Biomedical subjects

M Khanna

Publications and source records attributed to M Khanna.

At least 19 recordsLinked to original sources

PubMed1996

Engineering antibiotic producers to overcome the limitations of classical strain improvement programs.

Improvement of the antibiotic yield of industrial strains is invariably the main target of industry-oriented research. The approaches used in the past were rational selection, extensive mutagenesis, and biochemical screening. These approaches have their limitations, which are likely to be overcome by the judicious application of recombinant DNA techniques. Efficient cloning vectors and transformation systems have now become available even for antibiotic producers that were previously difficult to manipulate genetically. The genes responsible for antibiotic biosynthesis can now be easily isolated and manipulated. In the first half of this review article, the limitations of classical strain improvement programs and the development of recombinant DNA techniques for cloning and analyzing genes responsible for antibiotic biosynthesis are discussed. The second half of this article addresses some of the major achievements, including the development of genetically engineered microbes, especially with reference to beta-lactams, anthracyclines, and rifamycins.

Anthracyclines↗

PubMed1995

Substrate specificity, gene structure, and tissue-specific distribution of multiple human 3 alpha-hydroxysteroid dehydrogenases.

We have expressed in Escherichia coli functionally active proteins encoded by two human cDNAs that were isolated previously by using rat 3 alpha-hydroxysteroid dehydrogenase cDNA as the probe. The expressed proteins catalyzed the interconversion between 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol. Therefore, we name these two enzymes type I and type II 3 alpha-hydroxysteroid dehydrogenases. The type I enzyme has a high affinity for dihydrotestosterone, whereas the type II enzyme has a low affinity for the substrate. The tissue-specific distribution of these two enzymes was determined by reverse transcription polymerase chain reaction using gene-specific oligonucleotide primers. The mRNA transcript of the type I enzyme was found only in the liver, whereas that of the type II enzyme appeared in the brain, kidney, liver, lung, placenta, and testis. The structure and sequence of the genes encoding these two 3 alpha-hydroxysteroid dehydrogenases were determined by analysis of genomic clones that were isolated from a lambda EMBL3 SP6/T7 library. The genes coding for the type I and type II enzymes were found to span approximately 20 and 16 kilobase pairs, respectively, and to consist of 9 exons of the same sizes and boundaries. The exons range in size from 77 to 223 base pairs (bp), whereas the introns range in size from 375 bp to approximately 6 kilobase pairs. The type I gene contains a TATA box that is located 27 bp upstream of multiple transcription start sites. In contrast, the type II gene contains two tandem AP2 sequences juxtaposed to a single transcription start site.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Localization of multiple human dihydrodiol dehydrogenase (DDH1 and DDH2) and chlordecone reductase (CHDR) genes in chromosome 10 by the polymerase chain reaction and fluorescence in situ hybridization.

Multiple human dihydrodiol dehydrogenases and human chlordecone reductase belong to the aldoketo reductase superfamily. These two enzymes are involved in the metabolism of xenobiotics, such as polycyclic aromatic hydrocarbons and pesticides. Recently we have isolated three closely related genes encoding two dihydrodiol dehydrogenases (DDH1 and DDH2) and the chlordecone reductase (CHDR). Mapping of the location of the genes was performed using the polymerase chain reaction using gene-specific primers to amplify gene sequences in human/hamster hybrid DNA. All three genes were found to be located on chromosome 10. In situ hybridization using a lambda clone as the probe further confirmed regional localization at 10p14-p15.

Alcohol Oxidoreductases↗

PubMed1995

Distribution of 3 alpha-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans.

3 alpha-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3 alpha-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3 alpha-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activity was found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. We used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. One of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5'-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3 alpha-hydroxysteroid dehydrogenase and human chlordecone reductase. We are currently investigating the functional relationship between the enzymes encoded by these human cDNAs and 3 alpha-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Rifamycins: strain improvement program.

Rifamycins are primarily produced by Gram-positive bacterium Amycolatopsis mediterranei, which belongs to the order Actinomycetales. These antibiotics, apart from their application against pathogens of tuberculosis and leprosy, have also been found to be effective against several other pathogens including Mycobacterium avium and Pneumococcus. Because of the importance of rifamycin, the producer strain A. mediterranei has been genetically manipulated since 1957 in order to develop a strain that can either produce larger amounts of rifamycin or derivatives of rifamycin. In this article, the importance of the producer strain, traditional methods (mutations and recombination) of strain improvement, their limitations, and the development of a cloning vector and transformation methods that have made recombinant DNA techniques accessible for genetic manipulations of A mediterranei are discussed.

Actinobacteria↗

PubMed1995

Effect of corticosteroid treatment on various serological & bronchoalveolar lavage abnormalities in patients with sarcoidosis.

