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Terms modified by Pmol Selected AbstractsStructure,activity relationships of isoeugenol-based chlorophenylpiperazine derivatives on serotonergic/adrenergic receptor, platelet aggregation, and lipid peroxidationDRUG DEVELOPMENT RESEARCH, Issue 5 2010Kuo-Ping Shen Abstract Three isoeugenol-based eugenosedin chlorphenylpiperazine derivatives, Eu-A, Eu-B, and Eu-C, were synthesized and tested for their serotonergic, adrenergic antagonist, antioxidant, and anti-aggregation activities. In radioligand binding assays, all three agents displayed significant binding affinities on ,1, ,2, ,1, 5-HT1B, and 5-HT2A receptors. In human platelet, they inhibited epinephrine and 5-HT-induced aggregation, and in human platelet with ,2 and 5-HT2A receptors they had a competitive binding effect. Eu-B and Eu-C were more potent than Eu-A. All compounds had antioxidant effects derived from aryloxypropanolamine. Eu- A, Eu-B, or Eu-C (1, 3, 5,mg/kg iv) given to normotensive Wistar rats produced a dose-dependent decrease in mean arterial blood pressure and heart rate and when injected into the cisternum, Eu-A, Eu-B, or Eu-C (0.3, 0.03,µmol) increased blood pressure within 15,min. Pretreatment with any of the three agents inhibited clonidine (38,pmol)-induced hypotension. In vitro experiments, Eu-A, Eu-B, or Eu-C (1, 10, and 100,µM) competitively antagonized norepinephrine-, clonidine-, and 5-HT (10,8,10,4,M)-induced vasocontraction in isolated rat aorta, and competitively antagonized isoproterenol (10,8,10,4,M)-induced positive inotropic effects in a concentration-dependent manner in the isolated rat left atrium. In isolated rabbit ear arteries sensitized with 16,mM K+, all three agents antagonized 5-nonyloxytryptamine- and 5-HT-induced vasocontractions. These findings show that Eu-A, Eu-B, and Eu-C possess functional ,1, ,2, ,1, 5-HT1B, and 5-HT2A receptor blocking activities. In conclusion, the changes in the position of chloride at phenylpiperazine influenced the serotonergic receptor, adrenoceptor antagonistic activities, but not anti-aggregation and antioxidant activities. Drug Dev Res 71:1,9, 2010. © 2010 Wiley-Liss, Inc. [source] Enhancement of Anodic Response for DMSO at Ruthenium Oxide Film Electrodes as a Result of Doping with Iron(III)ELECTROANALYSIS, Issue 2 2003Brett Abstract The oxidation of dimethyl sulfoxide (DMSO) to dimethyl sulfone (DMSO2) is representative of numerous anodic oxygen-transfer reactions of organosulfur compounds that suffer from slow kinetics at noble metal electrodes. Anodic voltammetric data for DMSO are examined at various RuO2 -film electrodes prepared by thermal deposition on titanium substrates. The response for DMSO is slightly larger at RuO2 films prepared in a flame as compared with films prepared in a furnace; however, temperature is more easily controlled in the furnace. Doping of the RuO2 films with Fe(III) further improves the sensitivity of anodic response for DMSO. Optimal response is obtained at an Fe(III)-doped RuO2 -film electrode prepared using a deposition solution of 50,mM RuCl3 and 10,mM FeCl3 in a 1,:,1 mixture of isopropanol and 12,M HCl at an annealing temperature of 450,°C. The Levich plot (i vs. ,1/2) and Koutecky-Levich plot (1/i vs. 1/,1/2) of amperometric data for the oxidation of DMSO at an Fe(III)-doped RuO2 -film electrode configured as a rotated disk are consistent with an anodic response controlled by mass-transport processes at low rotational velocities. Flow injection data demonstrate that Fe(III)-doped RuO2 -film electrodes exhibit detection capability for methionine and cysteine in addition to DMSO. Detection limits for 100-,L injections of the three compounds are ca. 3.2×10,4,mM, i.e., ca. 32,pmol. [source] Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detectionELECTROPHORESIS, Issue 22 2008Alicia M. Hitchcock Abstract This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1,pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50,mM phosphate buffer, pH 3.5, and reversed polarity at 30,kV with 0.3,psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue. [source] Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4,,6-diamidino-2-phenylindolELECTROPHORESIS, Issue 19 2007Stephanie A. Smith Dr. Abstract PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4,,6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4,pmol (0.3,ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250,pmol per band. [source] Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencingELECTROPHORESIS, Issue 12 2007Pengfeng Xiao Dr. Abstract A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1,M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10,,L of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis. [source] Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillaryELECTROPHORESIS, Issue 15 2006Mahmoud M. Yassine Abstract Preparative capillary zone electrophoresis separations of