Differences between cellular mechanisms of ATP-and noradrenaline-induced inhibition of visceral smooth muscles under conditions of selective and combined activation of M<Subscript>2</Subscript> or M<Subscript>3</Subscript> cholinoreceptors

Our studies of the role of phospholipase C in inhibitory synaptic action upon visceral smooth muscles demonstrated that, under conditions of carbachol (CCh)-induced pre-activation of cholinoreceptors, ATP-or noradrenaline (NA)-evoked relaxation of these muscles is mediated by the phospholipase C-independent pathway, while the phospholipase C-dependent pathway does not manifest itself as a mechanism that determines the inhibitory effect of the above transmitters. Under conditions of pre-activation of muscarinic cholinoreceptors, ATP-and NA-induced relaxation is continued due to activation of inositol trisphosphate (InsP₃)-sensitive receptors despite the fact that the pathway of inhibition is phospholipase C-independent. This is confirmed by complete depression of the inhibitory effects of ATP and NA against the background of CCh-induced contraction after pre-incubation of the studied preparations together with 100 μM 2-APB, a blocker of InsP₃ receptors. Selective blockings of either M₂ or M₃ cholinoreceptors are accompanied by a complete loss of the ability of the above blocker of InsP₃ receptors (2-APB) to suppress ATP-and NA-induced contraction of smooth muscles in the state of CCh-induced contraction. It can be hypothesized that, under conditions of selective pre-activation of M₂ or M₃ cholinoreceptors, the mechanisms of intracellular signalling mediating the inhibition events are modified. The InsP₃-dependent pathway that determines both adrenergic and purinergic inhibition of smooth muscles is switched off, and the inhibitory action of neurotransmitters is realized under such conditions through the InsP₃-independent pathway. Therefore, in our study we first found differences between cellular mechanisms underlying ATP-and NA-induced inhibition of smooth muscles under conditions of selective activation of either M₂ or M₃ cholinoreceptors and the mechanisms underlying the relaxing action of inhibitory neurotransmitters under conditions of combined synergistic activation of cholinoreceptors of both the above-mentioned subtypes. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 22–31, January–February, 2007.     

New data on the taxonomy,distribution, and ecology of the weevil genus <Emphasis Type="Italic">Otiorhynchus</Emphasis> germar (Coleoptera,Curculionidae)

The subgenus Pocusogetus Rtt. of the genus Otiorhynchus Germ. is revised. The subgenus includes O. rosti Strl., O. shapovalovi Davidian et Yunakov, O. obsulcatus Strl., O. fischtensis Rtt., and O. gusakovi sp. n. closely related to O. fischtensis (both from Mt. Fisht, the Western Caucasus). O. fischtensis is transferred from the subgenus Vicoranius Rtt., its lectotype is designated. A key to species of Pocusogetus is given. The systematic position of the subgenera Pocusogetus and Vicoranius in the genus Otiorhynchus is discussed. New data on the geographical distribution and ecology of the little-known species of the subgenera Obvoderus Rtt., Pseudoprovadilus Magnano, and Clypeorhynchus Yunakov et Arzanov are given. Some features of ecological differentiation between Otiorhynchus species in the alpine and subalpine zones of the Caucasus are discussed.     

Characterization of novel microsatellite loci in rare minnow (<Emphasis Type="Italic">Gobiocypris rarus</Emphasis>) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2–7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.     

An NMR study of the conformational mobility of steroid estrogen 7α-methyl-8α analogues

All the signals in the ¹H and ¹³C NMR spectra of some analogues of 7α-methyl-8α-and 6-oxa-8α-steroid estrogens were completely assigned. Considering the values of nuclear Overhauser effect and vicinal coupling constants, these steroids were shown to exhibit a fast, on the NMR time scale, conformational equilibrium arising due to the inversion of ring B. The conformer populations were obtained from a comparison of the experimental and theoretical values of the dihedral angles and the interproton distances. This conformational equilibrium was shown to depend on the nature of atom in position 6: for the 7α-methyl-6-oxa-8α analogues of the steroid estrogens, the population of the conformer with the pseudoaxial orientation of the 7α-methyl group was observed to be decreased compared with the 7α-methyl-8α analogue.     

The DNA-dependent protein kinase catalytic subunit is phosphorylated in vivo on threonine 3950, a highly conserved amino acid in the protein kinase domain

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.     

A new role for cathelicidin in ulcerative colitis in mice

Cathelicidin, an antimicrobial peptide of the innate immune system, modulates microbial growth, wound healing, and inflammation. However, its association with inflammatory bowel diseases (IBDs) is unknown. Our objective was to determine whether cathelicidin would exert a modulatory effect on the progression of IBD and, if so, investigate the mechanism of action through which this effect occurred. We evaluated the potential for a synthetic cathelicidin, the mouse cathelin-related antimicrobial peptide (mCRAMP), to prevent the initiation and promote the healing of lesions from inflammatory colitis that was experimentally induced in mice with dextran sulfate sodium (DSS). During the experiment, mCRAMP was given: (i) as a parallel treatment starting together with 3% DSS feeding, and (ii) as a posttreatment starting 7 days after 3% DSS feeding. The body weight, fecal microflora populations, clinical symptoms, and histologic findings of colonic tissues were measured. Relative gene expression of mucins (MUC1, MUC2, MUC3, and MUC4) in colonic tissues was determined by real-time polymerase chain reaction. Intrarectal administration of mCRAMP ameliorated DSS-induced colitis with negligible effects on mucosal healing. The peptide also significantly reduced the increased number of fecal microflora in colitis animals. It reversed the decline of colonic mucus thickness during colitis through upregulation of the expression of mucin genes. Treatment with mCRAMP also prevented colitis development by suppressing the induction of apoptosis by DSS. The current study demonstrates for the first time that intrarectal administration of cathelicidin may be a novel therapeutic option for IBDs.     

Reversal of multidrug resistance of vincristine-resistant gastric adenocarcinoma cells through up-regulation of DARPP-32

Here we investigated the roles of DARPP-32 in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. We constructed the eukaryotic expression vector of DARPP-32 and transfected it into human vincristine-resistant gastric adenocarcinoma cell line SGC7901/VCR. Up-regulation of DARPP-32 could significantly enhance the sensitivity of SGC7901/VCR cells towards vincristine, adriamycin, 5-fluorouracil and cisplatin, and could decrease the capacity of cells to efflux adriamycin. What's more, the results of subrenal capsule assay confirmed that DARPP-32 might play a certain role in MDR of gastric cancer. DARPP-32 could significantly down-regulate the expression of P-gp and zinc ribbon domain-containing 1 (ZNRD1), but not alter the expression of multidrug resistance-associated protein (MRP) or the glutathione S-transferase (GST). DARPP-32 could also significantly decrease the anti-apoptotic activity of SGC7901/VCR cells. Further study of the biological functions of DARPP-32 might be helpful for understanding the mechanisms of MDR in gastric cancer.