Magnetic-field-induced delocalized to localized transformation in GaAs:N.

The use of a high magnetic field (57 T) to study the formation and evolution of nitrogen (N) cluster and supercluster states in GaAs:N is demonstrated. A magnetic field is used to lift the conduction band edge and expose resonant N cluster states so that they can be directly experimentally investigated. The reduction of the exciton Bohr radius also results in the fragmentation of N supercluster states, enabling a magnetic field induced delocalized to localized transition. The application of very high magnetic fields thus presents a powerful way to probe percolation phenomena in semiconductors with bound and resonant isoelectronic cluster states.

The effect of microbial phytase and myo-inositol on performance and blood biochemistry of broiler chickens fed wheat/corn-based diets.

A total of 1,200 Ross broiler chickens were used in 2 separate feeding studies to explore the effect of myo-inositol (MYO) and phytase on performance and blood biochemistry of broilers fed diets formulated to be either adequate or insufficient in Ca and digestible P (dP). Supplementation of diets that were formulated to be insufficient in Ca and dP with MYO resulted in improved BW gain and feed conversion ratio in both experiments. However, these effects were most pronounced in the finisher phase, and moderate negative effects were observed during the starter period. Supplementation of the diet with microbial phytase improved BW gain and feed conversion ratio to a similar extent as was observed with MYO, and there was a degree of subadditivity between the 2 additives. Blood glucose concentrations were increased by both MYO and phytase, though possibly by different mechanisms, because insulin concentrations were not directly relatable to circulating glucose levels, especially when both MYO and phytase were applied simultaneously. The increase in blood glucose concentrations with MYO and phytase was most pronounced in the diet with a lower Ca and dP concentration. It can be concluded that dietary supplementation with MYO or phytase was effective in improving performance of commercial broiler chickens. However, further work is required to explore complex ontogenetic effects of MYO and possible involvement of both MYO and phytase in Na-dependent transport mechanisms.

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.

Multi-carbohydrase and phytase supplementation improves growth performance and liver insulin receptor sensitivity in broiler chickens fed diets containing full-fat rapeseed.

The effect of a combination of carbohydrase and phytase enzymes on growth performance, insulin-like growth factor 1 gene expression, insulin status, and insulin receptor sensitivity in broiler chickens fed wheat-soybean meal diets containing 6% (starter) and 12% (grower-finisher) of full-fat rapeseed (canola type; low glucosinolate, low erucic acid) from 1 to 42 d of age was studied. A total of 510 one-day-old male broiler chickens were randomly assigned to 3 dietary treatments, with 17 pens per treatment and 10 birds per pen. The dietary treatments consisted of a control diet and P- and Ca-deficient diets supplemented with either phytase (500 U/kg) or a combination of phytase and a multi-carbohydrase enzyme (Superzyme OM). The diets were pelleted at 78 degrees C and were fed ad libitum throughout the starter (9 d), grower (18 d), and finisher (15 d) phases of the experiment. Over the entire trial, growth performance of birds fed the phytase-supplemented diet did not differ from birds fed the control diet. The use of phytase in combination with a multicarbohydrase enzyme improved (P = 0.007) the feed conversion ratio from 1.90 to 1.84. Insulin liver receptor sensitivity increased by 9.3 and 12.3% (P = 0.004) for the phytase- and the carbohydrase-phytase-supplemented diets, respectively. There was no effect of phytase alone or carbohydrase and phytase supplementation on total plasma cholesterol, high-density lipoprotein cholesterol, and blood glucose levels. However, low-density lipoprotein cholesterol decreased (P = 0.007) for the phytase-carbohydrase treatment. Gene expression of insulin-like growth factor 1 tended to decrease by 32% (P = 0.083) after phytase-carbohydrase supplementation. The combination of carbohydrase and phytase enzymes may serve as an attractive means of facilitating nutrient availability for digestion and thus enhance the feeding value of wheat-soybean meal-based diets containing full-fat rapeseed. However, the extent to which the effects of enzyme addition on insulin receptors are associated with growth performance of broiler chicken requires further research.

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration.

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Hot plasma and black hole binaries in starburst galaxy M82.

High-resolution x-ray observations of the prototype starburst galaxy Messier 82 (M82) obtained with the advanced CCD (charge-coupled device) imaging spectrometer on board the Chandra X-ray Observatory provide a detailed view of hot plasma and energetic processes. Plasma with temperature of about 40,000,000 kelvin fills the inner 1 kiloparsec, which is much hotter than the 1,000,000 to 2,000,000 kelvin interstellar medium component in the Milky Way Galaxy. Produced by many supernova explosions, this central region is overpressurized and drives M82's prominent galactic wind into the intergalactic medium. We also resolved about 20 compact x-ray sources, many of which could be high-mass x-ray binary star systems containing black holes.

