Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance.

We sought to examine mechanisms responsible for increased vasoconstriction that occurs during development of nitroglycerin tolerance. Rabbits were treated for 3 days with nitroglycerin patches (0.4 mg/hr), and their aortic segments were studied in organ chambers. This treatment resulted in attenuated in vitro relaxations to nitroglycerin and increased contractile sensitivity to angiotensin II, serotonin, phenylephrine, KCl, and a direct activator of protein kinase C, the phorbol ester phorbol 12,13-dibutyrate. The protein kinase C antagonists calphostin C (100 nM) and staurosporine (10 nM) corrected the hypersensitivity to constrictors in tolerant vessels, yet had minimal effects on constrictions in control vessels. Paradoxically, constrictions caused by endothelin 1 were decreased in nitrate-tolerant vessels. Immunocytochemical analysis revealed intense endothelin 1-like and big endothelin 1-like immunoreactivity in the media of nitroglycerin-tolerant but not of control aortas. The enhanced vasoconstriction to angiotensin II, serotonin, KCl, and phenylephrine could be mimicked in normal vessels by addition of subthreshold concentrations of endothelin 1, and this effect was prevented by calphostin C. We propose that increased autocrine production of endothelin 1 in nitrate tolerance sensitizes vascular smooth muscle to a variety of vasoconstrictors through a protein kinase C-mediated mechanism.

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a C-13-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-C-13] glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using H-1 spin-echo NMR, while C-13[H-1-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.

The Role of protein kinase C modulation in the antiproliferative effects of bistratene A, bryostatin 1 and phorbol esters

PKC-mediated signalling pathways are important in cell growth and differentiation, and aberrations in these pathways are implicated in tumourigenesis. The objective of this project was to clarify the link between cell growth inhibition and PKC modulation.The PKC activators bryostatin 1 and 12-0-tetradecanoylphorbol-13-acetate (TPA) inhibited growth in A549 and MCF-7 adenocarcinoma cells with great potency, and induced HL-60 leukaemia cell differentiation. Bistratene A affected these cells similarly. Experiments were conducted to test the hypotheses that bistratene A exerts its effects via PKC modulation and that characteristics of cytostasis induced by bryostatin 1 and TPA depend upon PKC isozyme-specific events. After incubation of A549 cells with TPA or bistratene A, 2D phosphoprotein electrophoretograrns revealed three proteins phosphorylated by both agents. However, bistratene A was unable to induce the formation of cellular networks on the basement membrane substitute Matrigel, and staurosporine was unable to reverse bistratene A-induced [3H]thymidine uptake inhibition, unlike TPA. Bistratene A did not induce PKC translocation or downregulation, activate or inhibit A549 and MCF-7 cell cytosolic PKC or compete for phorbol ester receptors. Western blot analysis and hydroxylapatite chromatography identified PKC α, ε and ζ in these cells. Bistratene A was unable to activate any of these isoforms. Therefore the agent does not exert its antiproliferative effects by modulation of PKC activity. The abilities of bryostatin 1 and TPA (10nM-1μM) to induce PKC isoform translocation and downregulation were compared with antiproliferative effects. Both agents induced dose-dependent downregulation and translocation of PKC α and ε to particulate and nuclear cell fractions. PKC ζ was translocated to the particulate fraction by both agents in MCF-7 cells. The similarity of PKC isoform redistribution by these agents did not explain their divergent effects on cell growth, and the role of nuclear translocation of PKC in cytostasis was not confirmed by these studies. Alternative factors governing the characteristics of growth inhibition induced by these agents are discussed.

Temporal variation of delta C-13 in particulate organic matter and oyster Crassostrea gigas in Marennes-Oleron Bay (France): Effect of freshwater inflow

The temporal variability of delta(13)C in suspended particulate organic matter (POM) and oyster Crassostrea gigas along a salinity gradient was investigated from May 1992 to September 1993 within the estuarine bay of Marennes-Oleron (France). During this period the mean daily discharge of the Charente River exhibited large seasonal variation, with a high discharge from November 1992 to January 1993. Contrary to that at the river mouth and the marine littoral, delta(13)C in POM and in oysters at mid-estuary was affected by the high flood period. The delta(13)C values of POM decreased in mid-estuary and remained at low levels during the high discharge period, indicating an increasing contribution of terrestrial inputs to the estuarine POM pool. At the same site, a remarkable decrease of delta(13)C in oysters occurred between December 1992 and March 1993 (after a time lag compared to the ambient POM), indicating incorporation of terrestrial organic matter in oyster tissues during the high flood discharge. The lag between the delta(13)C decrease in POM and oysters is attributed to the time needed for oyster tissues to incorporate enough newly terrestrial light carbon to be recognized by the delta(13)C measure (about 1 to 2 mo). This time interval depends on tissue turnover time. The delta(13)C POM decrease (i.e. 1.3 parts per thousand) cannot explain entirely the decrease observed in oysters (i.e. 2.3 parts per thousand). In fact, the pattern exhibited by mid-estuarine oysters can be explained by the increasing contribution of terrestrial organic matter to their feeding, and the inability to preferentially utilize specific components of the estuarine POM that are C-13-enriched.