cytochrome,c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C14DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7,million plates/m were observed in 50-µm,id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1,g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome,c resulted in dramatic improvements in the efficiency and peak shape. Using 100,mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH,7.0 with a 100-µm,id capillary, the maximum sample loading capacity in a single run was 160,pmol (2.0,µg) of each protein. [source] Comparison of automated in-gel digest methods for femtomole level samplesELECTROPHORESIS, Issue 19-20 2003Erin J. Finehout Abstract A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used. [source] Bone turnover markers and sex hormones in men with idiopathic osteoporosisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2001P. Pietschmann Background In contrast to osteoporosis in postmenopausal women, osteoporosis in men has received much less attention. Patients and We determined various biochemical parameters of bone metabolism and sex hormones in 31 men with idiopathic osteoporosis and 35 age matched control subjects. Results In the men with osteoporosis, a significantly increased urinary excretion of deoxypyridinoline (5·3 ± 0·2 vs. 4·6 ± 0·2 nmol mmol,1 creatinine; P = 0·033) in addition to increased serum levels of the c-terminal telopeptide of type I collagen (2677 ± 230 vs. 2058 ± 153 pmol; P = 0·037) were found. While parameters of bone formation were not significantly different in the patients and controls, serum bone sialoprotein levels were significantly decreased in the patients (3·7 ± 0·8 vs. 12·4 ± 4·0 ng mL,1; P = 0·021). Moreover, in men with idiopathic osteoporosis, lower levels of estradiol (91·3 ± 5·8 vs. 114·6 ± 7·8 pmol L,1; P = 0·044), higher levels of sex hormone binding globulin (31·5 ± 3·1 vs. 24·2 ± 1·4 nmol L,1; P = 0·034) and a decreased free androgen index (42·6 ± 5·2 vs. 56·4 ± 5·9; P = 0·016) were seen. Serum estradiol levels correlated negatively with several parameters of bone resorption. Conclusions In men with idiopathic osteoporosis, bone resorption is increased and exceeds bone formation. The excessive bone resorption seen in idiopathic male osteoporosis may be due to decreased estradiol levels and low levels of bioavailable testosterone. [source] Blockade of caspase-1 increases neurogenesis in the aged hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2007Carmelina Gemma Abstract Adult hippocampal neurogenesis dramatically decreases with increasing age, and it has been proposed that this decline contributes to age-related memory deficits. Central inflammation contributes significantly to the decrease in neurogenesis associated with ageing. Interleukin-1, is a proinflammatory cytokine initially synthesized as an inactive precursor that is cleaved by caspase-1 to generate the biologically active mature form. Whether IL-1, affects neurogenesis in the aged hippocampus is unknown. Here we analysed cells positive for 5-bromo-2-deoxyuridine (BrdU; 50 mg/kg) in animals in which cleavage of IL-1, was inhibited by the caspase-1 inhibitor Ac-YVAD-CMK (10 pmol). Aged (22 months) and young (4 months) rats received Ac-YVAD-CMK for 28 days intracerebroventricularly through a brain infusion cannula connected to an osmotic minipump. Starting on day 14, animals received a daily injection of BrdU for five consecutive days. Unbiased stereology analyses performed 10 days after the last injection of BrdU revealed that the total number of newborn cells generated over a 5-day period was higher in young rats than in aged rats. In addition, there was a 53% increase in the number of BrdU-labelled cells of the aged Ac-YVAD-CMK-treated rats compared to aged controls. Immunofluorescence studies were performed to identify the cellular phenotype of BrdU-labelled cells. The increase in BrdU-positive cells was not due to a change in the proportion of cells expressing neuronal or glial phenotypes in the subgranular zone. These findings demonstrate that the intracerebroventricular administration of Ac-YVAD-CMK reversed the decrease in hippocampal neurogenesis associated with ageing. [source] Conversion of vitamin D3 to 1,,25-dihydroxyvitamin D3 in human skin equivalentsEXPERIMENTAL DERMATOLOGY, Issue 2 2000B. Lehmann Abstract: These results demonstrate for the first time that human keratinocytes under in vivo -like conditions have the capacity of the enzymatic hydroxylation of vitamin D3 to hormonally active calcitriol (1,,25(OH)2D3). Supplementation of the culture medium with bovine serum albumin (BSA) up to 1.5% (w/v) amplifies the conversion of vitamin D3 to 1,,25(OH)2D3. The maximum turnover rate of this reaction at 780 nM vitamin D3 in presence of 1.0% (w/v) BSA amounts to approximately 3 pmol 1,,25(OH)2D3 per 106 cells after 6 h of incubation. The hydroxylation of vitamin D3 to 1,,25(OH)2D3 is inhibited by the P-450 oxidase inhibitor ketoconazole. The generation of 1,,25(OH)2D3 from vitamin D3 has an apparent Michaelis