NADPH oxidase as the source of ROS produced under waterlogging in roots of mung bean

The objective of this study was to examine the role of NADPH oxidase on superoxide radical production under waterlogging in mung bean (Vigna radiata) cvs. T 44 (tolerant) and Pusa Baisakhi (PB) (susceptible), and wild species Vigna luteola. Two days of waterlogging caused decline in superoxide radical (O₂ ^·−) contents in all the genotypes, however, further waterlogging up to 8 d caused significant increase in O₂ ^·− contents. In control and revived plants O₂ ^·− contents were higher in PB, while under waterlogging stress T 44 and V. luteola showed greater increases in the O₂ ^·− contents. During waterlogging the increase in O₂ ^·− content was found to be due to the diphenylene iodonium chloridesensitive NADPH oxidase (NOX). This was further confirmed by the waterlogging induced increase in NOX activity, which was higher in tolerant genotypes T 44 and V. luteola compared with PB. Gene expression studies showed enhanced expression of NOX in the roots of waterlogged V. luteola and T 44, while little expression was observed in control or treated plants of PB. PCR band products were cloned and sequenced, and partial cDNAs of NOX was obtained. Results suggest that increase in O₂ ^·− content during waterlogging could be due to the induction of membrane linked NOX.     

Discovery of inhibitors of plasminogen activator inhibitor-1: structure-activity study of 5-nitro-2-phenoxybenzoic acid derivatives

Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral bioavailability in the rodents.     

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [Cajanus cajan (L.) Millsp.] cv. JKR105

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L⁻¹ 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.     

A universal RNAi-based logic evaluator that operates in mammalian cells

Molecular automata that combine sensing, computation and actuation enable programmable manipulation of biological systems. We use RNA interference (RNAi) in human kidney cells to construct a molecular computing core that implements general Boolean logic to make decisions based on endogenous molecular inputs. The state of an endogenous input is encoded by the presence or absence of 'mediator' small interfering RNAs (siRNAs). The encoding rules, combined with a specific arrangement of the siRNA targets in a synthetic gene network, allow direct evaluation of any Boolean expression in standard forms using siRNAs and indirect evaluation using endogenous inputs. We demonstrate direct evaluation of expressions with up to five logic variables. Implementation of the encoding rules through sensory up- and down-regulatory links between the inputs and siRNA mediators will allow arbitrary Boolean decision-making using these inputs.     

Bone marrow transplantation restores follicular maturation and steroid hormones production in a mouse model for primary ovarian failure

Recent studies suggest that bone marrow stem cells (BMSCs) are promising grafts to treat a variety of diseases, including reproductive dysfunction. Primary ovarian failure is characterized by amenorrhea and infertility in a normal karyotype female, with an elevated serum level of follicle-stimulating hormone (FSH) and a decrease level of estrogen caused by a mutation in FSH receptor (FSHR) gene. Currently, there is no effective treatment for this condition. The phenotype of FSHR (-/-) mouse, FORKO (follitropin receptor knockout), is a suitable model to study ovarian failure in humans. Female FORKO mice have elevated FSH, decreased estrogen levels, are sterile because of the absence of folliculogenesis, and display thin uteri and small nonfunctional ovaries. In this study, we determined the effects of BMSC transplantation on reproductive physiology in this animal model. Twenty four hours post BMSC transplantation, treated animals showed detectable estroidogeneic changes in daily vaginal smear. Significant increase in total body weight and reproductive organs was observed in treated animals. Hemotoxylin and eosin (H&E) evaluation of the ovaries demonstrated significant increase in both the maturation and the total number of the follicles in treated animals. The FSH dropped to 40-50% and estrogen increased 4-5.5 times in the serum of treated animals compared to controls. The FSHR mRNA was detected in the ovaries of treated animals. Our results show that intravenously injected BMSCs were able to reach the ovaries of FORKO mice, differentiate and express FHSR gene, make FSHR responsive to FSH, resume estrogen hormone production, and restore folliculogenesis.     

Antidepressant activity of standardized extract of Bacopa monniera in experimental models of depression in rats.