Mechanisms of action of two different natural mixtures of persistent organic pollutants (POPs) in ovarian follicles.

The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)beta protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were 'Mjosa' extracted from burbot liver, which contains a high level of PBDEs, and 'Marine mix', extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs). Follicular cells were exposed in vitro to 'Marine mix' and 'Mjosa mix' at doses of 3.6 and 1.4 microg ml(-1), respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity. Western blot analysis indicated down-regulation of AhR by 'Marine mix' and down-regulation of ERbeta by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both 'Marine mix' and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity. These results suggest that: (1) 'Marine mix' is a mixed-type CYP inducer; (2) 'Mjosa mix' is an inducer of ERbeta and CYP2B; and (3) both 'Marine mix' and 'Mjosa mix' stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19.

Compounds of PAH mixtures dependent interaction between multiple signaling pathways in granulosa tumour cells.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.

Do G-protein coupled estrogen receptor and bisphenol A analogs influence on Leydig cell epigenetic regulation in immature boar testis ex vivo?

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Interplay between estrogen-related receptors and steroidogenesis-controlling molecules in adrenals. In vivo and in vitro study.

Estrogen-related receptors (ERRs) α, ß and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRß/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 µg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 µg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRß/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules.

Young Accreting Compact Objects in M31: The Combined Power of NuSTAR, Chandra, and Hubble.

We present 15 high-mass X-ray binary (HMXB) candidates in the disk of M31 for which we are able to infer compact object type, spectral type of the donor star, and age using multiwavelength observations from NuSTAR, Chandra, and the Hubble Space Telescope. The hard X-ray colors and luminosities from NuSTAR permit the tentative classification of accreting X-ray binary systems by compact object type, distinguishing black hole from neutron star systems. We find hard-state black holes, pulsars, and non-magnetized neutron stars associated with optical point-source counterparts with similar frequency. We also find nine non-magnetized neutron stars coincident with globular clusters and an equal number of pulsars with and without point-source optical counterparts. We perform spectral energy distribution (SED) fitting for the most likely optical counterparts to the HMXB candidates, finding seven likely high-mass stars and one possible red helium-burning star. The remaining seven HMXB optical counterparts have poor SED fits, so their companion stars remain unclassified. Using published star formation histories, we find that the majority of HMXB candidates-X-ray sources with UV-bright point-source optical counterpart candidates-are found in regions with star formation bursts less than 50 Myr ago, and three are associated with young stellar ages (<10Myr). This is consistent with similar studies of HMXB populations in the Magellanic Clouds, M33, NGC 300, and NGC 2403.

Electron transfer dissociation of doubly sodiated glycerophosphocholine lipids.

The ability to generate gaseous doubly charged cations of glycerophosphocholine (GPC) lipids via electrospray ionization has made possible the evaluation of electron-transfer dissociation (ETD) for their structural characterization. Doubly sodiated GPC cations have been reacted with azobenzene radical anions in a linear ion trap mass spectrometer. The ion/ion reactions proceed through sodium transfer, electron-transfer, and complex formation. Electron-transfer reactions are shown to give rise to cleavage at each ester linkage with the subsequent loss of a neutral quaternary nitrogen moiety. Electron-transfer without dissociation produces [M + 2Na](+.) radical cations, which undergo collision-induced dissociation (CID) to give products that arise from bond cleavage of each fatty acid chain. The CID of the complex ions yields products similar to those produced directly from the electron-transfer reactions of doubly sodiated GPC, although with different relative abundances. These findings indicate that the analysis of GPC lipids by ETD in conjunction with CID can provide some structural information, such as the number of carbons, degree of unsaturation for each fatty acid substituent, and the positions of the fatty acid substituents; some information about the location of the double bonds may be present in low intensity CID product ions.

