Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: antigenicity, biophysics, and immunogenicity

The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.     

Melatonin Does Not Modulate Testicular Responsiveness to Altered Photoperiods. A Study During Different Phases of the Annual Gonadal Cycle in Roseringed Parakeets (Psittacula krameri)

The roseringed parakeet has been shown to exhibit a variable testicular responsiveness to both altered photoperiodic regimens and to treatment with melatonin during different phases of the annual gonadal cycle. Adult male roseringed parakeets were held under either natural photoperiods (NP), or long photoperiods (LP; 16L 8D), or short photoperiods (SP; 8L 16D) for a total period of 90 days. From day 46 onward, half of the total birds in each group were administered with the vehicle of melatonin, and the other birds were injected daily in the afternoon with melatonin (25 µg/ 100 g body wt.) till the end of the experiment. An identical experimental schedule was followed during the four different (preparatory, progressive, pre-breeding, and breeding) phases of the annual testicular cycle. The testicular activities in various bird groups were evaluated by volumetric, gravimetric, histometric and karyometric measurements, and by quantitative histological studies. The findings revealed that exogenous melatonin may exert either a suppressive influence or none at all on the testicular functions in relation to the photoperiodic schedule as well as to the reproductive phase of the concerned bird, but in no case modulates gonadal responsiveness to artificially altered photoperiods.     

Hemoglobin E distribution in ten endogamous population groups of Assam, India

Previous studies have reported a high incidence of hemoglobin E (HbE) in Northeast Indian populations. In the present study 10 endogamous populations of Assam belonging to two racial groups, Caucasoid and Mongoloid, were examined. The frequency of HbE gene (Hb beta E) in the Caucasoid caste populations is around 0.1, whereas the gene is highly prevalent in the Mongoloid populations, frequencies ranging between 0.2 and 0.6. Predominance of Hb beta E in the Tibeto-Burman speakers is contrary to observations made in Southeast Asia, where an association between Austro-Asiatic speakers and high prevalence of HbE exist. The highest occurrence of the gene in this area, which is on the far end of the proposed centre of distribution in Northern Kampuchea and Northeast Thailand, is also a deviation from the expected pattern of gene distribution. It is speculated that Hb beta E in the Tibeto-Burman populations of Assam arose by an independent mutation which contributed to the high frequencies of Hb beta E in the Northeast Indian populations.     

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl(4) to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl(4) supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.     

Production of some extracellular enzymes by a lignin peroxidase-producing brown rot fungus, Polyporus ostreiformis, and its comparative abilities for lignin degradation and dye decolorization.

Polyporus ostreiformis produced Mn peroxidase, acid protease, alpha-amylase, and lignin peroxidase, with maximum activities of 40, 8,300, and 4,200 U liter-1 and 50 nkat liter-1, respectively, in nitrogen-limited liquid media. The fungus removed only 18.6% lignin from rice straw in 3 weeks but effected 99% decolorization of Congo red dye in 9 days.     

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.     

Loss of cannabinoid receptor CB1 induces preterm birth

Background

Preterm birth accounting approximate 10% of pregnancies in women is a tremendous social, clinical and economic burden. However, its underlying causes remain largely unknown. Emerging evidence suggests that endocannabinoid signaling via cannabinoid receptor CB1 play critical roles in multiple early pregnancy events in both animals and humans. Since our previous studies demonstrated that loss of CB1 defers the normal implantation window in mice, we surmised that CB1 deficiency would influence parturition events.

Methods and Findings

Exploiting mouse models with targeted deletion of Cnr1, Cnr2 and Ptgs1 encoding CB1, CB2 and cyclooxygenase-1, respectively, we examined consequences of CB1 or CB2 silencing on the onset of parturition. We observed that genetic or pharmacological inactivation of CB1, but not CB2, induced preterm labor in mice. Radioimmunoassay analysis of circulating levels of ovarian steroid hormones revealed that premature birth resulting from CB1 inactivation is correlated with altered progesterone/estrogen ratios prior to parturition. More strikingly, the phenotypic defects of prolonged pregnancy length and parturition failure in mice missing Ptgs1 were corrected by introducing CB1 deficiency into Ptgs1 null mice. In addition, loss of CB1 resulted in aberrant secretions of corticotrophin-releasing hormone and corticosterone during late gestation. The pathophysiological significance of this altered corticotrophin-releasing hormone-driven endocrine activity in the absence of CB1 was evident from our subsequent findings that a selective corticotrophin-releasing hormone antagonist was able to restore the normal parturition timing in Cnr1 deficient mice. In contrast, wild-type females receiving excessive levels of corticosterone induced preterm birth.

Conclusions

CB1 deficiency altering normal progesterone and estrogen levels induces preterm birth in mice. This defect is independent of prostaglandins produced by cyclooxygenase-1. Moreover, CB1 inactivation resulted in aberrant corticotrophin-releasing hormone and corticosterone activities prior to parturition, suggesting that CB1 regulates labor by interacting with the corticotrophin-releasing hormone-driven endocrine axis.