Bacopa monniera Wettst. (syn. Herpestis monniera L.; Scrophulariaceae) is a commonly used Ayurvedic drug for mental disorders. The standardized extract was reported earlier to have significant anti-oxidant effect, anxiolytic activity and improve memory retention in Alzheimer's disease. Presently, the standardized methanolic extract of Bacopa monniera (bacoside A - 38.0+/-0.9) was investigated for potential antidepressant activity in rodent models of depression. The effect was compared with the standard antidepressant drug imipramine (15 mg/kg, ip). The extract when given in the dose of 20 and 40 mg/kg, orally once daily for 5 days was found to have significant antidepressant activity in forced swim and learned helplessness models of depression and was comparable to that of imipramine.     

In vitro evaluation of Bacopa monniera on anti-Helicobacter pylori activity and accumulation of prostaglandins.

Bacopa monniera is an Indian tratidional medicine widely used to improve intellectual functions. Earlier, we had reported the prophylactic and curative effects of standardized extract of Bacopa monniera (BME) in various gastric ulcer models. The effect was due to augmentation of the defensive mucosal factors like increase in mucin secretion, life span of mucosal cells and gastric antioxidant effect rather than on the offensive acid-pepsin secretion. The present study includes evaluation of standardized BME (bacoside A content--35.5 +/- 0.9) on other contributing factors towards ulcerogenesis. BME in the dose of 1000 microg/ml showed anti-Helicobacter pylori activity in vitrol and in the dose of 10 microg/ml increased in vitro of prostanoids (PGE and PGI2) in human colonic mucosal incubates. It may be concluded that these factors may contribute to antiulcerogenic activity of BME.     

Immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae against lethal infection in mice

The present study was carried out to evaluate the immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae , by expressing full length GroEL in heterologous host Escherichia coli BL21(DE3). PCR-amplified groEL was ligated in pQE 30 expression vector and subsequently transformed in E. coli DH5α strains. Cloning of groEL was confirmed by double digestion, followed by DNA sequencing. The His-tag containing recombinant GroEL was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of GroEL, the mice were immunized by injecting 40 μg GroEL protein per mouse intraperitoneally. The results showed a significant increase in antibody titre and lymphocyte proliferation in animals immunized with GroEL as compared with control. Further, there was an appreciable increase in interleukin-2 (IL-2) and IL-4 production in lymphocytes isolated from immunized mice as compared with control. To determine the efficacy of GroEL in eliciting protection, the mice were challenged with the lethal dose of S. pneumoniae A66 type 3 capsular strain intranasally after the seventh day of the last immunization. In the GroEL-immunized mice the onset of death was insignificantly delayed and all the mice died by the seventh day postinfection.     

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).     

Complete dissociation of gonadotropin receptor binding and signal transduction in mouse Leydig tumour cells. Obligatory role of glycosylation in hormone action.

Utilizing a clonal cell line of mouse testicular Leydig cells (MA-10 cells) the complete steroidogenic and other hormonal properties of chemically deglycosylated ovine lutropin (DG-LH) and human choriogonadotropin (DG-hCG) were evaluated. In these cells, with the LH receptor-steroidogenic mechanism tightly coupled and in which there are few, if any, spare receptors, both DG-LH and DG-hCG failed to elicit progesterone production, unlike fully glycosylated native LH and hCG. The receptor-binding activity of DG-LH and DG-hCG was 2-3 times that of LH and hCG in competition experiments with radiolabelled hormones. The typical phenomenon of rounding of MA-10 cells induced by LH and hCG was absent when cells were incubated with DG-LH or DG-hCG. This could be directly attributable to their failure to produce cyclic AMP as second messenger. DG-LH and DG-hCG inhibited cell shape changes and steroidogenesis caused by LH and hCG. The deglycosylated hormones were potent antagonists of the action of glycosylated hormones. Delaying DG-hCG (antagonist) addition for up to 1 h after initiation of hCG action was also very effective in preventing further activation of steroidogenesis. Similar effects were produced by addition of affinity-purified anti-hCG antibodies. In affinity cross-linking experiments, both hCG and DG-hCG bound to the same 90 kDa receptor. Studies with MA-10 cells thus provide unequivocal evidence that the presence of antennary sugars in LH and hCG (and perhaps in other similar hormones such as follicle-stimulating hormone and thyroid-stimulating hormone), is essential for signal transduction. Differences observed in the literature in other cellular systems may be attributed to differences in hormone-receptor-effector coupling.