constant (Km) of 2.3×10,6 M. The intrinsic conversion of vitamin D3 to biologically active 1,,25(OH)2D3 may be of importance for the regulation of proliferation and differentiation of keratinocytes. [source] Morphine-like substance in leech gangliaFEBS JOURNAL, Issue 8 2000Evidence, immune modulation Binding experiments followed by measurement of nitric oxide release revealed an opiate alkaloid high affinity receptor with no affinity to opioids, representing a new µ-subtype receptor in the brain of the leech Theromyzon tessulatum. In addition, evidence of morphine-like substances was found in immunocytochemical studies and HPLC coupled to electrochemical detection (500 mV and 0.02 Hz). Based on previous evidence of the involvement of morphine as an immune response inhibitor, we demonstrate that in leech ganglia injection of lipopolysaccharide (LPS; a potent immunostimulatory agent derived from bacteria) provoked an increase in the level of ganglionic morphine-like substances after a prolonged latency period of 24 h (from 2.4 ± 1.1 pmol per ganglion to 78 ± 12.3 pmol per ganglion; P < 0.005; LPS injected 1 µg·mL,1); this effect is both concentration- and time-dependent. Finally, we have demonstrated that morphine, after binding to its own receptor, inhibits leech immunocyte activation through adenylate cyclase inhibition and nitric oxide release. This report confirms that morphine is an evolutionarily stable potent immunomodulator. [source] The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MSJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2009James Glick Abstract Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, ( ± )-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [( ± )- anti -benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5,-ACCCG5CG7TCCG11CG13C-3,/5,-GCGCGGGCGCGGGT-3,) derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational ,hot spots' and have also been reported as chemical ,hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5, G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5, G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G7) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run. Copyright © 2009 John Wiley & Sons, Ltd. [source] Evaluation of a new matrix-free laser desorption/ionization method through statistic studies: comparison of the DIAMS (desorption/ionization on self-assembled monolayer surface) method with the MALDI and TGFA-LDI techniquesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2008Matthieu Bounichou Abstract This work demonstrates that the desorption/ionization on self-assembled monolayer surface (DIAMS) mass spectrometry, a recent matrix-free laser desorption/ionization (LDI) method based on an organic target plate, is as statistically repeatable and reproducible as matrix assisted laser desorption ionization (MALDI) and thin gold film-assisted laser desorption/ionization (TGFA-LDI) mass spectrometries. On lipophilic DIAMS of target plates with a mixture of glycerides, repeatability/reproducibility has been estimated at 15 and 30% and the relative detection limit has been evaluated at 0.3 and 3 pmol, with and without NaI respectively. Salicylic acid and its d6 -isomer analysis confirm the applicability of the DIAMS method in the detection of compounds of low molecular weight. Copyright © 2008 John Wiley & Sons, Ltd. [source] Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2007Je Won Park Abstract A method employing silicone oil density centrifugation, solid-phase extraction (SPE) cleanup, and LC-ESI-MS/MS analysis was developed for the rapid, selective, sensitive, and quantitative detection of an intracellular pool of short organic acid-CoA esters in actinomycetes. The detection limit was determined to be approximately 0.8 pmol (1.2 ng/ml) for each standard CoA-ester analyzed by the present LC-ESI-MS/MS method. A selected ion chromatogram for a typical fragment ion (m/z 428) specific to CoA-esters enabled the detection of eight intracellular CoA-esters involved in both primary and secondary metabolisms. The application of this method to bacterial metabolomic study is demonstrated by the profiling of the intracellular CoA-ester pools in the wild-type Streptomyces venezuelae strain producing polyketide antibiotics (methymycin and pikromycin), a polyketide synthase (PKS)-deleted S. venezuelae mutant, and a S. venezuelae mutant expressing the heterologous PKS genes. By quantifying the individual CoA-esterlevel in three different genotypes of the S. venezuela e strain, further insight could be gained into the role of CoA-estersin polyketide biosynthesis. This analytical approach can be extended to the quantification of the size and composition of in vivo CoA-ester pools in various microbes, and can provide a detailed understanding of the relationship between the in vivo CoA-ester pool and the production of pharmaceutically important polyketides. Copyright © 2007 John Wiley & Sons, Ltd. [source] Quantification of urinary N -acetyl- S - (propionamide)cysteine using an on-line clean-up system coupled with liquid chromatography/tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2005Chien-Ming Li Abstract Acrylamide has been reported to be present in high-temperature processed foods and normal processed food intake could lead to significant acrylamide exposure. Acrylamide in vivo can be conjugated with glutathione in the presence of glutathione transferase. This conjugation product is further metabolized and excreted as N -acetyl- S -(propionamide)cysteine (NASPC) in the urine. NASPC could be considered a biomarker for acrylamide exposure. The objective of this study was to develop a highly specific, rapid and sensitive method to quantify urinary NASPC, serving as a biomarker for acrylamide exposure assessment. Isotope-labeled [13C3]NASPC was successfully synthesized and used as an internal standard. This urine mixture was directly analyzed using a newly developed liquid chromatographic/tandem mass spectrometric method coupled with an on-line clean-up system. The detection limit for this method was estimated as <5 µg l,1(0.4 pmol) on-column. The method was applied to measure the urinary level of NASPC in 70 apparently health subjects. The results showed that the NASPC urinary level was highly associated with smoking. Smokers had a significantly higher urinary NASPC level (135 ± 88 µg g,1 creatinine) than non-smokers (76 ± 30 µg g,1 creatinine). A highly sensitive and selective LC/MS/MS isotope dilution method was successfully established. With an on-line clean-up system, this system is capable of routine high-throughput analysis and accurate quantitation of NASPC in urine. This could be a useful tool for health surveillance for acrylamide exposure in a population for future study. Copyright © 2005 John Wiley & Sons, Ltd. [source] Continuous flow isotope ratio mass spectrometry for the measurement of nanomole amounts of 13CO2 by a reverse isotope dilution methodJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2002J. Guitton Abstract A simple method for the determination of nanomole amounts of 13CO2 generated from an in vitro reaction is reported. The incubation medium contains a known amount of unlabeled sodium bicarbonate and the gaseous 13CO2 enriches the atmosphere upon which a measurement of the isotopic enrichment (13CO2/12CO2) is made corresponding to a reverse isotope dilution. The quantification of the 13CO2 was performed by gas chromatography/isotope ratio mass spectrometry. This assay was validated in terms of linearity, accuracy and precision using three different substrates which produce 13CO2 either by enzymatic reaction [13C]urea, sodium [13C]formate) or by chemical reaction (sodium [13C]bicarbonate). Four calibration curves were tested for each 13C-labeled substrate, allowing the quantification of 13CO2 from 25 pmol to 150 nmol. The dynamics of the assay were obtained as a function of the quantity of unlabeled sodium bicarbonate added to each sample. Copyright © 2001 John Wiley & Sons, Ltd. [source] Hypothalamic-Pituitary-Adrenal Responses to Centrally Administered Orexin-A are Suppressed in Pregnant RatsJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2003P. J. Brunton Abstract Orexins are hypothalamic neuropeptides that stimulate arousal and food intake but also activate the hypothalamic-pituitary-adrenal (HPA) axis. During late pregnancy in the rat, the responsiveness of the HPA axis to stressors is attenuated, and thus we investigated HPA axis responses to centrally administered orexin-A during pregnancy. Intracerebroventricular injection of orexin-A (0.5 µg, 140 pmol) significantly increased plasma adrenocorticotropic hormone and corticosterone concentration within 10 min in virgin female Sprague-Dawley rats, but had no effect in day 21 pregnant rats. Orexin-A significantly increased corticotropin-releasing hormone (CRH) mRNA expression, measured by in situ hybridization, in the paraventricular nucleus (PVN) of the virgin group but not in the pregnant group. Thus, the responsiveness of PVN CRH neurones to orexin-A, and hence the pituitary-adrenal axis, is markedly reduced in pregnancy. This may favour anabolic adaptations in pregnancy. [source] Lipid peroxidation: a possible role in the induction and progression of chronic periodontitisJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2005C. C. Tsai Objectives:, Reactive oxygen species (ROS) are implicated in the destruction of the periodontium during inflammatory periodontal diseases. The imbalance in oxidant/antioxidant activity may be a key factor in the damaging effects of ROS. This study aimed to determine the lipid peroxidation levels in gingival crevicular fluid and saliva, and glutathione (GSH) and glutathione peroxidase (GPx) in saliva in patients with chronic periodontitis. Methods:, Gingival crevicular fluid and saliva were collected from 13 patients and 9 healthy control subjects during the preliminary study, and from 21 patients during the subsequent study. Lipid