Anil K. Rastogi

In vitro cultivation and characterization of axenic amastigotes of Leishmania.

The establishment of axenic cultures of the amastigote stage of Leishmania is important to understand the mechanisms regulating the differentiation, survival and pathogenicity of the parasite with a view to develop and identify molecular and chemotherapeutic targets. Recent developments in axenic culture and the characterization of amastigotes of different species of Leishmania are discussed.

Oxy free radical system in heart failure and therapeutic role of oral vitamin E

Twenty patients of heart failure and ten matched healthy controls were included in the trial. Out of these 20 patients of heart failure, 12 patients were also studied prospectively. Plasma levels of superoxide anion and malonyldialdehyde were increased while the levels of superoxide dismutase, catalase and glutathione reductase were decreased in patients of heart failure as compared to control subjects. The alteration in oxidative stress and antioxidant system did not correlate with the age and sex of patients or the etiology of heart failure. With the increasing severity of heart failure the malonyldialdehyde and superoxide anion increased significantly and catalase, glutathione reductase and superoxide dismutase levels decreased. The group of heart failure patients with ejection fraction < 40% (n = 7) exhibited significantly higher levels of malonyldialdehyde than those with an ejection fraction > 40% (n = 13). The superoxide anion and malonyldialdehyde levels were significantly higher in patients of heart failure in the pre-treatment state as compared to those in post-treatment state. Conversely catalase, glutathione reductase and superoxide dismutase were higher in the post-treatment period as compared to their values before treatment. The addition of vitamin E in doses of 400 mg once a day orally for 4 weeks significantly reduced the malonyldialdehyde and superoxide anion levels and produced an elevation of the antioxidant enzymes. Thus, there is an apparent normalisation of the indices of oxidative stress following treatment of heart failure and a markedly improved response on vitamin E supplementation which may be more beneficial.

Insulin like activity in (-) epicatechin.

SummaryWater extract of the bark of plant ofPterocarpus marsupium Roxb is used as an antidiabetic drug in indigenous medicine in India. (−) Epicatechin, its active principle, has been found to be insulinogenic. The presentin vitro study reports some insulin like activities of (−) epicatechin. Like insulin, (−) epicatechin stimulates oxygen uptake in fat cells and tissue slices of various organs, increases glycogen content of rat diaphragm in dose-dependent manner with corresponding increase in U14-C glucose uptake, and inhibits theophylline induced lipolysis in isolated fat pads in dose-dependent manner. Experiments on competitive binding of125I-insulin and (−) epicatechin to liver cell plasma membrane indicate that insulin does not share binding site with (−) epicatechin. (−) Epicatechin at a concentration of up to 1 mM does not effect the release of glucagon from the isletsin vitro. Thus, (−) epicatechin has insulinogenic as well as insulin like properties.

Hypoglycemie activity of Pterocarpus marsupium wood

Abstract Feeding of the ethyl acetate-soluble fraction of an absolute ethanol extract of Pterocarpus marsupium wood for 5 days significantly lowered blood sugar levels with a corresponding increase in the blood insulin level in alloxan-diabetic rats.

Characterization of intracellular metabolites of axenic amastigotes of Leishmania donovani by 1H NMR spectroscopy.

The intracellular metabolites of long-term in vitro cultured axenic amastigotes of Leishmania donovani (strain Dd8) were determined and compared with those of promastigotes and intracellular amastigotes, employing proton NMR spectroscopy. The presence of two new metabolites, i.e. betaine and beta-hydroxybutyrate were reported. Betaine was detected in all the three stages being highest in the promastigotes while beta-hydroxybutyrate could be detected only in promastigotes and axenic amastigotes. Among other metabolites, succinate and valine were found in higher quantities in intracellular amastigotes and axenic amastigotes than in promastigotes. Acetoacetate was present only in axenic and intracellular amastigotes. The comparative metabolite profile of different parasite forms reveals that axenic amastigotes seem to represent an intermediate stage between promastigotes and intracellular amastigotes in spite of their strong resemblance to intracellular amastigotes in morphology, infectivity, biochemical studies and even in the manifestation of amastigote specific A2 protein.

Protective effect of Picroliv from Picrorhiza kurroa against Leishmania donovani infections in Mesocricetus auratus.

