Inhibition of heterochromatin condensation of human Y chromosome by distamycin-A

Distamycin-A, an oligopeptide antibiotic with a N-methylpyrrole ring system and propionamide side chain, preferentially forms stable bonds with AT rich double stranded DNA. When introduced to cell cultures, it inhibits condensation of the heterochromatic region of the Y chromosome. The frequency of metaphases showing inhibition of heterochromatin condensation of the Y chromosome was found to be dependent on the treatment time and concentration of distamycin-A in the culture medium. When distamycin-A was added to a concentration of 100 micrograms/ml at the start of the culture (72 hours), the frequency of Y heterochromatin decondensation was found to be 48%, 30% and 6% in amniotic fluid, lymphocyte and fibroblast cultures respectively. The highest frequency of metaphases with decondensed Y heterochromatin were observed when distamycin-A treatment was carried out for the last 24 hours prior to harvest, the frequencies being 94%, 72% and 59% in amniotic fluid, lymphocyte and fibroblast cultures respectively. Increase in the concentration of distamycin-A from 25 micrograms/ml to 50 micrograms/ml during the last 24 hours of culture increased the incidence of metaphases with Y heterochromatin decondensation from 51% to 69% in amniotic fluid, 40 to 49% in lymphocyte and 29% to 31% in fibroblast cultures. Highest frequency of metaphases with Y heterochromatin decondensation were observed when the cultures were exposed to distamycin-A at a concentration of 100 micrograms/ml for the last 24 hours of culture.     

An approach for immunodiagnosis of clinical cases of filariasis

In this communication an immunodiagnostic approach has been adopted for detection of antigen and antibody in amicrofilaeamic Mf(-) patients by countercurrent immuno electrophoresis (CCIE) and immunodiffusion (ID). Using Setaria cervi and Immune Complex (IC) antigens, out of fifteen clinical cases the number of positive patients in CCIE were twelve and ten respectively. Sixty percent of the Mf(-) cases were positive in antigen detection against both the homologous and heterologous antibody. In ID nine Mf(-) cases gave precipitin bands against S. cervi antigen while with IC antigens ten patients were positive. In similar experiments, it was found that out of fifteen Mf(-) cases nine and eleven patients were positive in antigen detection against microfilaraemic Mf(+) sera and S. cervi antibody respectively. All the Mf(+) cases were positive in both antibody and antigen detection. From the standpoint of immunodiagnosis the data were analysed by two-way analysis of variance study and a newly developed system using Binomial distribution. The sera from the control group were negative in all the immunodiagnostic tests.     

Frequency of micronuclei induced in peripheral lymphocytes by trimethyltin chloride

The clastogenicity of trimethyltin chloride was evaluated in human peripheral blood lymphocytes with micronucleus counts (MNC) as the endpoint. Two concentrations (0.5 micrograms and 1.0 microgram) of trimethyltin chloride were added to lymphocytes of healthy male and female subjects of different age groups, in mitogen-stimulated and serum-supplemented culture medium (RPMI 1640, Gibco) for 48 h at 37 degrees C. A significant increase in micronucleus counts was observed with both doses, which was more pronounced with the lower dose. ANOVA in male and female donors revealed significant differences between age groups (P less than 0.001), chemical concentrations (P less than 0.001) and for the interaction of these 2 factors (P less than 0.05 in females only). However, no regular increase or decrease in MNC frequencies was observed with the donor's age. Higher frequencies of MNC enhancement were observed in male individuals than in females.     

Leishmania donovani: amastigote inhibition and mode of action of berberine

Berberine, an alkaloid from Berberis aristata Linnaeus, may be a useful drug for the treatment of visceral leishmaniasis. In both the 8-day and long-term models of Leishmania donovani infection in hamsters, it markedly diminished the parasitic load and proved to be less toxic than pentamidine. It rapidly improved the hematological picture of infected animals. Like pentamidine, it inhibited in vitro multiplication of amastigotes in macrophage culture and their transformation to promastigotes in cell free culture. Manometric studies showed that both drugs had inhibitory action on both the endogenous and the glucose-stimulated respiration of amastigotes. They inhibited incorporation of [14C]adenine, [14C]uracil, and [3H]thymidine into nucleic acids, and of [14C]leucine into the protein of amastigotes, indicating an inhibitory action on macromolecular biosynthesis. They also decreased deoxyglucose uptake. Using spectrophotometric, spectrofluorimetric, and circular dichroism techniques, berberine was found to interact in vitro with nuclear DNA from L. donovani promastigotes.     