Influence of carbon sources and C/N ratio on EPS production in anaerobic sequencing batch biofilm reactors for wastewater treatment

The objective of this work was to evaluate the influence of different carbon sources and the carbon/nitrogen ratio (C/N) on the production and main composition of insoluble extracellular polymers (EPS) produced in an anaerobic sequencing batch biofilm reactor (ASBBR) with immobilized biomass in polyurethane foam. The yield of EPS was 23.6 mg/g carbon, 13.3 mg/g carbon, 9.0 mg/g carbon and 1.4 mg/g carbon when the reactor was fed with glucose, soybean oil. fat acids, and meat extract, respectively. The yield of EPS decreased from 23.6 to 2.6 mg/g carbon as the C/N ratio was decreased from 13.6 to 3.4 gC/gN, using glucose as carbon source. EPS production was not observed under strict anaerobic conditions. The results suggest that the carbon source, microaerophilic conditions and high C/N ratio favor EPS production in the ASBBR used for wastewater treatment. Cellulose was the main exopolysaccharide observed in all experimental conditions. (C) 2009 Elsevier Ltd. All rights reserved.

Cement stabilization of runoff residuals: A study of stabilization/solidification of urban rainfall-runoff residuals in type 1 Portland cement by XRD and Si-29 NMR analysis

Urban rainfall-runoff residuals contain metals such as Cr, Zn, Cu, As, Pb and Cd and are thus reasonable candidates for treatment using Portland cement-based solidification-stabilization (S/S). This research is a study of S/S of urban storm water runoff solid residuals in Portland cement with quicklime and sodium bentonite additives. The solidified residuals were analyzed after 28 days of hydration time using X-ray powder diffraction (XRD) and solid-state Si-29 nuclear magnetic resonance (NMR) spectroscopy. X-ray diffraction (XRD) results indicate that the main cement hydration products are ettringite, calcium hydroxide and hydrated calcium silicates. Zinc hydroxide and lead and zinc silicates are also present due to the reactions of the waste compounds with the cement and its hydration products. Si-29 NMR analysis shows that the coarse fraction of the waste apparently does not interfere with cement hydration, but the fine fraction retards silica polymerization.

Genetic Transformation of Passionflower and Evaluation of R(1) and R(2) Generations for Resistance to Cowpea aphid borne mosaic virus

We report on the production and evaluation of passionflower transgenic lines for resistance to Cowpea aphid borne mosaic virus (CABMV). Genetic transformation was done using Agrobacterium tumefaciens and transgene integration was confirmed by Southern blot analyses, resulting in nine transgenic lines for `IAC 275` and three for `IAC 277`. Transgenic lines were clonally propagated and evaluated for resistance to CABMV After the third inoculation, under higher inoculum pressure, only propagated plants of the transgenic line T16 remained asymptomatic, indicating a high resistance to infection with CABMV. This transgenic line was self-pollinated and the RI generation was evaluated together with the RI generation of another resistant transgenic line (T2) identified previously. Plants were inoculated with CABMV by means of viruliferous Myzus nicotianae. All 524 T2R(1) plants became infected, whereas 13 of 279 T16R(1) remained asymptomatic after four successive inoculations. A TI6R(2) generation was obtained and plants were inoculated with CABMV mechanically or by aphids. After successive inoculations, 118 of 258 plants were symptomless, suggesting that the resistance to CABMV was maintained in the plant genome as the homozygous condition was achieved. Five selected resistant TI6R(2) plants which contained the capsid protein gene are being crossed for further analyses.

Regiospecific synthesis of 5-trichloromethyl-1H-pyrazole and 1H-pyrazole-5-carboxylic ester derivatives

Reaction of 1,1,1-trichloro-4-methoxy-3-penten-2-one (1) with hydrazines (2a-h) (NH2NHR, R = H, Me, t-Bu, Ph, 4-NO2-C6H4, C6F5, CO2Me, CONH2) under differing conditions regiospecifically affords different pyrazole derivatives, 3-methyl-5-trichloromethyl-5-hydroxy-4,5-dihydropyrazoles (3a, d-h), 3-methyl-5-trichloromethyl-1H-pyrazoles (4a,b,d-g) and 5-carboxyethyl-3-methyl-1H-pyrazoles (5a-e). The structural assignments were based on the analysis of their H-1/C-13 NMR and ESI-MS data.