peroxidation level, GSH level and GPx activity were determined by spectrophotometric assay. Results:, The preliminary study found that when comparing patients to healthy controls, the gingival crevicular fluid samples produced the following results, respectively: higher lipid peroxidation concentration (µm) (by sites: 167.55 vs. 53.71, p < 0.0001; by subjects: 151.99 vs. 50.66, p < 0.005) and total amount (pmol) (by sites: 93.02 vs. 8.47, p < 0.0001, by subjects: 80.44 vs. 7.84, p < 0.0005). In saliva samples, lower GSH concentration (µm) (373.04 vs. 606.67, p < 0.05), higher lipid peroxidation concentration (µm) (0.66 vs. 0.13, p < 0.0005), and no difference in GPx activity were found in patients than in those of healthy controls. The subsequent study showed statistically significant (p < 0.05) improvement of clinical periodontal parameters (plaque index, gingival index, probing attachment level, probing pocket depth and gingival crevicular fluid volume), decreases in gingival crevicular fluid lipid peroxidation levels (concentration and total amount) at the sites after the completion of phase 1 periodontal treatment. Similarly, the periodontal treatment resulted in a significant decrease of lipid peroxidation concentrations (p < 0.05), increase in GSH concentration (p < 0.001), and no change in GPx activity in saliva samples. Conclusion:, The increased levels of lipid peroxidation may play a role in the inflammation and destruction of the periodontium in periodontitis. [source] Clinical measurement of thrombin generation by calibrated automated thrombography requires contact factor inhibitionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2004R. Luddington Summary.,Background: Measurement of thrombin generation by calibrated automated thrombography (CAT) could fulfill the requirements of a global test of coagulability and is potentially applicable to routine clinical laboratory practice. The purpose of this study was to determine if corn trypsin inhibitor (CTI) could be used to abolish contact factor activation in this assay, thus allowing accurate measurement of low tissue factor (TF) concentration-triggered thrombin generation on samples taken in a routine clinical setting. Methods: The endogenous thrombin potential (ETP) was measured by CAT. Results: The study demonstrated that addition of CTI after plasma separation is not sufficient and blood must be drawn into tubes containing CTI if in-vitro contact factor-activated thrombin generation is to be abolished. Contact factor-activated thrombin generation is completely inhibited at a CTI concentration of 18.3 µg mL,1 whole blood. Increasing the CTI concentration above this level does not lead to suppression of the TF-triggered ETP. At a TF concentration of 2 pmol, ETPs were significantly lower in the presence of CTI (P < 0.001). The difference (no CTI minus CTI) between results ranged from ,,1 to 2159 nM min,1 (median ,,754). Whilst the low concentration TF-ETP assay was not optimized to distinguish degrees of coagulability between patient samples, there was a significant difference in ETP between normal and hemophilia samples and samples from patients with a clinical prothrombotic tendency. Conclusions: CTI can be applied to ETP measurement by CAT. This permits the use of CAT in a low TF-triggered thrombin generation assay without concern for the effect of interference from in-vitro contact factor activation and the optimum reagent conditions for using CAT as a global test of coagulability in clinical practice can now be defined. [source] Determination of serum glucose by horseradish peroxidase-catalysed imidazole chemiluminescence coupled to a micro-flow-injection systemLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 5 2007Osamu Nozaki Abstract The reactivity of flow-injection (FI)-horseradish peroxidase (HRP)-catalysed imidazole chemiluminescence (CL) was studied for continuous determination of hydrogen peroxide (H2O2) and serum glucose with immobilized glucose oxidase. Light emission by the HRP-catalysed imidazole CL was obtained when immobilized HRP, alkaline imidazole (in Tricine solution, pH 9.3) and H2O2 were reacted at room temperature. The optimal pH for the CL reaction was 9.3 and the optimal concentration of imidazole was 100 µmol/L. When no imidazole was added, the light intensity of the same H2O2 specimen decreased to a level that could not be quantitatively determined. The spectrum of the light emitted by imidazole CL was in the range 400,600 nm with a peak at 500 nm. The calibration equation for determination of H2O2 was y = 9860x2 + 3830x + 11 700, where y = light intensity (RLU) and x = concentration of H2O2 (µmol/L). The detection limit of H2O2 was 5 pmol, and the reproducibility of the H2O2 assay was 2.3% of the coefficient of variation (H2O2 48 µmol/L, n = 13). The CL method was successfully applied to assay glucose after on-line generation of H2O2 with the immobilized glucose oxidase column, resulting in good reproducibility (CV = 3.3% and 1.0% for the standard glucose and the control serum, respectively). Copyright © 2007 John Wiley & Sons, Ltd. [source] Reactivation of horseradish peroxidase with imidazole for continuous determination of hydrogen peroxide using a micro,ow injection,chemiluminescence detection systemLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 4 2003Osamu Nozaki Abstract A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H2O2). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H2O2) possible by micro,ow injection based on horseradish-catalysed luminol chemiluminescence. For reproducible determination of H2O2 with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H2O2 must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro,ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within-run reproducibility, CV = 4.0%) and the detection limit for H2O2 was 5 pmol. Copyright © 2003 John Wiley & Sons, Ltd. [source] Electrogenerated chemiluminescence of luminol for oxidase-based fibre-optic biosensorsLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 2 2001Christophe A. Marquette Abstract The luminol electrochemiluminescence has been exploited for the development of several fibre-optic biosensors allowing the detection of hydrogen peroxide and of substrates of H2O2 -producing oxidases. Electro-optical flow injection analysis of glucose, lactate, cholesterol and choline are thus described. To perform the experiments, a glassy carbon electrode was polarized at a fixed potential. Luminol was then electrochemically oxidized and could react in the presence of hydrogen peroxide to produce light. Several parameters had to be optimized to obtain reliable optical biosensors. An optimum applied potential of +425 mV between the glassy carbon electrode and the platinum pseudo-reference electrode was determined, allowing the best signal: noise ratio to be obtained. It was also necessary to optimize the experimental conditions for the immobilization of the different oxidases involved (preactivated membranes, chemically activated collagen membranes, photopolymerized matrix). For each biosensor developed, the optimum reaction conditions have been studied: buffer composition, pH, temperature, flow rate and luminol concentration. Under optimal conditions, the detection limits (S/N,=,3) were 30,pmol, 60,pmol, 0.6,nmol and 10,pmol for lactate, glucose, cholesterol and choline, respectively. The miniaturization of electrochemiluminescence-based biosensors has been realized using screen-printed electrodes instead of a glassy carbon macroelectrode, with choline oxidase as a model H2O2 -generating oxidase. Copyright © 2001 John Wiley & Sons, Ltd. [source] Interaction between azathioprine and aminosalicylates: an in vivo study in patients with Crohn's diseaseALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2002O. Dewit Background: The inhibition of thiopurine methyltransferase activity, one of the enzymes responsible for azathioprine metabolism, by aminosalicylates has been described in an in vitro study. This could result in a higher risk of bone marrow depression when using the two drugs together. Aim: To investigate the in vivo interaction between azathioprine and aminosalicylates in quiescent Crohn's disease by measuring 6-thioguanine nucleotide levels, thiopurine methyltransferase activity and the plasma levels of the acetylated metabolite of 5-aminosalicylic acid. Methods: Sixteen patients taking a stable dose of azathioprine, plus sulfasalazine or mesalazine, were enrolled and completed the study. They were not taking any drugs interfering with azathioprine metabolism. Four visits every 4 weeks were held over a 3-month period. Aminosalicylate administration was withdrawn after the second visit. At each visit, the blood cell count, inflammatory parameters, levels of 6-thioguanine nucleotide and the acetylated metabolite of 5-aminosalicylic acid and thiopurine methyltransferase activity were determined. Results: After aminosalicylate withdrawal, mean 6-thioguanine nucleotide levels decreased significantly from 148 pmol (57,357 pmol) to 132 pmol (56,247 pmol) per 8 × 108 red blood cells (P=0.027), without significant changes in thiopurine methyltransferase activity or biological parameters. Conclusions: This in vivo study favours the existence of an interaction between azathioprine and aminosalicylates through a mechanism which remains unclear. This drug,drug interaction should be taken into account when using azathioprine and aminosalicylates simultaneously. [source] Inhibitory effect of oxytocin on accelerated colonic motility induced by water-avoidance stress in ratsNEUROGASTROENTEROLOGY & MOTILITY, Issue 8 2009M. Matsunaga Abstract, Recent studies have indicated that brain and gut activities are interrelated and exposure to several stressors, such as water-avoidance stress, stimulates the motor function of the gut through corticotropin-releasing factor (CRF)-signalling pathways in the brain. Central oxytocin is known to attenuate stress