Plant latex thrombin-like cysteine proteases alleviates bleeding by bypassing factor VIII in murine model

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.     

Response of the Italian agile frog (Rana latastei) to a Ranavirus, frog virus 3: a model for viral emergence in naïve populations

Ranavirus (family Iridoviridae) is a genus of pathogens of poikilotherms, and some ranaviruses may play a role in widespread mortality of amphibians. Ecology of viral transmission in amphibians is poorly known but can be addressed through experimentation in the laboratory. In this study, we use the Ranavirus frog virus 3 (FV3) as an experimental model for pathogen emergence in naive populations of tadpoles. We simulated emerging disease by exposing tadpoles of the Italian agile frog (Rana latastei), to the North American Ranavirus FV3. We demonstrated that mortality occurred due to viral exposure, exposure of tadpoles to decreasing concentrations of FV3 in the laboratory produced dose-dependent survival rates, and cannibalism of virus-carrying carcasses increased mortality due to FV3. These experiments suggest the potential for ecological mechanisms to affect the level of exposure of tadpoles to Ranavirus and to impact transmission of viral pathogens in aquatic systems.     

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.     

Lipid function in plant cell polarity

The establishment and maintenance of cell polarity play pivotal roles during plant development. During the past five years, proteins that are required for different aspects of plant cell polarity have been identified. However, the functions of lipids and their interactions with proteins that mediate polarity remained largely unaddressed. Recent genetic studies have discovered cell and tissue polarity mutants that have defects in sterol composition, glycosylphosphatidylinositol-anchored proteins, glycosylphosphatidylinositol biosynthesis and phospholipid signalling. Analyses of the affected gene products have provided a first glance at the roles of lipids in cell polarity signalling, as well as in the trafficking and anchoring of polar proteins.     

Human molecular chronotyping in sight?

Recent research on mouse models has taken us closer to deciphering the molecular clock mechanism that defines an individual's 'body time'. How feasible will it be to create a molecular timetable that allows determination of individual body time from tissue harvested at a single time point?     

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.     

Vasopressin type 1A receptor up-regulation by cyclosporin A in vascular smooth muscle cells is mediated by superoxide

Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.     

Robust circadian rhythmicity of Per1 and Per2 mutant mice in constant light,and dynamics of Per1 and Per2 gene expression under long and short photoperiods

The Per1 and Per2 genes are components of the mammalian circadian clock. Mutations in these genes alter phase resetting in response to a nocturnal light pulse, and Per2 mutant mice are known to become arrhythmic in constant darkness. We show that under constant light conditions, Per2 mutant mice exhibit robust activity rhythms as well as body temperature rhythms with a period length that is less than 24 h. In Per1 mutants, the period length of both activity and body temperature rhythms is longer than 24 h in constant light. Per1 mutants prolong their period length (tao) when illuminance is increased, whereas Per2 mutants shorten their endogenous period. Additionally, the authors show that the circadian pattern of Per1 and Per2 gene expression in mice is modified under different photoperiods and that there is a mutual influence of these genes on their timing of expression. We propose that, in mice, the phase relationship between Per1 and Per2 gene expression might be critical for transducing day length information to the organism. Per1 could be part of a morning oscillator tracking dawn, and Per2 could be part of an evening oscillator tracking dusk.     

Transcriptional activation of cytochrome P450 CYP2C45 by drugs is mediated by the chicken xenobiotic receptor (CXR) interacting with a phenobarbital response enhancer unit

Cytochromes P450 (CYP)-2C enzymes fulfill an important role in xenobiotic metabolism and therefore have extensively been studied in rodents and humans. However, no CYP2C genes have been described in avian species to date. In this paper, we report the cloning, functional analysis, and regulation of chicken CYP2C45. The sequence shares up to 58% amino acid identity with CYP2Cs in other species. The overexpression of CYP2C45 in chicken hepatoma cells leghorn male hepatoma (LMH) led to increased scoparone metabolism. CYP2C45 regulation was studied in LMH cells at the mRNA level and in reporter gene assays using a construct containing 2.6 kb of its 5'-flanking region. Exposure of LMH cells to phenobarbital or metyrapone led to a 95- or 210-fold increase in CYP2C45 mRNA and a 140- or 290-fold increase in reporter gene expression, respectively. A phenobarbital response enhancer unit (PBRU) of 239 bp containing a DR-4 nuclear receptor binding site was identified within the 2.6-kb fragment. Site-specific mutation of the DR-4 revealed the requirement of this motif for CYP2C45 induction by drugs. The chicken xenobiotic receptor CXR interacted with the PBRU in electromobility shift and transactivation assays. Furthermore, the related nuclear receptors, mouse PXR and mouse CAR, transactivated this enhancer element, suggesting evolutionary conservation of nuclear receptor-DNA interactions in CYP2C induction.