Effect of daily oral prednisolone treatment was studied in 29 patients with pulmonary sarcoidosis. Twenty normal control subjects were also studied. Pretreatment absolute lymphocyte counts and proportion of lymphocytes in peripheral blood were significantly lower in patients with sarcoidosis as compared to normal controls. Total cell count and the proportion of lymphocytes in bronchoalveolar lavage (BAL) fluid were significantly higher in sarcoidosis. The proportion of CD3+ and CD4+ was significantly lower in peripheral blood and higher in BAL fluid in patients with sarcoidosis. Immunoglobulins (IgG, A and M) and complements (C3, C4 and CH50) were significantly higher both in peripheral blood and BAL fluid. Patients with sarcoidosis were treated with daily oral prednisolone (30 mg/day). Repeat studies were performed after an interval of 4-6 months in 20 patients with sarcoidosis. A significant increase in absolute lymphocyte counts in peripheral blood and decrease in the proportion of lymphocytes in BAL fluid occurred with prednisolone treatment. Proportion of CD3+, CD4+ and B cells increased in peripheral blood and decreased in BAL fluid. Complement and immunoglobulin levels revealed a significant reduction in peripheral blood and BAL fluid. It is concluded that patients with sarcoidosis have peripheral blood lymphopaenia and lymphocytic alveolitis. They have increased levels of complement and immunoglobulins both in the peripheral blood and BAL fluid. All these abnormalities show significant improvement with prednisolone treatment.

Adult↗

PubMed1995

Immunologic & pulmonary function abnormalities in tropical pulmonary eosinophilia.

Twenty six patients (24 males and 2 females) with tropical pulmonary eosinophilia (TPE) were studied. Arterial blood gas analysis, pulmonary functions, peripheral blood examination and bronchoalveolar lavage (BAL) were performed. Peripheral blood and BAL fluid analyses were performed in healthy volunteers. Pulmonary functions revealed a mild restrictive ventilatory defect with airways obstruction. Mild hypoxaemia was observed on arterial blood gas analysis. Serum immunoglobulins IgG (P < 0.01), IgA (P < 0.001) and IgM (P < 0.001) were significantly raised as compared to normal controls. Serum complement (C3) level was higher, however, it was not significantly different as compared to normal controls. Serum haemolytic component of the complement (CH50) was significantly higher (P < 0.001) in patients with TPE compared to normal control subjects. The immunoglobulins IgG, IgA and IgM in the BAL fluid were significantly (P < 0.001) increased as compared to normal controls. The fibronectin (FN) level was also significantly increased (P < 0.001) in the BAL fluid. It is concluded that patients with TPE have mild restrictive ventilatory defect with airways obstruction and mild hypoxaemia. They have eosinophilic alveolitis with increased levels of immunoglobulins in the peripheral blood and BAL fluid. The significance of elevated FN in the BAL fluid is not clear and serial estimations may have to be done in order to clarify its role in the pathogenesis of fibrosis in chronic TPE.

Adolescent↗

PubMed1994

Structure of a gene coding for human dihydrodiol dehydrogenase/bile acid-binding protein.

The structure and sequence of the gene (DD/BABP) encoding a human dihydrodiol dehydrogenase/bile acid-binding protein (DD/BABP) were determined by analysis of genomic clones. Several overlapping clones containing parts of the gene were isolated from a lambda EMBL3 SP6/T7 library and characterized by restriction mapping and DNA sequencing. The gene spans approx. 16kb and consists of nine exons. The sizes of the exons range from 77 to 167 bp, whereas the intron sizes range from 375 to 3430 bp. The transcription start point (tsp) is located at -56 bp of the first ATG codon, as determined by primer extension. Neither a TATA box nor a CAT box was found upstream from the tsp. Southern blot analysis of the human genomic DNA, using the cDNA as the probe, revealed several additional hybridizing DNA bands, suggesting the existence of multiple related genes.

Amino Acid Sequence↗

PubMed1994

Distribution and ontogeny of 3 alpha-hydroxysteroid dehydrogenase in the rat brain.

3 alpha-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3 alpha-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3 alpha-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activities were found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. No sexual dimorphism was found in either the concentration levels or the activities of the brain 3 alpha-hydroxysteroid dehydrogenase. At the time of birth, the rat brain already expresses a significant level of 3 alpha-hydroxysteroid dehydrogenase; the levels of brain 3 alpha-hydroxysteroid dehydrogenase activity in rats continue to rise during the first week after their birth, and reach a plateau thereafter.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA.

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase.

Adsorption↗

PubMed1992

Purification and amino-terminal sequence of the dengue virus-induced cytotoxic factor.

The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DV-infected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 25 kDa. Anti-CF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of 19 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus.

Amino Acid Sequence↗

PubMed1991

Breakdown of the blood-brain barrier during dengue virus infection of mice.

