Regulation of an 8-kDa peptide involved in testosterone production by luteinizing hormone in rat Leydig cells

Results of Western blot analysis carried out with an interstitial cell extract from male guinea pig and ovarian extract from immature female rats administered equine chorionic gonadotropin (eCG) provide supportive evidence to our earlier suggestion that an 8-kDa peptide is involved in acquisition of steroidogenic capacity by the rat Leydig cells. It was found that though the signal was observed in other tissues such as liver, kidney and lung which do not produce gonadal hormones, the peptide was modulated only by lutenizing hormone (LH) in the rat Leydig cells.

A stereospecific approach to cis-anti-cis triquinanes

A methodology for the synthesis of compounds containing the cis-anti-cis fused triquinane system has been developed starting from tricyclo[5.2. 1.0(2.6)]deca-4,8-dien-3-(exo)ol 6 involving Ireland ester Claisen rearrangement and intramolecular diazo ketone cyclopropanation reactions as key steps.

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions. Proteins 2011;79:3108-3122. (C) 2011 Wiley-Liss, Inc.

l-pGlu-(2-propyl)-l-His-l-ProNH2 attenuates 4-aminopyridine-induced epileptiform activity and sodium current: a possible action of new thyrotropin-releasing hormone analog for its anticonvulsant potential

L-PGlu-(2-proPyl)-L-His-L-ProNH(2) (NP-647) is a CNS active thyrotropin-releasing hormone (TRH) analog with potential application in various CNS disorders including seizures. In the present study, mechanism of action for protective effect of NP-647 was explored by studying role of NP-647 on epileptiform activity and sodium channels by using patch-clamp methods. Epileptiform activity was induced in subicular pyramidal neurons of hippocampal slice of rat by perfusing 4-aminopyridine (4-AP) containing Mg(+2)-free normal artificial cerebrospinal fluid (nACSF). Increase in mean firing frequency was observed after perfusion of 4-AP and zero Mg(+2) (2.10+/-0.47 Hz) as compared with nACSF (0.12+/-0.08 Hz). A significant decrease in mean firing frequency (0.61+/-0.22 Hz), mean frequency of epileptiform events (0.03+/-0.02 Hz vs. 0.22+/-0.05 Hz of 4-AP+0 Mg), and average number of action potentials in paroxysmal depolarization shift-burst (2.54+/-1.21 Hz vs. 8.16+/-0.88 Hz of 4-AP +0 Mg) was observed. A significant reduction in peak dV/dt (246+/-19 mV ms(-1) vs. 297 18 mV ms-1 of 4-AP+0 Mg) and increase (1.332+/-0.018 ms vs. 1.292+/-0.019 ms of 4-AP+0 Mg) in time required to reach maximum depolarization were observed indicating role of sodium channels. Concentration-dependent depression of sodium current was observed after exposure to dorsal root ganglion neurons to NP-647. NP-647 at different concentrations (1, 3, and 10 mu M) depressed sodium current (15+/-0.5%, 50+/-2.6%, and 75+/-0.7%, respectively). However, NP-647 did not show change in the peak sodium current in CNa18 cells. Results of present study demonstrated potential of NP-647 in the inhibition of epileptiform activity by inhibiting sodium channels indirectly. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

An inventory of the ethnobotanicals used as anti-diabetic by a rural community of Dhemaji district of Assam, Northeast India

Ethnopharmacological relevance: Traditional remedies used for treating diabetic ailments are very important in the primary health care of the people living in rural Dhemaji district of Assam, north-east India. Novel information gathered from the current survey is important in preserving folk indigenous knowledge. Materials and methods: Interviews were conducted amongst 80 households comprising of 240 individuals using semi-structured questionnaires. The focus was on plants used in treating diabetes mellitus. Results: The current survey documented 21 plant species (20 families) which are reportedly used to treat diabetes mellitus by the rural people in the study area. To the best of our knowledge, Amomum linguiforme, Cinnamomum impressinervium, Colocasia esculenta, Dillenia indica, Euphorbia ligularia, Garcinia pedunculata, Solanum indicum, Sterculia villosa and Tabernaemontana divaricata are recorded for the first time based on globally published literature as medicinal plants used for treating diabetes mellitus and related symptoms. Conclusions: The wide variety of plants that are used to treat diabetes mellitus in this area supports the traditional value that medicinal plants have in the primary health care system of the rural people of Dhemaji district of Assam. The finding of new plant uses in the current study reveals the importance of the documentation of such ethnobotanical knowledge. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: Critical roles of IFN gamma