responses, including CRF expression in the brain. Here, we examined whether central oxytocin attenuated the acceleration of colonic motility induced by water-avoidance stress. A force transducer was attached to the distal colon of male rat, and the colonic motility and faecal pellet output were recorded while the rats were exposed to water-avoidance stress. Intracerebroventricular (i.c.v.) injections of oxytocin (5, 50 and 500 pmol) and the oxytocin receptor antagonist tocinoic acid (25 ,g) were administered before exposure to water-avoidance stress, and the effect of oxytocin on colonic motor function was determined. Centrally administered oxytocin inhibited the accelerated colonic motility induced by water-avoidance stress. The effective dose ranged between 5 and 50 pmol on i.c.v. injection. Oxytocin also decreased the number of CRF-positive cells in the paraventricular nucleus and corticosterone release. The inhibitory effect of oxytocin on accelerated colonic motility was blocked by pretreatment with oxytocin receptor antagonist. Furthermore, centrally administered tocinoic acid enhanced the acceleration of colonic motility. These results suggested that endogenous central oxytocin may contribute to the regulation of colonic function and inhibit the brain CRF-signalling pathways targeting the gut, resulting in the inhibition of stress-induced colonic contractions. [source] Utility of azathioprine metabolite measurements in post-transplant recurrent autoimmune and immune-mediated hepatitisPEDIATRIC TRANSPLANTATION, Issue 6 2004Carolina Rumbo Abstract:, Patients with post-transplant immune-mediated hepatitis (IMH) and recurrent autoimmune hepatitis (RAIH) have a poor outcome and a higher need for retransplantation. Azathioprine (AZA) is used as adjunctive immunosuppression after transplantation; optimizing its dose may be a key point in preserving graft function. Complications of high AZA dosing make dose escalation potentially problematic. Our aim was to correlate AZA metabolite levels with therapeutic effects, toxicity, and adherence to medication in children with IMH and RAIH. Charts of 14 patients were retrospectively reviewed. The post-transplant diagnosis was based on liver biopsy and autoimmune markers. AZA was prescribed after establishing the post-transplant diagnosis. AZA was started at 1.1 (1.0,1.8) mg/kg/day. Routine biochemical studies, tacrolimus levels, 6-thioguanine (6-TG) and 6-methylmercaptopurine levels were assessed every 8 wk. AZA dose was routinely adjusted to achieve 6-TG levels between 235 and 450 pmol per 8 × 108 RBC. A total of 92 samples from 14 patients were reviewed. Four patients were excluded because of non-adherence. AZA dose was increased by 245% resulting in eight of 10 patients in the target range; no hepatic or bone marrow toxicity was observed. ALT levels and steroid requirements were significantly reduced (p < 0.05). The AZA dose required to achieve target 6-TG levels was significantly greater in children <10 yr. AZA metabolite testing in children post-liver transplant is useful in assessing adherence to medication and it is potentially helpful in optimizing medication dosing. In younger children the AZA dose requirements were two to four times higher than previously reported standard doses. [source] Adipokinetic hormone (Pyrap-AKH) enhances the effect of a pyrethroid insecticide against the firebug Pyrrhocoris apterusPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 4 2010Dalibor Kodrík Abstract BACKGROUND: Adipokinetic hormones (AKHs) are insect neuropetides controlling stress situations including those elicited by insecticide treatment. The effect of Pyrap-AKH on the mortality of the firebug Pyrrhocoris apterus (L.) treated with the insecticide permethrin (Ambush 25 EC) was studied. RESULTS: Coinjection of 50 ng permethrin with 80 pmol Pyrap-AKH induced a significant 2.3-fold increase in bug mortality compared with the insecticide alone. The results were confirmed by topical coapplication of both agents (400 ng and 80 pmol respectively). Injections of 50 and 100 ng permethrin elicited a significant increase in the AKH level in CNS and the haemolymph. The results indicate an involvement of AKH in stress response to permethrin. The enhanced effect of insecticide by AKH treatments probably results from the stimulatory role in bug metabolism: carbon dioxide production was increased 3.5- and 2.5-fold respectively 1 and 3 h after permethrin treatment, and 4.3- and 3.4-fold after the permethrin plus AKH cotreatment, compared with the control. CONCLUSION: The elevation of metabolism could intensify the permethrin action by its faster penetration into tissues and by stimulation of biochemically active cells, and could be a reason for enhanced action of permethrin after its cotreatment with Pyrap-AKH. Copyright © 2009 Society of Chemical Industry [source] Genetic and hormonal control of melanization in reddish,brown and albino mutants in the desert locust Schistocerca gregariaPHYSIOLOGICAL