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Biomedical subjects

K Venkateswarlu

Publications and source records attributed to K Venkateswarlu.

At least 55 records · Page 3Linked to original sources
PubMed1997

In-vitro activity of D0870, a new triazole antifungal drug, in comparison with fluconazole and itraconazole against Aspergillus fumigatus and Candida krusei.

The activity of the new triazole antifungal D0870 was compared with those of itraconazole and fluconazole against Candida krusei and Aspergillus fumigatus, two fungi showing inherent tolerance of fluconazole. The activity of D0870 resembled that of itraconazole against whole cells of C. krusei, but it was less effective against A. fumigatus. However, the effect on sterol biosynthesis, in terms of the sterol type accumulating and IC50 for in-vitro biosynthesis, appeared similar in both species. The superior antifungal effect of D0870 over fluconazole appeared related to better inhibition of ergosterol biosynthesis in A. fumigatus, but in C. krusei this did not account for the entire difference which may result mainly from reduced efflux of drug.

Antifungal Agents↗
PubMed1997

Fluconazole tolerance in clinical isolates of Cryptococcus neoformans.

Eleven isolates of Cryptococcus neoformans were investigated to determine the biochemical basis of their tolerance to fluconazole. The MICs of fluconazole for three isolates with low-level resistance were 3- to 6-fold higher than those for sensitive isolates, while the MICs for four isolates with high-level resistance were 100- to 200-fold higher than those for sensitive isolates. The level of ergosterol present in the isolates varied, and those which had relatively low levels of ergosterol were resistant to amphotericin B. Changes in the affinity of the target enzyme (sterol 14alpha-demethylase) and decreases in the cellular content of fluconazole seemed to be responsible for the resistance in isolates with low-level and high-level resistance, respectively.

Amphotericin B↗
PubMed1997

Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.

Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

Azoles↗
PubMed1997

Itraconazole resistance in Aspergillus fumigatus.

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

Adult↗
PubMed1996

Evidence for cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium.

The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448-450 and 452-454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH dependent and inhibited by carbon monoxide. Km values for activity showed a difference between the cellular fractions with a Km of 89 microM for microsomal P-450 and 400 microM for cytosolic P-450. The Vmax values observed were 0.83 nmol min-1 (nmol microsomal P-450)-1 and 0.4 nmol min-1 (nmol cytosolic P-450)-1. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.

Basidiomycota↗
PubMed1996

Biochemical characterisation of ketoconazole inhibitory action on Aspergillus fumigatus.

The effect of ketoconazole on growth, sterol composition, in vitro sterol biosynthesis and P450-CO complex formation and its interaction with microsomal P450 was determined. On solid medium and in liquid medium ketoconazole inhibited Aspergillus fumigatus growth completely at 5 x 10(-5) M and 50% of the growth at 1.3 x 10(-5) M and 2.1 x 10(-5) M respectively. A close relationship between accumulation of 14 alpha-methyl sterols (eburicol, obtusifoliol and 14 alpha-methyl fecosterol) and depletion of ergosterol with growth arrest was observed in ketoconazole treated cultures. The half inhibitory concentration for in vitro ergosterol biosynthesis and half saturating concentration for type II binding spectrum of ketoconazole were calculated as 73.8 +/- 6.3 nM and 0.13 +/- 0.04 microM respectively. CO displacement studies revealed inhibition of CO-P450 complex formation by ketoconazole.

Antifungal Agents↗
PubMed1996

Reduced accumulation of drug in Candida krusei accounts for itraconazole resistance.

Due to intrinsic resistance Candida krusei is emerging as a systemic pathogen in AIDS patients undergoing fluconazole therapy, but acquired resistance to itraconazole has not been studied biochemically. We report here studies on the basis for azole resistance and sterol composition in C. krusei. An itraconazole-resistant isolate showed reduced susceptibility to azole drugs in in vitro growth inhibition studies. Accumulation of 14 alpha-methyl-3,6-diol under azole treatment was associated with growth arrest. In vitro ergosterol biosynthesis and type II binding studies suggested no alteration in the affinity to azole drugs of the target enzyme, the cytochrome P-450 sterol 14 alpha-demethylase, in the resistant isolate. Resistance was associated with a decreased intracellular content of drug in the resistant isolate.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays.

D0870 was 12 to 15 times more active than fluconazole in experiments to determine the MIC for growth arrest for two isolates of Candida albicans. A biochemical comparison of in vitro sterol biosynthesis in cell extracts showed only a twofold superiority of D0870 over fluconazole. A large differentiation (10-fold) in 50% saturating concentrations obtained by examining the binding of the azoles to microsomal P-450 was observed in a type II binding spectrophotometric assay, possibly reflecting the differential affinity for more than one P-450 enzyme. Additional mechanisms besides affinity for the target enzyme sterol 14 alpha-demethylase, such as differential intracellular accumulation of drug, may contribute to the differences in antifungal activity.

Antifungal Agents↗
PubMed1995

Resistance to fluconazole in Candida albicans from AIDS patients correlated with reduced intracellular accumulation of drug.

Mucosal candidosis is an almost inevitable consequence of AIDS. Resistance to fluconazole therapy associated with enhanced tolerance, detectable in microbiological estimation of sensitivity, occurs in up to 10% of cases with late-stage AIDS. We report here our biochemical analysis of the basis of resistance in a study of two susceptible and two resistant isolates. Resistance was not associated with a change in the target enzyme sterol 14 alpha-demethylase, as indicated by equivalent levels of fluconazole inhibition of activity in extracts from all four isolates, or by mutations in sterol delta desaturase as previously observed in Saccharomyces cerevisiae and Ustilago maydis. Reduced cellular content of fluconazole in the resistant isolates of between six to ten-fold was observed which could account for their resistant phenotype.

