Differential role of nitric oxide in regional sympathetic responses to stimulation of NTS A2a adenosine receptors

Our previous studies showed that preganglionic adrenal (pre-ASNA), renal (RSNA), lumbar, and postganglionic adrenal sympathetic nerve activities (post-ASNA) are inhibited after stimulation of arterial baroreceptors, nucleus of the solitary tract (NTS), and glutamatergic and P2x receptors and are activated after stimulation of adenosine A1 receptors. However, stimulation of adenosine A2a receptors inhibited RSNA and post-ASNA, whereas it activated pre-ASNA. Because the effects evoked by NTS A2a receptors may be mediated via activation of nitric oxide (NO) mechanisms in NTS neurons, we tested the hypothesis that NO synthase (NOS) inhibitors would attenuate regional sympathetic responses to NTS A2a receptor stimulation, whereas NO donors would evoke contrasting responses from pre-ASNA versus RSNA and post-ASNA. Therefore, in chloralose/urethane-anesthetized rats, we compared hemodynamic and regional sympathetic responses to microinjections of selective A2a receptor agonist (CGS-21680, 20 pmol/50 nl) after pretreatment with NOS inhibitors Nomega-nitro-L-arginine methyl ester (10 nmol/100 nl) and 1-[2-(trifluoromethyl)phenyl]imidazole (100 pmol/100 nl) versus pretreatment with vehicle (100 nl). In addition, responses to microinjections into the NTS of different NO donors [40 and 400 pmol/50 nl sodium nitroprusside (SNP); 0.5 and 5 nmol/50 nl 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA NONOate, also known as NOC-18), and 2 nmol/50 nl 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate, also known as NOC-15)], the NO precursor L-arginine (10-50 nmol/50 nl), and sodium glutamate (500 pmol/50 nl) were evaluated. SNP, DETA NONOate, and PAPA NONOate activated pre-ASNA and inhibited RSNA and post-ASNA, whereas l-arginine and glutamate microinjected into the same site of the NTS inhibited all these sympathetic outputs. Decreases in heart rate and depressor or biphasic responses accompanied the neural responses. Pretreatment with NOS inhibitors reversed the normal depressor and sympathoinhibitory responses to stimulation of NTS A2a receptors into pressor and sympathoactivatory responses and attenuated the heart rate decreases; however, it did not change the increases in pre-ASNA. We conclude that NTS NO mechanisms differentially affect regional sympathetic outputs and differentially contribute to the pattern of regional sympathetic responses evoked by stimulation of NTS A2a receptors.     

Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and delta1-opioid receptor agonists is mediated by iNOS

Although ischemia-induced late preconditioning (PC) is known to be mediated by inducible nitric oxide (NO) synthase (iNOS), the role of this enzyme in pharmacologically induced late PC remains unclear. We tested whether targeted disruption of the iNOS gene abrogates late PC elicited by three structurally different NO donors [diethylenetriamine/NO (DETA/NO), nitroglycerin (NTG), and S-nitroso-N-acetyl-penicillamine (SNAP)], an adenosine A1 receptor agonist [2-chloro-N6-cyclopentyladenosine (CCPA)], and a delta1-opioid receptor agonist (TAN-670). The mice were subjected to a 30-min coronary occlusion followed by 24 h of reperfusion. In iNOS knockout (iNOS-/-) mice, infarct size was similar to wild-type (WT) controls, indicating that iNOS does not modulate infarct size in the absence of PC. Pretreatment of WT mice with DETA/NO, NTG, SNAP, TAN-670, or CCPA 24 h before coronary occlusion markedly reduced infarct size. In iNOS-/- mice, however, the late PC effect elicited by DETA/NO, NTG, SNAP, TAN-670, and CCPA was completely abrogated. Furthermore, in WT mice pretreated with TAN-670 or CCPA, the selective iNOS inhibitor 1400W also abolished the delayed PC properties of these drugs; 1400W had no effect in WT mice. These data demonstrate that iNOS plays an obligatory role in NO donor-induced, adenosine A1 receptor agonist-induced, and delta1-opioid receptor agonist-induced late PC, underscoring the critical role of this enzyme as a common mediator of cardiac adaptations to stress.     