Miscibility and specific interactions in blends of poly(4-vinylphenol) and poly(2-ethoxyethyl methacrylate)

The properties of the hydrogen-bonded polymer blends of poly(4-vinylphenol) and poly(2-ethoxyethyl methacrylate) are presented. Spectroscopic techniques such as C-13 solid-state NMR and FT-IR are used to probe specific interactions of the blends at various compositions. Spectral features from both techniques revealed that site-specific interactions are present, consistent with a significant degree of mixing of the blend components. Changes in chemical shift and line shape of the phenolic carbon and carbonyl resonances in the C-13 CPMAS spectra of the blends as a function of composition are interpreted as resulting from changes in the relative intensities of two closely overlapped signals. A quantitative measure of hydrogen-bonded carbonyl groups using C-13 NMR has been obtained which agreed well with the results from FT-IR analyses. It is also shown that C-13 NMR can be used to measure the fraction of hydroxyl groups associated with carbonyl groups, which was not possible previously using FT-IR due to extensive overlapping of bands in the hydroxyl stretching region. The results of measurements of H-1 T-1 and 1H T-1 rho indicate that PVPh and PEEMA are intimately mixed on a scale less than 2-3 nm.

Copolymer hydrogels of 2-hydroxyethyl methacrylate with n-butyl methacrylate and cyclohexyl methacrylate: synthesis, characterization and uptake of water

The bulk free radical copolymerizations of 2-hydroxyethyl methacrylate (HEMA) with n-butyl methacrylate (BMA) or cyclohexyl methacrylate (CHMA) were studied over the composition mole fraction interval of 0-1 for HEMA in the monomer feed. The C-13 NMR (125 MHz) spectra of the copolymers were analysed to determine the copolymer composition and the stereochemical configuration of the copolymers. The terminal model reactivity ratios of HEMA and BMA were found to be r(HEMA) = 1.73 and r(BMA) = 0.65 and for HEMA and CHMA, r(HEMA) = 1.26 and r(CHMA) = 0.31. The BMA and CHMA homopolymers were found to be predominantly syndiotactic with isotacticity parameters of theta(BB) = 0.18 and theta(CC) = 0.19, respectively. The copolymers were also found to be predominantly syndiotactic, indicating a strong tendency for racemic additions of the monomers in the formation of the copolymers. The diffusion of water into cylinders of poly(HEMA-co-BMA) and poly(HEMA-co-CHMA) was studied over a range of copolymer compositions and was found to be Fickian. The diffusion coefficients of water at 37 degrees C were determined from swelling measurements and were found to vary from 1.72 x 10(-11) m(2) s(-1) for polyHEMA to 0.97 x 10(-11) m(2) s(-1) for poly(HEMA-co-BMA) having a mole fraction F-HEMA = 0.80 and to 0.91 x 10(-11) m(2) s(-1) for a poly(HEMA-co-CHMA) also having F-HEMA = 0.80. The mass of water absorbed at equilibrium relative to the mass of dry polymer varied from 58.8 for polyHEMA to 27.2% for poly(HEMA-co-BMA) having F-HEMA = 0.85 and to 21.3% for poly(HEMA-co-CHMA) having F-HEMA = 0.80. (C) 1999 Elsevier Science Ltd. All rights reserved.

Level-one highest weight representations of U-q[g1(1 vertical bar 1)] and associated vertex operators

We study the level-one irreducible highest weight representations of U-q[gl(1\1)] and associated q-vertex operators. We obtain the exchange relations satisfied by these vertex operators. The characters and supercharacters associated with these irreducible representations are calculated'. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

Mesoionic pyridopyrimidinylium and pyridooxazinylium olates and non-mesoionic pyridopyrimidinones. Structures in the solid state, solution, and matrices

X-Ray crystal structures, C-13 NMR spectra and theoretical calculations (B3LYP/6-31G*) are reported for the mesoionic (zwitterionic) pyridopyrimidinylium- and pyridooxazinyliumolates 2a, 3a and 5a,b as well as the enol ether 11b and the enamine 11c. The 1-NH compounds like 1a, 2a and 3a exist in the mesoionic form in the crystal and in solution, but the OH tautomers such as 1b and 2b dominate in the gas phase as revealed by the Ar matrix IR spectra in conjunction with DFT calculations. All data indicate that the mesoionic compounds can be regarded as intramolecular pyridine-ketene zwitterions (cf. 16 --> 17) with a high degree of positive charge on the pyridinium nitrogen, a long pyridinium N-CO bond (ca. 1.44-1.49 Angstrom), and normal C=O double bonds (ca. 1.22 Angstrom). All mesoionic compounds exhibit a pronounced tilting of the olate C=O groups (the C=O groups formally derived from a ketene) towards the pyridinium nitrogen, giving NCO angles of 110-118 degrees. Calculations reveal a hydrogen bond with 6-CH, analogous to what is found in ketene-pyridine zwitterions and the C3O2-pyridine complex. The 2-OH tautomers of type 1b, 2b, and 11 also show a high degree of zwitterionic character as indicated by the canonical structures 11 12.