The prevalent drugs for treatment of kala azar viz. sodium stibogluconate (SSG) and pentamidine cause severe toxic side effects and acute immunosuppression in the treated individuals. Picroliv, a standardized mixture of iridoid glycosides, prepared from the alcoholic extract of the root and rhizome of Picrorhiza kurroa has shown strong hepatoprotective activity against several models of hepatotoxicity. Therefore, the present study was undertaken with an objective to study the effects of Picroliv (12.5 mg/kg x 7 days oral) alone and in combination with SSG on parasitemia, lipid peroxidation and hepatic marker enzymes of golden hamsters during Leishmania donovani infection. The results indicated a marked hepatoprotective effect of Picroliv in terms of biochemical markers, and a significant antileishmanial activity implying that it can be utilized as an adjunct to chemotherapy or in combination therapy of kala azar along with sodium stibogluconate, thus enhancing the efficacy of antileishmanials.

Molecular typing of HLA class I and class II antigens in Indian kala-azar patients

Summary HLA has been shown to be associated with many diseases. To find out whether host genetic factors like the HLA are involved in susceptibility to kala‐azar (visceral leishmaniasis) in India, we formulated an association study with genetically related controls. All samples were typed by PCR SSOP (sequence specific oligonucleotide probes) for HLA class I (A and B) and class II (DR) antigens. The test of association we used was the transmission disequilibrium test (TDT). No significant evidence for association with any of the three HLA loci was obtained.

Heme metabolism in promastigotes of Leishmania donovani

Promastigotes of Leishmania donovani (Dd-8 strain) showed presence of important key enzymes of heme synthesizing (d-aminolevulinic acid synthase and ferrochelatase) and degrading (heme oxygenase and biliverdin reductase) systems, classical leishmanicidal drugs viz allopurinol, amphotericin B, pentamidine and CDRI compound 93/202 inhibited the heme oxygenase activity of the parasite, whereas, δ-aminolevulinic acid synthase activity practically remained unaffected. The Km, Vmax ad pH values of heme oxygenase of promastigotes were found to be 1666 μM hemin, 625 nmol of bilirubin formed h-1 mg protein-1 and 7.5 respectively. The findings suggest the presence and importance of heme metabolism in the de novo synthesis of different hemoproteins of the Leishmania parasite as well as the detoxification and its defence against biological insults.

In vitro insulin action on different ATPases of erythrocyte membranes in normal and diabetic rats

SummaryThein vitro effect of porcine insulin on Na++K+-, Ca2+- and Mg2+-ATPases of the rat erythrocyte membrane of normal and alloxan-induced diabetic rats was investigated. Na++K+- and Ca2+-stimulated enzyme activities were significantly decreased in diabetic rats in comparison to normal animals. The specific activities of both these ATPases in the latter group were markedly reduced on pre-incubating the ghosts with insulin. Similar treatment of the erythrocyte membranes of diabetic animals, however, resulted in a significant increase of these activities. These qualitatively different effects of the hormone in the two groups increased progressively with hormone concentration and duration of pre-incubation. Mg2+-stimulated ATPase activity was not significantly affected in diabetes or by insulin.

Relative resistance of promastigotes of a virulent and an avirulent strain of Leishmania donovani to hydrogen peroxide.

Sensitivity of promastigotes of Leishmania donovani (Dd-8, virulent: UR-6 avirulent strain) to hydrogen peroxide has been studied in vitro conditions. There was a dose-dependent toxic effect of H2O2 on both strains. The promastigotes of Dd-8 strain, however, were more resistant compared to UR-6 strain. The cell response was not affected by the forms of H2O2 stress, for instance, single-dose exposure with reagent H2O2 or equivalent amount generated over a period of 1 h. Dd-8 promastigotes removed H2O2 from medium at a higher rate (6.43 nmol/min/ 3 x 10(7) cell/ml) compared to UR-6 promastigotes (3.17 nmol/min/3 x 10(7) cells/ml). Hydrogen peroxide metabolism by Dd-8 cells was significantly inhibited by aminotriazole, azide, and thiol reagents, while that of UR-6 cells was sensitive only to thiol reagents. The results are consistent with the negligible activity of glutathione peroxidase and nondetectable catalase in UR-6 promastigotes; both enzyme activities were present in Dd-8 promastigotes. These findings suggest that the ability of the parasite to resist H2O2 toxicity may have a potential role in its virulence.