Expression of peroxisome proliferator-activated receptors is regulated by gonadotropins and steroid hormones in in vitro porcine ovarian follicles.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors which are involved in the regulation of different processes such as lipid metabolism, inflammation, angiogenesis, tissue remodeling and steroidogenesis. Our previous data described the ovarian expression of PPAR isoforms (-α, -γ and -ß), and the increase of PPAR-α and PPAR-γ in porcine ovarian follicles during the estrous cycle. The current studies were undertaken to test the hypothesis that gonadotropin and steroid hormones can regulate the expression of PPAR isoforms in the ovary. Medium follicles (4 - 6 mm) at 10 - 12 days of the estrous cycle were obtained from mature crossbred gilts. Ovarian follicles were exposed to gonadotropins FSH and LH at 50, 100 and 150 ng/ml, and to steroid hormones such as progesterone (P4), testosterone (T) and estradiol (E2) at 10-8, 10-7, 10-6 M for 24 hours. Then mRNA and protein expression of PPAR-α/γ/ß via real time PCR and Western blot, respectively, were measured. We observed that FSH increased both mRNA and protein expression of all PPAR isoforms, while LH only increased PPAR-α/γ. We have also noted that P4 and E2 significantly increased expression of PPAR-α/γ without having an effect on ß isoform, while T had no effect on all PPARs expression. Our study clearly showed that local regulators of ovarian activity, both gonadotropin and steroid hormones are regulators of PPAR isoforms expression in porcine ovarian follicles.

Differential accumulation of low-chlorinated (Delor 103) and high-chlorinated (Delor 106) biphenyls in human placental tissue and opposite effects on conversion of DHEA to E2.

OBJECTIVE: Polychlorinated biphenyls (PCBs) and related compounds elicit a diverse spectrum of toxic responses. Additionally, they are able to pass through the human placenta. The aim of the presented data was to compare the action of low-chlorinated (Delor 103) and (Delor 106) high-chlorinated biphenyls on placental steroidogenesis. METHODS: Explants of human placental tissue were used to test differences in PCBs accumulation and influence on placental steroidogenesis. Delor 103 or 106, were added daily for six days at a dose of 200 pg from day 0 to day 6 of culture. The media in the control and experimental groups were changed every day, and collected and frozen for steroid analysis by RIA. Determinations of PCBs of tissue and medium were analysed by GC/MS/MS. RESULTS: Delor 103 was found at a higher level in the tissue than Delor 106. The first day of exposure to Delor 103 had no effect on the conversion of dehydroepiandrosterone (DHEA) to estradiol (E2) while there was a 2-fold decrease in E2 secretion from days 3 to 6. Conversely, Delor 106 caused an immediate increase in E2 secretion, which was maintained at higher levels throughout the exposure period. CONCLUSION: Differences between the accumulation of lower chlorinated and higher chlorinated biphenyls in human placental tissue and in the properties of the congeners can have multiple effects that may intensify or counteract the effects on uterine contraction by PCBs.

Primary and tumor mouse Leydig cells exposed to polychlorinated naphthalenes mixture: Effect on estrogen related-receptors expression, intracellular calcium level and sex hormones secretion.

We report the effects of polychlorinated napthalanes (PCNs) on the mRNA expression of estrogen-related receptors (ERRs) α, ß and γ, calcium (Ca2+) concentration, and sex steroid secretion in mouse primary and tumor Leydig cells. The cells were exposed to a mixture of PCNs (10nM) alone or in combination with one of sex steroid receptor antagonists; 182,780 (ICI; 10µM); hydroxyflutamide (HF; 10(-4)M) and G-coupled estrogen receptor antagonist (G15; 10nM) respectively. The expression of mRNAs and protein for ERRα, ß, and γ was detected in primary and tumor Leydig cells. The expression of ERRs was always lower in primary Leydig cells. Exposure of Leydig cells to PCNs significantly increased the expression of ERRs mRNA irrespective of the cell type. Concomitantly, an increased concentration of Ca2+ and sex steroids was revealed in exposed cells. After ICI, HF or G15 was added no changes in expression of ERRs was found. In Leydig cells changes in ERRs expression at mRNA level are clearly linked to changes in Ca2+ level and steroid secretion. Estrogen and androgen receptors are not involved in PCNs action in Leydig cells. The effect of PCNs on mouse Leydig cells is independent on the cell of origin (primary or tumor).

Classic NSAID and selective cyclooxygenase (COX)-1 and COX-2 inhibitors in healing of chronic gastric ulcers.