ENTOMOLOGY, Issue 1 2010KOUTARO MAENO The genetic and hormonal control of body colouration is investigated using two recessive genetic mutant strains, the reddish,brown (RB) mutant and an albino mutant, as well as a normal (pigmented) strain of the desert locust Schistocerca gregaria. The colour patterns of the RB nymphs are similar to those of a normal strain, although the intensity of the melanization is weaker in the former. Reciprocal crosses between the RB and albino mutants produce only normal phenotypes in the F1 generation. In the F2 generation, the normal, RB and albino phenotypes appear in a ratio of 9 : 3 : 4, indicating that two Mendelian units might determine the appearance of dark body colour and the intensity of melanization, respectively. In other words, at least two steps of regulation might be involved in the expression of body colour. Injections of [His7]-corazonin, a neuropeptide inducing dark colour in this locust, fail to induce dark colour in albino nymphs but show a dose-dependent darkening in RB nymphs in the range, 10 pmol to 1 nmol. Some RB nymphs become indistinguishable from normal individuals after injection of the peptide. Implantation of corpora cardiaca (CC) taken from RB mutants into other RB individuals induces darkening in the latter and CC from RB, albino and normal strains have similar dark colour-inducing activity when implanted into albino Locusta migratoria. These results suggest the possibility that the RB mutant gene regulates the intensity of melanization, possibly through controlling the pathway of pigment biosynthesis associated with [His7]-corazonin. [source] Topical application of Pya -AKH stimulates lipid mobilization and locomotion in the flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)PHYSIOLOGICAL ENTOMOLOGY, Issue 1 2002Dalibor Kodrík Abstract Two different methods of applying Pya -AKH to long-winged (macropterous) females of the firebug, Pyrrhocoris apterus (Linnaeus) (Heteroptera) were compared: both injection and topical application increased the levels of lipids in the haemolymph and stimulated locomotor activity. Lipid mobilization was maximal when 10 pmol was applied by injection or 40,100 pmol by topical application, with the first significant responses occurring 1.5 h after injection and 2 h after topical application. The highest elevations of lipid concentration in the haemolymph were comparable between the treatments, i.e. 14.36 ± 3.59 mg/mL for injection and 14.43 ± 4.07 mg/mL for topical application. However, these maximal elevations were achieved at different times: 3 h after the injection and 7 h after the topical application. Injection of 10 and 40 pmol of Pya -AKH stimulated locomotor activity with maximal activity 3 h later but, surprisingly, injection of 80 pmol showed no effect initially and than a slight inhibitory effect after 6,8 h. Increased locomotor activity was found after topical application of Pya -AKH, but the response was lower than after injection and appeared later, 5,9 h after the hormone application. In addition, the greatest increase in walking activity required topical application of 300 pmol and was still less dramatic than the response to injection. The stimulatory effect of Pya -AKH on locomotion was positively correlated with its effect on lipid mobilization only for injection of the hormone. It is argued that a stress caused by injection could play a role in the appearance of the complex response to adipokinetic hormone. [source] The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography,electrospray tandem mass spectrometryPHYTOCHEMICAL ANALYSIS, Issue 6 2003Konstantinos Petritis Abstract Varying concentrations of cyclopropane-1,1-dicarboxylic acid (CDA), an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, added to the solid culture medium of tomato nodal shoot segments resulted in a reduction in the level of endogenous ethylene according to the concentration of inhibitor applied. Following treatment with inhibitor, plants were homogenised and the concentrations of CDA and of 1-aminocyclopropane-1-carboxylic acid (ACC) were measured simultaneously in the resulting juice using an HPLC-ESI/MS-MS method. The levels of CDA and ACC measured in the plant tissues were associated with the concentration of inhibitor added to the solid medium. The HPLC-ESI/MS-MS method described produced limits of detection of 0.8 pmol for ACC and of 4 pmol for CDA. Copyright © 2003 John Wiley & Sons, Ltd. [source] Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2009William R. Alley Jr. Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip-based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C18 LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C18 chips. On the other hand, hydrophobic glycopeptides were better retained on C18 chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1,pmol). The performance of both media also appeared comparable when analyzing a four-protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||