Synthetic inhibitors of human leukocyte elastase Part 1--Sulphated polysaccharides

Ten dextran sulphates and six chitosan sulphates of variable Mr and extent of sulphate substitution have been examined for their ability to inhibit human leukocyte elastase (HLE). All were potent partial non-competitive inhibitors of this enzyme, highest activity being obtained with compounds of large molecular weight and maximum sulphate incorporation (Ki = 5.0 X 10(-10)M]. In all cases, the dextran sulphates were more effective inhibitors than chitosan sulphates of similar size and charge, but both classes were inactive against bovine trypsin, chymotrypsin and porcine pancreatic elastase at concentrations less than 10(-4)M. The data suggest that drug binding to HLE occurs by stereospecific electrostatic interactions at site(s) removed from the catalytic reaction centre.     

Postharvest production of ochratoxin A by Aspergillus ochraceus and Penicillium viridicatum in barley with different protein levels

The production of ochratoxin A (OA) in barley by Aspergillus ochraceus and Penicillium viridicatum was measured at 12 and 25 degrees C. The grain had been fertilized with various amounts of nitrogen fertilizer (0, 90, or 240 kg/ha) and contained (at crop maturity) 9.1, 10.4, or 12.0% protein, respectively. The production of OA by both fungi increased as the protein concentration increased. Glutamic acid and proline were enriched relative to other amino acids as the protein concentration increased. The differences in OA production could not be explained by a differential effect of protein or amino acids on fungal growth in barley. However, glutamic acid and proline enhanced OA production in liquid cultures of both A. ochraceus and P. viridicatum.     

Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.     

Interaction between two group IV metals-lead and zirconium-in bone marrow cells ofMus musculus in vivo

The interaction between two group IV metals, the highly toxic lead and the relatively inactive and low toxic zirconium, was studied in the bone marrow chromosomes ofMus musculus in vivo. Low and high doses of zirconium oxychloride were fed orally to the experimental mice (i) 2 h before, (ii) 2 h after or (iii) together with different doses of lead nitrate. Protection against lead-induced clastogenicity was observed only when the lower dose of zirconium was administered prior to lead. All other combinations gave an additive or synergistic effect as was seen by significant increases in the frequencies of chromosomal aberrations.     

Allosteric modulation of Leishmania donovani plasma membrane Ca(2+)-ATPase by endogenous calmodulin.

The plasma membrane of the human pathogen Leishmania donovani possesses a high-affinity transmembrane Ca(2+)-ATPase that has its catalytic site oriented toward the cytoplasmic milieu (Ghosh, J., Ray, M., Sarkar, S., and Bhaduri, A. (1990) J. Biol. Chem. 265, 11345-11351). When the enzyme is studied in its more authentic, physiologically relevant, membrane-associated form, it exhibits pronounced sigmoidal kinetics with Ca2+ (K0.5 approximately 700 nM) in a trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid buffering system that effectively complexes all available Mg2+. Addition of exogenous Mg2+ (60 microM) completely abolishes sigmoidicity and establishes strictly hyperbolic kinetics, and the Km for Ca2+ reduces to 100 nM. Mg2+ can be replaced by heterologous calmodulin. The exclusive dependence of the enzyme on only Ca2+ for its activity and its positive allosteric modulation by Mg2+ distinguish this enzyme from other well-characterized plasma membrane Ca(2+)-ATPases. Employing this Ca(2+)-ATPase as the assay system, a soluble endogenous activating protein factor was purified that, by several criteria, corresponds to authentic calmodulin. The parasite calmodulin shifts the kinetics to hyperbolic kinetics, increases the Vmax 2-fold, and most important lowers the Km (approximately 100 nM) to a physiological level. The interaction with endogenous calmodulin thus converts the enzyme from a totally inactive to a fully active state.