal phosphate was incubated with the enzyme, tritiated pyridoxamine phosphate was formed. Analysis of the chirality of the methylene group at C-4' indicated that, during abortive transamination, protonation occurred from the 4'-si face of the coenzyme, the same stereochemical result as that obtained for several bona fide transaminase enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Carboxy-Lyases↗
PubMed1990

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination.

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1990

Streptomyces L-methionine decarboxylase: purification and properties of the enzyme and stereochemical course of substrate decarboxylation.

L-Methionine decarboxylase from Streptomyces species ATCC 21020 has been purified to near homogeneity, characterized, and compared to the enzyme from the fern Dryopteris filix-mas [Stevenson, D.E., Akhtar, M., & Gani, D. (1990) Biochemistry (first paper of three in this issue)]. The enzyme catalyzes the decarboxylation of a range of alkylamino acid substrates, but the substrate specificity is different from that for the fern enzyme. In accord with the properties of the fern enzyme, the Streptomyces enzyme is also a homodimer of Mr 100,000 +/- 5000 and requires PLP for activity. At low pH where the value of Vmax for both enzymes is maximal and essentially pH independent, kcat for the Streptomyces enzyme with (2S)-methionine as substrate is slightly higher (60 s-1) than the value for the eukaryotic protein (50 s-1). The pH optimum for V/K is much higher than that for the fern enzyme although many features of the pH dependence are similar, including the shape of the curve for the pH dependence of Km. When the decarboxylations of (2S-methionine, (2S)-norleucine, and (2R)-S-ethyl-L-cysteine were conducted on a preparative scale in protium and deuterium oxide, unlabeled and deuteriated amines were formed. 1H NMR spectroscopic analysis of the stereochemistry at C-1 of the camphanamide derivatives of the products [Stevenson, D. E., Akhtar, M., & Gani, D. (1990) Biochemistry (first paper of three in this issue)] indicated that each conversion was stereospecific and occurred with retention of configuration at C-2 of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxy-Lyases↗
PubMed1990

Adenosine for paroxysmal supraventricular tachycardia: dose ranging and comparison with verapamil. Assessment in placebo-controlled, multicenter trials. The Adenosine for PSVT Study Group.

OBJECTIVE: To assess the safety and efficacy of intravenous adenosine in terminating acute episodes of paroxysmal supraventricular tachycardia. DESIGN: Two prospective, double-blind, randomized, placebo-controlled trials to evaluate dose response in patients receiving adenosine and to compare the effects of adenosine with those of verapamil. PATIENTS: A total of 359 patients with a tachycardia electrocardiographically consistent with paroxysmal supraventricular tachycardia were entered into the two protocols. Patients subsequently found to have arrhythmias other than paroxysmal supraventricular tachycardia were excluded from the efficacy analysis. INTERVENTIONS: The first protocol compared sequential intravenous bolus doses of 3, 6, 9, and 12 mg of adenosine to equal volumes of saline. In the second protocol, patients received either 6 mg and, if necessary, 12 mg of adenosine or 5 mg and, if necessary, 7.5 mg of verapamil. MEASUREMENTS AND MAIN RESULTS: When data are expressed in terms of cumulative response in eligible patients, intravenous adenosine terminated acute episodes of paroxysmal supraventricular tachycardia in 35.2%, 62.3%, 80.2%, and 91.4% of patients who received maximum doses of 3, 6, 9, and 12 mg, respectively, in a four-dose sequence, whereas 8.9%, 10.7%, 14.3%, and 16.1% of patients responded to four sequential placebo doses (P less than 0.0001). In the second trial, cumulative response rates after 6 mg followed, if necessary, by 12 mg of adenosine were 57.4% and 93.4%, and after 5 mg followed, if necessary, by 7.5 mg of verapamil were 81.3% and 91.4%. The average time after injection to termination of tachycardia by adenosine was 30 seconds. Adenosine caused adverse effects in 36% of patients, but they lasted less than 1 minute and were usually mild. CONCLUSIONS: Adenosine in graded doses up to 12 mg rapidly and effectively terminates acute episodes of paroxysmal supraventricular tachycardia in which the atrioventricular node is an integral part of the re-entrant circuit. The overall efficacy of adenosine is similar to that of verapamil, but its onset of action is more rapid. Adverse reactions to adenosine are common but are minor and brief.

Acute Disease↗
PubMed1990

Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme.

The aminoglycoside phosphotransferase gene from a butirosin-producing strain of Bacillus circulans was cloned in a high-expression vector (pKK223-3) to give the recombinant plasmid pMS5. Escherichia coli harbouring the plasmid, E. coli JM103[pMS5], was characterized, and several features of the expression of the phosphotransferase were studied. The phosphotransferase activity was best expressed in a medium lacking glucose, and the highest levels of the enzyme were found between 12 and 24 h of growth. The induction of the phosphotransferase expression with isopropyl beta-D-thiogalactopyranoside (inducer) was found to be undesirable as the overproduction of the enzyme led to the killing of the bacteria. The subcellular location of the phosphotransferase, and also the site in vivo of the phosphorylation of neomycin, was found to be in the cytoplasm. The phosphotransferase was purified to homogeneity in good yield (17 mg of purified protein/3 litres of culture) and was shown to be a monomer of Mr 30,000-32,000. The N-terminal amino acid sequence was in agreement with that predicted from the gene sequence and confirmed the absence of any signal sequence. The regiospecificity of the phosphotransferase reaction was studied by m.s. and by 1H-, 13C- and 31P-n.m.r. using ribostamycin as the substrate, and it was found that the antibiotic was phosphorylated at the 3'-hydroxy group.