AIDS-Related Opportunistic Infections↗
PubMed1995

Effective concentrations of nitrophenolics toward growth yield of selected microalgae and cyanobacteria isolated from soil.

The effective concentrations (EC), which cause a certain percentage of inhibition in growth yield, of four nitrophenolics (o-nitrophenol, m-nitrophenol, p-nitrophenol (PNP), and 2,4-dinitrophenol) and the major metabolites of PNP reduction (p-nitrosophenol and p-aminophenol) toward growth of two microalgae (Chlorella vulgaris and Scenedesmus bijugatus) and two cyanobacteria (Nostoc muscorum and Nostoc linckia), all isolated from soil, were determined following linear regression analysis. The EC50 values of the selected toxicants toward the test organisms were in the range of 32 to 227 micrograms ml-1. Such a wide range in EC figures emphasizes the need for a prior determination of EC values of a toxicant before establishing its toxicity pattern toward a nontarget microorganism.

2,4-Dinitrophenol↗
PubMed1993

Nontarget effects of carbaryl and its hydrolysis product, 1-naphthol, towards Anabaena torulosa.

The toxic effects of carbaryl and its hydrolysis product, 1-naphthol, singly and in combination, towards a filamentous diazotrophic cyanobacterium, Anabaena torulosa, were determined. The toxicity criteria employed, viz., packed cell volume, chlorophyll a, phycocyanin, carotenoids, heterocyst differentiation and nitrogen fixation, were greatly affected by the toxicants above 25 micrograms/ml concentrations. However, 1-naphthol was more toxic to the diazotroph than was carbaryl. The toxicity was more pronounced when carbaryl and 1-naphthol were in combinations even at lower concentrations.

Anabaena↗
PubMed1992

Interactions of monocrotophos and quinalphos with Anabaena torulosa isolated from rice soil.

The interaction of insecticide combinations of monocrotophos and quinalphos on Anabaena torulosa, by the criteria of absorbance (OD) and packed cell volume (PCV) of the culture, content of chlorophyll a, phycocyanin, carotenoids, total protein, DNA and RNA, heterocyst differentiation and nitrogen fixation, were assessed. In general, monocrotophos and quinalphos, in combination, interacted significantly yielding three different responses viz., additive, antagonistic or synergistic. The nature of the interaction found with OD and PCV was nearly the same for a particular concentration of the insecticides. However, no consistent interaction was observed with respect to carotenoids and phycocyanin. The three types of interaction were noticed for total protein, DNA and RNA. Interestingly, the insecticide combinations at lower concentrations yielded all interaction responses for heterocyst frequency and nitrogenase activity. But, higher concentrations, in combination, resulted in synergism for heterocyst differentiation and nitrogen fixation.

Anabaena↗
PubMed1991

Conserved upstream sequence elements in plant 5S ribosomal RNA-encoding genes.

As a basis for further comparative studies, nuclear 5S rRNA gene repeats from two plants of the Solanaceae family, tobacco (Nicotiana rustica) and tomato (Lycopersicon esculentum), were isolated and sequenced. The more abundant 5S rRNA gene repeat in tobacco is 430 bp long, while a second less common variant is 521 bp long. In contrast, the 5S rRNA gene repeat from tomato is only 355 bp long. The spacer sequences from these gene repeats, as well as from other published plant nuclear 5S rRNA genes, were compared for repeating or conserved sequence elements. The results indicate that often observed, but non-conserved, repeating sequence elements probably arise spontaneously by unequal crossover with no functional significance. However, three conserved sequence elements immediately upstream of the coding sequence; a C residue at -1, a G + C-rich element centered at -13, and an A + T-rich element centered at -26 resemble regulatory features which have been identified in other types of genes.

Base Sequence↗
PubMed1991

A conserved core structure in the 18-25S rRNA intergenic region from tobacco, Nicotiana rustica.

To identify conserved and functionally important features in the intergenic sequences of ribosomal DNAs, the nucleotide sequence of the 18-25S rRNA intergene region in tobacco rDNA was determined and compared to that of other higher plants. Unlike previous comparisons of more diverse organisms, sufficient sequence homology is retained in the higher plants to examine the evolutionary changes which make these regions diverse. Estimates of the secondary structure permit the identification of a 'core-like' structure which appears to maintain the processed sites in close proximity and can be identified in the more divergent sequences.

Base Sequence↗
PubMed1991

Evidence for T-DNA mediated gene targeting to tobacco chloroplasts.

The integration of foreign DNA into plant cells by Agrobacterium mediated transformation is random and normally directed at the nucleus. Here we present evidence that such transformation can be used to introduce foreign genes into higher plant chloroplasts by site-specific homologous recombination. A binary vector was made in vitro that included chloroplast ribosomal DNA (rDNA) within T-DNA borders, and transgenic tobacco plants obtained using this construct were analyzed for the targeted recombination by hybridization, polymerase chain reaction (PCR) and DNA sequencing.

Base Sequence↗
PubMed1990

Stimulation of ammonification and nitrification in soils by the insecticides monocrotophos and quinalphos.

The application (up to 5 kg ha-1) of monocrotophos or quinalphos to four types of agricultural soils significantly stimulated the mineralization of peptone-nitrogen and the oxidation of ammonium-nitrogen. In soils treated with 2.5 kg ha-1 of either insecticide, the rate of ammonification and nitrification was fairly rapid after 2 and 4 weeks of incubation. The enhancement of both transformations, mediated by microorganisms, was significantly more in the quinalphostreated soils. The results suggest that a balance between the effect of insecticides on insect pests and their impact on beneficial microbial activities in soil must be determined.

Ammonia↗