A breakdown of the blood-brain barrier occurred in mice inoculated intracerebrally (i.c.) or intraperitoneally (i.p.) with dengue virus type 2 (DEN2). This resulted in leakage of protein-bound Evans blue dye and 51Cr-labelled erythrocytes into the brain tissue. The leakage increased with time after infection and coincided with an increase of a DEN2-induced cytokine, the cytotoxic factor (CF), in the spleens of such mice. The titres of virus in the brain increased exponentially in i.c. inoculated mice but the virus was not detected in brains of mice given DEN2 by the i.p. route. Similar breakdown of the blood-brain barrier also occurred in mice inoculated intravenously with CF; the damage was dose-dependent and the vascular integrity was restored during the 3 h period after inoculation. Treatment of mice with antihistamine drugs, blocking H1 or H2 receptors, decreased the DEN2-induced protein leakage by up to 50% in i.c. inoculated mice and up to 92% in those inoculated i.p. Indomethacin, a prostaglandin synthetase inhibitor, had no effect. In i.c. inoculated mice protein leakage was inhibited by about 60% by treatment with CF-specific (CFA) or DEN2-specific antisera (DEN2A) whereas protection was complete with the combined treatment with both antisera. On the other hand, in i.p. inoculated mice the inhibition of protein leakage was 80 to 89% with CFA. These findings show a breakdown of the blood-brain barrier leading to cerebral oedema during DEN2 infection which is mediated via the release of histamine by a virus-induced cytokine.

Animals↗

PubMed1991

Obligatory role of Ca2+ in the cytotoxic activity of dengue virus-induced cytotoxin.

The role of calcium ions (Ca2+) in the cytotoxic activity of the dengue type 2 virus (DV)-induced macrophage (M phi) cytotoxin (CF2) was investigated in the present study. The findings show that CF2 prepared in Ca(2+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca(2+)-free medium but killed the cells suspended in the medium with Ca2+. Substitution with calcium chloride restored the cytotoxic activity of CF2 the optimal dose being 10(-4) M concentration. CF2 induced an influx of Ca2+, as assayed by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, viz. M phi and T lymphocytes. The cytotoxic activity of CF2 as well as the CF2-induced influx of 45Ca was inhibited by treatment of the target cell with the calcium channel blocking drugs verapamil and nifedipine. Thus, the presence of Ca2+ is obligatory for the cytotoxic activity of CF2 and cell death is associated with increased intracellular Ca2+.

Animals↗

PubMed1991

Presence of Ca2+ is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor.

The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2(+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2(+)-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10(-7) M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (M phi) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in M phi and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.

Animals↗

PubMed1990

Effect of dengue virus-induced cytotoxin on capillary permeability.

Capillary permeability is increased in cases of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) but its genesis is not known. Dengue type 2 virus (DV) induces production of a cytokine (CF2) by mouse macrophages. The present study was undertaken to investigate the effect of CF2 on capillary permeability. It was observed that intraperitoneal inoculation of CF2 in mice increased the capillary permeability in a dose-dependent manner, as shown by leakage of intravenously injected radioactive iodine (125I) or Evan's blue dye in the peritoneal cavity. Peak leakage occurred at 30 min and the vascular integrity was restored by 1-2 h. The increase in capillary permeability was abrogated by pretreatment of mice with avil (H1 receptor blocker) but not by ranitidine (H2 receptor blocker). The findings thus show that DV-induced CF2 increases the capillary permeability via release of histamine.

Animals↗

PubMed1990

Increased capillary permeability mediated by a dengue virus-induced lymphokine.

The mechanism of increased capillary permeability, seen in cases of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), is not known. Dengue type 2 virus (DV) is known to induce production of a lymphokine, the cytotoxic factor (CF), by the T lymphocytes of mouse spleen. The data presented here show that intraperitoneal inoculation of CF in mice results in increased capillary permeability in a dose-dependent manner, as shown by leakage of intravenously injected radiolabelled iodine (125I) or Evans blue dye. Peak leakage occurred 30 min after inoculation of CF and the vascular integrity was restored by 2 hr. The increase in capillary permeability was abrogated by pretreatment of mice with anti-CF antibodies, avil (H1 receptor blocker) or ranitidine (H2 receptor blocker). The findings thus show that a DV-induced lymphokine, the CF, increases the capillary permeability via release of histamine.

Animals↗

PubMed1989

Proteinase-like activity in the cytotoxic factor produced by T cells during dengue virus infection.

Dengue type 2 virus (DV)-induced cytotoxic factor (CF) or the virus-primed spleen cell capable of secreting CF were treated with various proteinase inhibitors and their activity was assayed. It was observed that the cytotoxic activity of CF was inhibited significantly, in a dose-dependent manner, by pretreatment with bovine pancreatic trypsin inhibitor (BPTI) and phenylmethylsulphonyl fluoride (PMSF), to a lesser extent by soya-bean trypsin inhibitor (SBTI) and leupeptin and not at all by 1,10-phenanthroline (OP). Similar effects were observed by pretreatment of DV-primed spleen cells. Amidolytic activity of CF or its purified fractions was assayed using twelve chromogenic peptide substrates and all the substrates were hydrolysed to the varying extent. The amidolytic activity of CF was also inhibited by pretreatment with proteinase inhibitors. Thus, CF could be a proteinase with the distinction of having a broad spectrum of activity.

Animals↗

PubMed1983

Fetus in fetu.

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Abdominal Neoplasms↗