Retigabine holds KV7 channels open and stabilizes the resting potential

The anticonvulsant Retigabine is a K_V7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric K_V7.2/K_V7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine’s action remains unknown, previous studies have identified the pore region of K_V7 channels as the drug’s target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric K_V7.2/K_V7.3 channel has at least two open states, which we named O₁ and O₂, with O₂ being more stable. The O₁ state was reached after short membrane depolarizations, whereas O₂ was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the K_V7.2/K_V7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O₂ state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of K_V7.2/K_V7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered.     

In vivo and in vitro evaluation of pharmacological activities of Adenia trilobata (Roxb.)

Adenia trilobata, locally known as akandaphal in Bangladesh, has some traditional uses. Leaves and stems extracted with pure methanol (MEATL, MEATS) and fractioned by n-hexane (NFATL, NFATS), which was subjected to qualitative phytochemical analysis. The qualitative phytochemical analysis of four extracts showed the presence of secondary metabolites such as alkaloid, carbohydrate, glycosides, flavonoids, phenols, flavonol, and saponins. All four extracts of A. trilobata, exhibited a strong antioxidant activity while a moderately (MEATS = 328 μg/mL) to weakly toxic (NFATL = 616.85 μg/mL) LC₅₀ observed in brine shrimp lethality bioassay. In thrombolytic test, MEATL (18.54 ± 2.18%; P < 0.01) and MEATS (25.58 ± 4.76%; P < 0.0001) showed significant percentage of clot lysis in human blood. The in vivo analgesic activity carried by acetic acid test and formalin test, while the antidiarrheal activity assayed by two standard methods e.g., castor oil-induced diarrhea and castor oil-induced gastrointestinal motility. Both, in vivo model, showed an extremely significant (P < 0.0001) dose-dependent manner percentage of inhibition in comparison to the control group. Present results suggested, A. trilobata could be a potential source for antioxidative, cytotoxic, thrombolytic, analgesic, antidiarrheal agents which require further study to identify the mechanism of A. trilobata.     

Adaptive capability as indicated by endocrine and biochemical responses of Malpura ewes subjected to combined stresses (thermal and nutritional) in a semi-arid tropical environment

A study was conducted to assess the effect of combined stresses (thermal and nutritional) on endocrine and biochemical responses in Malpura ewes. Twenty eight adult Malpura ewes (average body weight 33.56 kg) were used in the present study. The ewes were divided into four groups viz., GI (n = 7; control), GII (n = 7; thermal stress), GIII (n = 7; nutritional stress) and GIV (n = 7; combined stress). The animals were stall fed with a diet consisting of 60% roughage and 40% concentrate. GI and GII ewes were provided with ad libitum feeding while GIII and GIV ewes were provided with restricted feed (30% intake of GI ewes) to induce nutritional stress. GII and GIV ewes were kept in climatic chamber at 40°C and 55% RH for 6 h a day between 1000 hours and 1600 hours to induce thermal stress. The study was conducted for a period of two estrus cycles. The parameters studied were Hb, PCV, glucose, total protein, total cholesterol, ACP, ALP, cortisol, T₄, T₃, and insulin. Combined stress significantly (P < 0.05) affected all parameters studied. Furthermore, the results revealed that, compared to thermal stress, nutritional stress had a less significant effect on the parameters studied. However, when both these stresses were coupled, they had a severe impact on all the parameters studied in these ewes. It can be concluded from this study that two stressors occurring simultaneously may impact severely on the biological functions necessary to maintain homeostasis in sheep.     