Protection of PC12 cells from hydrogen peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108

A number of studies indicate that free radicals are involved in the neurodegeneration in Parkinson's and Alzheimer's diseases. EPS2, an exopolysaccharide with a mean molecular weight of 1.3 x 10(5) Da, was isolated by ion-exchange and sizing chromatography from the culture of Keissleriella sp. YS4108, a marine filamentous fungus. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The protective effects of EPS2 on peroxide hydrogen (H2O2)-induced cell lesion, level of lipid peroxidation, antioxidant enzyme activities were investigated in the rat pheochromocytoma line PC12 cells. Following a 1-h exposure of the cells to H2O2, a significant reduction in cell survival and activities of glutathione peroxidase (GSH-Px) and catalase (CAT), as well as increased levels in malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. However, preincubation of the cells with EPS2 prior to H2O2 exposure elevated the cell survival and GSH-Px and CAT activities, and decreased the level of MDA and LDH activity in a dose-dependent manner. In conclusion, EPS2 possesses pronounced protective effects against H2O2-induced cell toxicity. The finding is of a higher value in searching for new therapeutic agent for treating oxidative damage-derived neurodegenerative disorders.     

Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making

AIMS: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making. METHODS AND RESULTS: Xylanase was purified 3.5-fold to homogeneity with a recovery yield of 32.8%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 25.6 kDa. The purified xylanase had an optimum pH of 6.2, and it was stable over pH 5.6-10.3. The optimal temperature of xylanase was 75 degrees C and it was stable up to 65 degrees C at pH 6.2. Study was further carried out to investigate the effect of the purified xylanase on the properties of wheat bread and its staling during storage. CONCLUSIONS: The purified xylanase from T. lanuginosus CAU44 was stable up to 65 degrees C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specific bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry.     

The p domain of norovirus capsid protein forms a subviral particle that binds to histo-blood group antigen receptors

Norovirus is the most important cause of nonbacterial acute gastroenteritis. We have shown previously that the isolated P domain containing the hinge forms a dimer and binds to histo-blood group antigen (HBGA) receptors with a low affinity (M. Tan, R. S. Hegde, and X. Jiang, J. Virol. 78:6233-6242, 2004). Here, we reported that the P domain of VA387 without the hinge forms a small particle with a significantly increased receptor binding affinity. An end-linked oligopeptide containing one or more cysteines promoted P-particle formation by forming intermolecular disulfide bridges. The binding sensitivity of the P particle to HBGAs was enhanced >700-fold compared to the P dimer, which was comparable to that of virus-like particles. The binding specificity of the P particle was further confirmed by strong binding to the Caco-2 cells, a human colon carcinoma cell line. This binding enhancement was observed in the P particles of both norovirus GI and GII strains. The P particle is estimated to contain 12 P dimers, in which the P2 subdomain builds up the outer layer, while the P1 subdomain forms the internal core. Taken together, our data indicate that the P domain is involved not only in dimerization but also in polymerization of the protein during the capsid assembling. The enhanced receptor binding of the P particle reflects the intrinsic feature of the viral capsid. The easy production of the P particle and its strong binding to HBGAs suggest that the P particle is useful in studying pathogenesis and morphogenesis of norovirus and candidates for antiviral or vaccine development.     

Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10mM SrCl(2) for 0.5-5h, activation rates (88.4+/-4.1 to 91.2+/-2.7%) did not differ (mean+/-S.E.; P>0.2), but rate of blastulation (57.3+/-3.5%) and cell number per blastocyst (45.0+/-2.4) were the highest after treatment for 2.5h. When treated with 1-20mM SrCl(2) for 2.5h, the activation rate and cell number per blastocyst were higher (P<0.02) after 10mM SrCl(2) treatment than other treatments. The best activation and development were obtained with Ca(2+)-free Sr(2+) medium, but the activation rate was low (37.7+/-1.6%) in Ca(2+)-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher (P<0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6mM SrCl(2), and many lysed, with increased intensity of Sr(2+) treatment. The presence of CB in SrCl(2) medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr(2+) treatment alone, with 34% blastulating, but rates of activation and blastulation increased (P<0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less (P<0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl(2) treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5h with 10mM SrCl(2) in Ca(2+)-free medium supplemented with 5 microg/mL of CB.     

Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis isolated from the gastrointestinal tracts of healthy chickens

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121 degrees C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37 degrees C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.