Mycobacterium indicus pranii (MIP) is approved for use as an adjuvant (Immuvac/Cadi-05) in the treatment of leprosy. In addition, its efficacy is being investigated in clinical trials on patients with tuberculosis and different tumors. To evaluate and delineate the mechanisms by which autoclaved MIP enhances anti-tumor responses, the growth of solid tumors consisting of Sp2/0 (myeloma) and EL4 (thymoma) cells was studied in BALB/c and C57BL/6 mice, respectively. Treatment of mice with a single intra-dermal (i.d.) injection of MIP 3 days after Sp2/0 implantation greatly suppresses tumor growth. MIP treatment of tumor bearing mice lowers Interleukin (IL)6 but increases IL12p70 and IFN? amounts in sera. Also, increase in CD8+ T cell mediated lysis of specific tumor targets and production of high amounts of IL2 and IFN? by CD4+ T cells upon stimulation with specific tumor antigens in MIP treated mice is observed. Furthermore, MIP is also effective in reducing the growth of EL4 tumors; however, this efficacy is reduced in Ifn?-/- mice. In fact, several MIP mediated anti-tumor responses are greatly abrogated in Ifn?-/- mice: increase in serum Interleukin (IL)12p70 amounts, induction of IL2 and lysis of EL4 targets by splenocytes upon stimulation with specific tumor antigens. Interestingly, tumor-induced increase in serum IL12p70 and IFN? and reduction in growth of Sp2/0 and EL4 tumors by MIP are not observed in nonobese diabetic severe combined immunodeficiency mice. Overall, our study clearly demonstrates the importance of a functional immune network, in particular endogenous CD4+ and CD8+ T cells and IFN?, in mediating the anti-tumor responses by MIP.

Luteinizing hormone regulates inhibin-alpha subunit expression through multiple signaling pathways involving steroidogenic factor-1 and beta-catenin in the macaque corpus luteum

We employed different experimental model systems to define the role of GATA4, beta-catenin, and steroidogenic factor (SF-1) transcriptional factors in the regulation of monkey luteal inhibin secretion. Reverse transcription polymerase chain reactions and western blotting analyses show high expression of inhibin-alpha, GATA4, and beta-catenin in corpus luteum (CL) of the mid-luteal phase. Gonadotropin-releasing hormone receptor antagonist-induced luteolysis model suggested the significance of luteinizing hormone (LH) in regulating these transcriptional factors. Inducible cyclic AMP early repressor mRNA expression was detected in the CL and no change was observed in different stages of CL. Following amino acid sequence analysis, interaction between SF-1 and beta-catenin in mid-stage CL was verified by reciprocal co-immunoprecipitation experiments coupled to immunoblot analysis. Electrophoretic mobility shift analysis support the role of SF-1 in regulating luteal inhibin-alpha expression. Our results suggest a possible multiple crosstalk of Wnt, cAMP, and SF-1 in the regulation of luteal inhibin secretion.

Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey

Background: In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. Methods: Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. Results and discussion: The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs pSrc (Y-416)] and PDE as well as decreased expression of SR-B1correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy. Conclusions: The results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.

Anti-obese activity of Butea monosperma (Lam) bark extract in experimentally induced obese rats

To study the efficacy of ethanolic extract of B. monosperma bark in cafeteria and atherogenic diet fed rats and monosodium glutamate (MSG) obese rats, different doses (200, 400 and 800 mg/kg) of ethanolic extract of B. monosperma bark showed dose dependent decrease in body weight, daily food intake, glucose, lipids, internal organs' weight and fat pad weight in cafeteria and atherogenic diet fed rats and monosodium glutamate obese rats. The results suggested that B. monosperma has significant anti-obese activity.

The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the alpha-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.