Peganine hydrochloride dihydrate an orally active antileishmanial agent

Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bioassay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (±8.07)% against established VL in hamsters at a dose of 100 mg/kg b.wt.     

Pre- and post-challenge immuno-haematological changes in Labeo rohita juveniles fed gelatinised or non-gelatinised carbohydrate with n-3 PUFA

The combined effect of dietary carbohydrate type and n-3 PUFA (EPA+DHA) on pre- and post-challenge haemato-immunological responses in Labeo rohita juveniles was studied. Fish were fed for 67days with six different test diets containing either gelatinised (G) or non-gelatinised (NG) corn (43%) with three levels of n-3 PUFA (0.5%, 1.0% and 2.0%). During the pre-challenge period, significantly higher (P<0.05) NBT, serum lysozyme activity, total protein and globulin content was recorded in the NG carbohydrate fed groups. Highest NBT value was recorded in the groups fed with 1.0% n-3 PUFA, whereas the highest serum lysozyme activity (P<0.05) was recorded at either 0.5% or 2.0% n-3 PUFA fed groups in both the pre- and post-challenge period. Feeding of NG corn significantly increased the total leucocyte count, lysozyme activity, A/G ratio and decreased the total erythrocyte count, haemoglobin, serum total protein and globulin content of L. rohita juveniles during the post-challenge period. Similarly, feeding of n-3 PUFA at any level significantly increased the immunological parameters like lysozyme activity or A/G ratio, whereas total leukocyte count increased due to feeding of either 0.5% or 1.0% n-3 PUFA. The NBT and albumin values remained similar in both the pre- and post-challenge period. After challenge with Aeromonas hydrophila, the highest survival was recorded in the NG carbohydrate fed groups, whereas the lowest survival was recorded in the highest level of n-3 PUFA fed group irrespective of dietary carbohydrate type. Thus, a high level of G carbohydrate as well as n-3 PUFA is found to be immunosuppressive in L. rohita juveniles. NG carbohydrate supplemented with 1.0% n-3 PUFA is found to be optimum to enhance the immunity in L. rohita juveniles.     

Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart

The precise molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. Toll-like receptor-4 (TLR-4) engages lipopolysaccharide (LPS) and activates signaling pathways leading to the expression of proinflammatory cytokines implicated in myocardial dysfunction. We determined whether TLR-4 was necessary for LPS-induced myocardial dysfunction in vivo. The effects of LPS on left ventricular (LV) function were studied in mice with defective TLR-4 signaling (C3H/HeJ, TLR-4 deficient) and wild-type mice (C3HeB/FeJ). Mice (n = 5/group) were injected with LPS or diluent, and LV function was examined by using two-dimensional echocardiography and conductance catheters. LPS significantly decreased all indexes of LV function in wild-type mice when compared with controls; LV function was not depressed in the LPS-treated TLR-4-deficient mice relative to controls. LPS increased myocardial nitric oxide synthase-2 expression and cGMP only in wild-type mice. This study suggests that TLR-4 mediates the LV dysfunction that occurs in LPS-induced shock. Therefore, TLR-4 might be a therapeutic target for attenuating the effects of LPS on the heart.     

Lymphocyte plasma membranes II. Cytochemical localization of 5′-nucleotidase in rat lymphocytes

Cytochemical studies of thymic and splenic lymphocytes from rats showed that 5′-nucleotidase was restricted to the plasma membranes. Isolated plasma membranes contained the highest specific activity of 5′-nucleotidase of any cellular fractions. The results indicate that this enzyme can be used as a plasma membrane marker for lymphocytes.     

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.     

Induction of Apoptosis in G1/S Blocked HeLa Cells by R-Roscovitine: A Preliminary Study

HeLa cells are human cervical cancer cells with HR HPV-18 genes integrated in the genome. The functions of tumor suppressor proteins p53 and pRB are abrogated and cell cycle regulation becomes nonfunctional. The aim of the present study was to investigate whether the CDK inhibitor R-Roscovitine would allow the G₁/S blocked HeLa cells to enter into mitosis prematurely and induce apoptosis. HeLa cells blocked in G₁/S border were treated with different concentrations of Roscovitine for 4 and 18 h respectively. Induction of apoptosis was studied by FACS and DNA fragmentation. Presence of γH2AX in the treated cells was studied by confocal microscopy. Expression levels of CASP3, CDKN1A i.e. p21 (Cip1/Waf1) and Bcl2 were studied by semi-quantitative RT-PCR to analyze the role played by these proteins in Roscovitine induced apoptosis in G₁/S blocked HeLa cells. Results indicate that the Roscovitine allowed the thymidine blocked HeLa cells to enter into mitosis prematurely. Presence of γH2AX loci in treated cells indicates DNA damage in prematurely mitotic cells. Analysis of DNA fragmentation and chromatin condensation confirmed apoptosis as the possible mechanism of Roscovitine induced cell death. Our results also reveal that Roscovitine induced apoptosis is associated with the overexpression of CASP3, p21 (cip1/waf1) and Bcl2.     

PprA Contributes to Deinococcus radiodurans Resistance to Nalidixic Acid,Genome Maintenance after DNA Damage and Interacts with Deinococcal Topoisomerases

PprA is known to contribute to Deinococcus radiodurans'' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA''s role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans''s capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.     

Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum

The effect of varying the solute species on the crystallization of the Ca2(+)-ATPase from rabbit muscle reticulum (SR) is reported. We have found that substitution of KCl with salts of organic acids in the crystallization protocol reported by Pikula et al. has a profound effect on the size of two-dimensional crystalline arrays. Crystalline arrays of up to 3 microns diameter have been obtained by incubating purified calcium ATPase in standard crystallization medium but with 0.8 M sodium propionate substituted for KCl. These two-dimensional (2-D) arrays display a reduced tendency to stack in addition to having larger planar dimensions. Increasing the KCl concentration does not have the same effect on stacking or crystal growth that sodium propionate has. The production of 2-D sheets has some dependence on the hydrocarbon chain length of the salt because crystals formed in propionate were larger and less stacked than those formed in acetate or formate. There seems to be no dependence on cation. These observations suggest that in addition to reducing the forces that lead to stacking of the sheets, propionate may facilitate incorporation of the detergent-solubilized protein into the 2-D sheet.