Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Premiers stades de l'oogenèse de Rhabdophaga saliciperda (Cecidomyiidae,Diptera)

The females of Rhabdophaga saliciperda have in their somatic cells 8 chromosomes and the males 6. The type of sex determination is therefore: X₁X₁X₂X₂—♀; X₁X₂—♂. The cells of the germinal line have 46 chromosomes, but a variation of their number was observed. In the oogonia and spermatogonia the number of heterochromatic chromosomes may exceed the number of E chromosomes, i.e. 8. In the beginning of the growth stage of the oocytes an incorporation of somatic cells was observed. The nuclei of these somatic cells persist in the cytoplasm of the oocytes until the maturation divisions. The possibility of their participation in the reconstruction of the nucleus of the mature egg is envisaged. The metaphase of the I segmentation division has a complex character. During prophase of the first meiotic division the E chromosomes form 4 bunches of 6–8 chromosomes each. Some univalents may also be present. The 8 S chromosomes form 4 regular bivalents. The 4 groups of E chromosomes persist until metaphase I. During metaphase I a phenomenon of expulsion of the majority of E chromosomes from the metaphase spindle was observed. The 4 bivalents remain in the equatorial plain of the spindle with some E Chromosomes. After this expulsion 2 groups of chromosomes are formed. In connection with them 2 spindles develop. An irregular distribution of E chromosomes follows without their division. The bivalents are probably separated in regular manner. These 2 spindles correspond to the I maturation division. The II maturation division was not observed because of lack of respective stages.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.     

Proton-acceptor properties and capability for mutarotation of some glucosylamines in methanol

N-(m-Nitrophenyl)-beta-D-glucopyranosylamine (Gln), N-(N-methylphenyl)-beta-D-glucopyranosylamine (Glm), N-beta-D-glucopyranosylpyrazole (Glp), and N-beta-D-glucopyranosylimidazole (Gli) have been synthesized. Their basicity constants, pKb, determined in methanol were, respectively, 14.99, 14.36, 15.04, and 9.74. The derivatives of secondary amines (Glm, Glp, and Gli) did not mutarotate in methanol in the presence of 3,5-dinitrobenzoic acid and hydrochloric acid. The heats of formation and entropies were calculated by the AM1 and PM3 methods for the glucosylamines and their cations under consideration of two plausible protonation centers. Thermodynamic parameters for the proton transfer in the reaction: glucosylamine + CH3OH2+ = glucosylamineH+ + CH3OH were determined and the protonation center in the glucosylamine molecule was identified. The mechanism of mutarotation of the glucosylamines is discussed and the conclusion made that formation of an acyclic immonium cation is not a satisfactory condition for the reaction to proceed.     

Synthesis of,and Conformational Studies on, 2-Trifluoromethyl,Substituted Benzimidazole Ribofuranosides

Abstract

The 1-β-D-ribofuranosides of several 2-trifluoromethyl benzimidazoles were prepared by the fusion method, and their conformations, particularly about the glycosidic bond, determined by ¹H NMR spectroscopy.     

Constitutive activity of Erk1/2 and NF-κB protects human endometrial stromal cells from death receptor-mediated apoptosis

Apoptosis in the human endometrium plays an essential role for endometrial receptivity and early implantation. A dysbalance of pro- and anti-apoptotic events in the secretory endometrium seems to be involved in implantation disorders and consecutive pregnancy complications. However, little is known about the mechanisms regulating apoptosis-sensitivity in the human endometrium. Therefore this study was performed to identify molecular mechanisms underlying the resistance toward apoptosis in human endometrial stromal cells (ESCs). Human ESCs were isolated from hysterectomy specimens and used as undifferentiated cells or after decidualization in vitro. Cells were incubated with an activating anti-Fas antibody, tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF-α and inhibitors of protein- and RNA-syntheses, a caspase-inhibitor and inhibitors of extracellular signal regulated kinase (Erk)1/2, nuclear factor (NF)-κB and Akt. Apoptosis was measured by flow cytometric detection of hypodiploid nuclei. Caspase-activity was detected by luminescencent assays. Several pro- and anti-apoptotic molecules and the activation of Erk1/2, NF-κB and Akt were analyzed by in-cell Western assays or flow cytometry. Inhibition of protein- and RNA-syntheses differentially sensitized human ESCs for death receptor-mediated apoptosis in a caspase-dependent manner, based on the up-regulation of the death receptors Fas and TRAIL-R2. The constitutive activity of Erk1/2 and NF-κB could be identified as a reason for the apoptosis-resistance of human ESCs. These results suggest the pro-survival signaling pathways Erk1/2 and NF-κB as key regulators of the sensitivity of human ESCs for death receptor-mediated apoptosis. The modulation of these pathways might play an important role in the physiology of implantation.     

Co-expression pattern of dopamine beta-hydroxylase (DβH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.)

Co-expression of dopamine β-hydroxylase (DβH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45–120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DβH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DβH immunoreactive nerve fibers (DβH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DβH, while some DβH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DβH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DβH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.     

Conversion of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F₁ progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5–0.9 mm, 1.0–1.4 mm, and ≥1.5 mm), and transferred into 190–2 regeneration medium with different growth regulators: 0.5 mg L^?1 kinetin (KIN) and 0.5 mg L^?1 1-naphthaleneacetic acid (NAA); 1 mg L^?1 zeatin (ZEA) and 0.5 mg L^?1 NAA; or 1 mg L^?1 dicamba (DIC), 1 mg L^?1 picloram (PIC), and 0.5 mg L^?1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0–1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L^?1 NAA and 0.5 mg L^?1 KIN, while the fewest germinated on medium with 1 mg L^?1 DIC, 1 mg L^?1 PIC, and 0.5 mg L^?1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.