Antihyperlipidemic potential of diosmin in Swiss Albino mice with high-fat diet induced hyperlipidemia

The aim of this study was to investigate the antihyperlipidemic potential of Diosmin (DS) in mice fed with a high-fat diet (HFD). Animals were divided in five groups (n = 6). The total duration of the study was 90 days split into two intervals. During the first 45-day interval, mice were administered with HFD, whereas during the second 45-day interval they were co-administered HFD plus DS or the standard drug atorvastatin. DS was administered at the dose of 100 and 200 mg/kg;p.o. DS treatment to HFD-induced hyperlipidemic mice caused significant decrements in the levels of total cholesterol, triglycerides, LDL-C and VLDL-C. Moreover, DS resulted in significant increase in the levels of HDL-C and improvements in total protein levels, whereas it caused remarkable decreases in SGOT, SGPT and ALP enzymatic activities in hyperlipidemic mice. Histopathological examination of hyperlipidemic mice revealed a disorganized hepatic tissue, fatty changes, and mononuclear cell infiltration, which were all ameliorated by DS administration. The results revealed that DS possesses potential ameliorating benefits again.st hyperlipidemia induced by HFD on lipid profile, liver function enzymes and hepatic histoarchitecture. Further investigations are highly recommended and clinical trials are warranted in order to assess the efficacy and to fully dissect the mode-of-action underpinning the observed antihyperlipidemic effect of DS.     

PGE2-mediated inhibition of T cell p59fyn is independent of cAMP

We recently observed that prostaglandinE₂(PGE₂)-mediated suppression of Tcell functions could result from an attenuation ofp59^fyn protein tyrosine kinaseactivity. The present study evaluated the effects of an adenylatecyclase agonist (forskolin) and antagonist (SQ-22536), as well as thoseof cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cellp59^fyn kinase activity. The studyallowed us to assess whetherPGE₂-mediated activation ofadenylate cyclase by itself or the elevation in intracellular cAMPlevels is an integral event in the modulation of anti-CD3-linkedp59^fyn activation in T cells. Theexperiments were carried out with splenic T cells from maleSprague-Dawley rats. A 30-50% suppression in theautophosphorylation and the kinase activity ofp59^fyn in T cells incubated withPGE₂ or forskolin was observed.Pretreatment of T cells with SQ-22536 prevented significantPGE₂-mediated inhibition of T cellp59^fyn kinase activity. Incontrast, no change in p59^fynautophosphorylation and kinase activity in T cells treated with cAMPanalogues was observed. These data suggest thatPGE₂-mediated suppression ofp59^fyn autophosphorylation andkinase activity in T cells is dependent on the activation of adenylatecyclase and independent of the elevation in cAMP levels.

     

Crystallization of a fragment of human fibronectin: Introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine

Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7–10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 Å Bragg spacings. A mutant FN7–10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed. © 1994 Wiley-Liss, Inc.     

An Immuno-informatics driven Epitope study from the molecular interaction of JEV non-structural (NS) proteins with Ribophorin (RPN)

Japanese encephalitis (JE) is an acute viral infection of the central nervous system where the JE virus infects the lumen of the endoplasmic reticulum (ER) and rapidly accumulates substantial amount of seven different nonstructural proteins (NS). These NS proteins tend to bind on a glycoprotein receptor, ribophorin (RPN) resulting in the malfunctioning of ER in host cells, subsequently triggering an unfolded protein response. Therefore, it is of interest to predict the best possible antigenic determinants in the NS protein capable of eliciting immune response as a strategy to combat JE. Hence, it is our interest to explore the most potent NS protein among all showing the best possible molecular interaction with the RPN receptor present on ER. However, the structures of these NS protein and RPN are currently unknown. Thus, we modeled their structures using the established homology modeling techniques in the MODELLER 9v10 software. The molecular docking of NS proteins with RPN was subsequently completed using the Discovery Studio 2.5 software suite. The docked conformations of RPN with NS were further analyzed and its graphical interpretations were presented for identifying the most potential NS protein for efficient epitope activity. Further, the B cell epitopes were mapped using BCPred and the predicted epitope regions are documented. The data presented in this report provides useful insights towards the design and development of potential epitopes to generate a vaccine candidate against JEV.