Prostaglandins (PG) derived from COX-1 are essential for the maintenance of mucosal integrity but COX-2 isoform synthesizes PG at a site of inflammation. Recently, COX-2 mRNA expression was demonstrated at the ulcer edge during healing of chronic gastric ulcers but the role for expression of COX-2 and its products such as PGE(2) and cytokines including interleukin (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in ulcer healing remains unknown. In this study, Wistar rats with gastric ulcers produced by serosal application of acetic acid (ulcer area 28 mm(2)) received daily treatment either with: (1) vehicle (saline); (2) NS-398 (10 mg/kg-d i.g.) and Vioxx (5 mg/kg-d i.g.), both, highly specific COX-2 inhibitors; (3) meloxicam (5 mg/kg-d i.g.), a preferential inhibitor of COX-2; (4) resveratrol (10 mg/kg-d i.g.), a specific COX-1 inhibitor; (5) indomethacin (5 mg/kg-d i.g); and (6) aspirin (ASA; 50 mg/kg-d i.g.), non-selective inhibitors of both COX-1 and COX-2. At day 3, 7, and 14 after ulcer induction, the animals were sacrificed and the area of gastric ulcers was determined by planimetry and histology, gastric blood flow (GBF) at ulcer base and margin was measured by H(2) clearance technique, and blood was withdrawn for measurement of plasma IL-1beta and TNFalpha levels. The mucosal biopsy samples were taken for the determination of PGE(2) generation by RIA and expression of COX-1, COX-2, IL-1beta, and TNFalpha mRNA by RT-PCR. In vehicle-treated rats, gastric ulcers healed progressively and at day 14 the healing was completed, accompanied by a significant rise in the GBF at ulcer margin. The IL-1beta, TNFalpha, and COX-1 mRNA were detected in intact and ulcerated gastric mucosa, whereas COX-2 mRNA were upregulated only in ulcerated mucosa with peak observed at day 3 after ulcer induction. The plasma IL-1beta level was significantly increased at day 3 and 7 but then declined at day 14 to that measured in vehicle-controls. Indomethacin and ASA, which suppressed PGE(2) generation both in the non-ulcerated and ulcerated gastric mucosa, significantly delayed the rate of ulcer healing and this was accompanied by the fall in GBF at ulcer margin and further elevation of plasma IL-1beta and TNFalpha levels, which was sustained up to the end of the study. Treatment with NS-398 and Vioxx, which caused only a moderate decrease in the PGE(2) generation in the non-ulcerated gastric mucosa, delayed ulcer healing and attenuated significantly the GBF at ulcer margin and PGE(2) generation in the ulcerated tissue, while raising the plasma IL-1beta and TNFalpha similarly to those observed in indomethacin- and ASA-treated rats. Resveratrol, which suppressed the PGE(2) generation in both non-ulcerated and ulcerated gastric mucosa, prolonged ulcer healing and this was accompanied by the fall in the GBF at the ulcer margin and a significant increase in plasma IL-1beta and TNFalpha levels. We conclude that (1) classic NSAID delay ulcer healing due to suppression of endogenous PG, impairment in GBF at ulcer area, and excessive cytokine expression and release, and (2) this deleterious effect of classic NSAID on the healing of pre-existing ulcers can be reproduced by selective COX-1 and COX-2 inhibitors, suggesting that both COX isoforms are important sources of PG that appear to contribute to ulcer healing.

Involvement of gastrin in gastric secretory and protective actions of N-alpha-methyl histamine.

N alpha-methylhistamine (N alpha-MH) is one of an unusual metabolite of histamine that was found in Helicobacter pylori-infected stomachs and is believed to interact with specific histamine H(1), H(2) and H(3)-receptors to stimulate gastric acid secretion and gastrin release from isolated G-cells but the effects of N alpha-MH on gastric mucosal integrity have been little studied. This study was designed; (1) to compare the effect of exogenous N alpha-MH with that of standard histamine on gastric secretion and plasma gastrin levels in rats equipped with gastric fistula (series A); and (2) to assess the action of N alpha-MH on gastric lesions induced by 100% ethanol (series B) in rats with or without removal of antral portion of the stomach (antrectomy). Rats of series B were pretreated intragastrically (i.g.) or intraperitoneally (i.p.) with N alpha-MH or histamine (0.1-2 mg/kg) 30 min prior to 100% ethanol (1.5 ml, i.g.) with or without: (1) vehicle (saline); (2) RPR 102681 (30 mg/kg i.p.), to block CCK-B/gastrin receptors; and (3) ranitidine (40 mg/kg s.c.) to inhibit histamine H(2)-receptors. The area of gastric lesions was determined planimetrically, gastric blood flow (GBF) was assessed by H(2)-gas clearance method and venous blood was collected for determination of plasma gastrin levels by radioimmunoassay (RIA). N alpha-MH and histamine dose-dependently increased gastric acid output (series A); the dose increasing this secretion by 50% (ED(50)) being 2 and 5 mg/kg i.g or i.p., respectively, and this effect was accompanied by a significant rise in plasma gastrin levels. Both, N alpha-MH and histamine attenuated dose-dependently the area of gastric lesions induced by 100% ethanol (series B) while producing significant rise in the GBF and plasma immunoreactive gastrin increments. These secretory, protective, hipergastrinemic and hyperemic effects of N alpha-MH and histamine were completely abolished by antrectomy, whereas pretreatment with RPR 102681 attenuated significantly the N alpha-MH and histamine-induced protection against ethanol damage and accompanying hyperemia. Ranitidine, that produced achlorhydria and a further increase in plasma gastrin levels, failed to influence the N alpha-MH- and histamine-induced protection and accompanying rise in the GBF. We conclude that (1) N alpha-MH stimulates gastric acid secretion and exhibit gastroprotective activity against acid-independent noxious agents in the manner similar to that afforded by histamine; and (2) this protection involves an enhancement in the gastric microcirculation and release of gastrin acting via specific CCK-B/gastrin receptors but unexpectedly, appears to be unrelated to histamine H(2)-receptors.