Amino Acid Sequence↗
PubMed1990

Fine-needle aspiration biopsy of seminoma and dysgerminoma: cytologic, histologic, and electron microscopic correlations.

Cytomorphologic features in a series of fine-needle aspiration biopsies from eight patients with seminoma or dysgerminoma were reviewed, and the findings were further correlated with histopathologic and ultrastructural appearances. Three cell types were encountered. Type I was least differentiated and least frequent. Type II was most frequent and had well-developed cytoplasm rich in organelles. Type III was similar to type II but also contained abundant cytoplasmic glycogen. In cytology smears, type III cells contained large punched-out cytoplasmic vacuoles corresponding to the areas of glycogen deposition. The background contained a variable mixture of inflammatory cells including lymphocytes, plasma cells, epithelioid cells, and epithelioid granulomas. The significance of these findings in the diagnosis of seminoma or dysgerminoma and the distinction from other germ-cell and non-germ-cell tumors is briefly discussed.

Adolescent↗
PubMed1990

Fine-needle aspiration biopsy of mucus-producing medullary carcinoma of thyroid: report of a case with cytologic, histologic, and ultrastructural correlations.

Cytologic features of aspiration smears from a mucus-producing medullary carcinoma of the thyroid are described. Two patterns of mucin production by the tumor cells were noted. In some cells, mucin was present as numerous small cytoplasmic vacuoles with ultrastructural morphology characteristic of mucin granules. Other cells had larger, more well-defined cytoplasmic mucin vacuoles; on ultrastructural examination, they were found to be intracytoplasmic lumina. Although mucin production has been demonstrated in several histologic studies of medullary carcinoma, it has not been previously recognized in fine-needle aspiration biopsies from this neoplasm.

Biopsy, Needle↗
PubMed1990

Fine-needle aspiration biopsy diagnosis of endodermal sinus tumor: histologic and ultrastructural correlations.

Fine-needle aspiration biopsies from five patients with endodermal sinus tumors (ESTs) were reviewed, and the findings were correlated with histologic and ultrastructural appearances. In the aspiration smears, two types of tumor cells were seen, forming clusters of variable sizes. Type A cells had distinct cell borders, and their cytoplasm contained only occasional vacuoles. Type B cells had ill-defined cell borders and formed syncytial clusters; their cytoplasm was characterized by large numbers of rounded vacuoles. The background contained patches of mucoid material and macrophages with foamy cytoplasm. Eosinophilic hyaline cytoplasmic bodies and irregular deposits of intercellular basement membrane-like material were recognized easily in aspiration smears. These features correlated well with histologic and ultrastructural appearances. The significance of these findings in the fine-needle aspiration biopsy diagnosis of EST and its distinction from other germ-cell and non-germ-cell tumors is discussed.

Adult↗
PubMed1990

Familial glomerulopathy with proximal tubular dysfunction: a new syndrome?

Two female siblings with nephrotic syndrome and proximal tubular dysfunction are described. One, aged 15 months, presented with steroid-resistant nephrotic syndrome and was found to have partial Fanconi syndrome and hypothyroidism. At the age of 21 months her renal function started deteriorating rapidly and she died of septicaemia 5 months later. The second sibling, aged 3 years, had steroid-resistant nephrotic syndrome with proximal renal tubular acidosis and hyperaminoaciduria. The renal biopsies in both showed unusual changes in glomerular basement membrane along with IgM and C3 deposition. It is suggested that these siblings may be suffering from a hitherto undescribed clinico-pathological entity.

Child, Preschool↗
PubMed1990

Studies on estrogen biosynthesis using radioactive and stable isotopes.

The conversion of androgens into estrogen involves three distinct generic reactions which are catalyzed by a single P450 enzyme (aromatase or P450(aromatase)). The first step in the process is the conversion of 19-methyl into a hydroxymethyl group which requires NADPH + O2, thus representing the well-known hydroxylation process. The next stage, converting the -CH2OH into -CHO, also requires NADPH + O2 and may be rationalized either through a second hydroxylation reaction producing a gem-diol, CH(OH)2 (which dehydrates to the aldehyde), or via another route. The final stage in the process again uses NADPH + O2, culminating in the release of C-19 as formate. Our extensive studies using precursors containing 2H, 3H, and 18O have shown that the carbonyl oxygen of the 19-aldehyde group is the one that was introduced in the first step as the hydroxyl group. The aldehydic oxygen along with another, from O2, used in the third step of the process, is incorporated into the released formate. It was found that at each stage of the process, oxygen atoms were introduced or transferred as "whole numbers." In light of these data, mechanisms in which H2O is used to promote the C-10-C-19 bond cleavage or those in which the conversion of the 19-oxoandrostenedione into estrogen is considered to occur via the sequence -CHO----(-)CH(OH)2----estrogen are eliminated. In addition, our mechanistic analysis makes it unlikely that 1 beta-, 2 beta-, or 10 beta-hydroxysteroids serve as intermediates in estrogen biosynthesis. We consider a free radical mechanism for the hydroxylation process.

Aromatase↗
PubMed1990

Unexplained syncope.

There is a large patient population that experiences syncope. Although the exact mechanism is not known, arrhythmias and vasodepressor mechanisms probably account for most cases of unexplained syncope. Fortunately, patients with unexplained syncope can be successfully treated once the correct diagnosis has been established.

Humans↗