Exogenous and endogenous ghrelin in gastroprotection against stress-induced gastric damage.

Ghrelin, identified in the gastric mucosa has been involved in control of food intake and growth hormone (GH) release but little is known about its influence on gastric secretion and mucosal integrity. The effects of ghrelin on gastric secretion, plasma gastrin and gastric lesions induced in rats by 75% ethanol or 3.5 h of water immersion and restraint stress (WRS) were determined. Exogenous ghrelin (5, 10, 20, 40 and 80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by ethanol and WRS and this was accompanied by the significant rise in plasma ghrelin level, gastric mucosal blood flow (GBF) and luminal NO concentrations. Ghrelin-induced protection was abolished by vagotomy and attenuated by suppression of COX, deactivation of afferent nerves with neurotoxic dose of capsaicin or CGRP(8-37) and by inhibition of NOS with L-NNA but not influenced by medullectomy and administration of 6-hydroxydopamine. We conclude that ghrelin exerts a potent protective action on the stomach of rats exposed to ethanol and WRS, and these effects depend upon vagal activity, sensory nerves and hyperemia mediated by NOS-NO and COX-PG systems.

Ischemic preconditioning, the most effective gastroprotective intervention: involvement of prostaglandins, nitric oxide, adenosine and sensory nerves.

Various organs, including heart, kidneys, liver or brain, respond to brief exposures to ischemia with an increased resistance to severe ischemia/reperfusion and this phenomenon is called "preconditioning". No study so far has been undertaken to check whether such short, repeated gastric ischemic episodes protect gastric mucosa against severe damage caused by subsequent prolonged ischemia/reperfusion and, if so, what could be the mechanism of this phenomenon. The ischemic preconditioning was induced by short episodes of gastric ischemia (occlusion of celiac artery from one to five times, for 5 min each) applied 30 min before prolonged (30 min) ischemia followed by 3 h of reperfusion or 30 min before topical application of strong mucosal irritants, such as 100% ethanol, 25% NaCl or 80 mM taurocholate. Exposure to regular 30-min ischemia, followed by 3-h reperfusion, produced numerous severe gastric lesions and significant fall in the gastric blood flow and prostaglandin E(2) generation. Short (5-min) ischemic episodes (1-5 times) by itself failed to cause any gastric lesions, but significantly attenuated those produced by ischemia/reperfusion. This protection was accompanied by a reversal of the fall in the gastric blood flow and prostaglandin E(2) generation and resembled that induced by classic gastric mild irritants. These protective and hyperemic effects of standard preconditioning were significantly attenuated by pretreatment with cyclooxygenase-2 and cyclooxygenase-1 inhibitors, such as indomethacin, Vioxx, resveratrol and nitric oxide (NO)-synthase inhibitor, N(G)-nitro-L-arginine (L-NNA). The protective and hyperemic effects of standard preconditioning were restored by addition of 16,16 dm prostaglandin E(2) or L-arginine, a substrate for NO synthase, respectively. Gastroprotective and hyperemic actions of standard ischemic preconditioning were abolished by pretreatment with capsaicin-inactivating sensory nerves, but restored by the administration of exogenous CGRP to capsaicin-treated animals. Gene and protein expression of cyclooxygenase-1, but not cyclooxygenase-2, were detected in intact gastric mucosa and in that exposed to ischemia/reperfusion with or without ischemic preconditioning, whereas cyclooxygenase-2 was overexpressed only in preconditioned mucosa. We conclude that: (1) gastric ischemic preconditioning represents one of the most powerful protective interventions against the mucosal damage induced by severe ischemia/reperfusion as well as by topical mucosal irritants in the stomach; (2) gastric ischemic preconditioning resembles the protective effect of "mild irritants" against the damage by necrotizing substances in the stomach acting via "adaptive cytoprotection" and involves several mediators, such as prostaglandin derived from cyclooxygenase-1 and cyclooxygenase-2, NO originating from NO synthase and sensory nerves that appear to play a key mechanism of gastric ischemic preconditioning.