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Biomedical subjects

Q Xu

Publications and source records attributed to Q Xu.

At least 127 records · Page 7Linked to original sources
PubMed2000

Purification of the novel endonuclease, Hpy188I, and cloning of its restriction-modification genes reveal evidence of its horizontal transfer to the Helicobacter pylori genome.

We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one-base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R-M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome.

Amino Acid Sequence↗
PubMed2000

Expression of isolated C-type carbohydrate recognition domains.

A galactose-binding lectin from the venom of the snake Trimeresurus stejnegeri consists of isolated carbohydrate recognition domains, belonging to group VII of the C-type animal lectins. As a first step toward determining the tertiary structure of the galactose-specific lectin, we produced the lectin in Escherichia coli. By in vitro refolding and affinity chromatography, modest amounts (8 mg/liter) of active recombinant proteins were obtained. The recombinant protein was homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Its amino acid sequence without the initiated methionine at the N-terminus was also characterized by mass spectrometry. The data of hemagglutination and enzyme-linked lectin binding assays demonstrated that the recombinant lectin showed similar sugar-binding activity as the native protein. In addition, fluorescence spectroscopy and circular dichroism also showed obviously their structural similarity.

Animals↗
PubMed2000

NO(2)(+) nitration mechanism of aromatic compounds: electrophilic vs charge-transfer process

The nitration of methylnaphthalenes with NO(2)BF(4) and NOBF(4) was examined in order to shed light on the controversial aromatic nitration mechanism, electrophilic vs charge-transfer process. The NO(2)(+) nitration of 1,8-dimethylnaphthalene showed a drastic regioselectivity change depending on the reaction temperature, where ortho-regioselectivity at -78 degrees C and para-regioselectivity at 0 degrees C were considered to reflect the electrophilic and the direct or alternative charge-transfer process, respectively, because the NO(+) nitration through the same reaction intermediates as in the NO(2)(+) nitration via a charge-transfer process resulted in para-regioselectivity regardless of the reaction temperature. The NO(2)(+) nitration of redox potential methylnaphthalenes higher than 1,8-dimethylnaphthalene gave a similar ortho-regioselectivity enhancement to 1,8-dimethylnaphthalene at lower temperature, thus reflecting the electrophilic process. On the other hand, the NO(2)(+) nitration of redox potential methylnaphthalenes lower than 1,8-dimethylnaphthalene showed para-regioselectivity similar to the NO(+) nitration, indicating the direct or alternative charge-transfer process. In the presence of strong acids where the direct charge-transfer process will be suppressed by protonation, the ortho-regioselectivity enhancement was observed in the NO(2)(+) nitration of 1,8-dimethylnaphthalene, suggesting that the direct charge-transfer process could be the main process to show para-regioselectivity. These experimental results imply that the NO(2)(+) nitration proceeds via not only electrophilic but also direct charge-transfer processes, which has been considered to be unlikely because of the high energy demanding process of a bond coordination change between NO(2)(+) and NO(2). Theoretical studies at the MP2/6-31G(d) level predicted ortho- and para-regioselectivity for the NO(2)(+) nitration via electrophilic and charge-transfer processes, respectively, and the preference of the direct charge-transfer process over the alternative one, which support the experimental conclusion

Journal Article↗
PubMed2000

Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori.

The genetic organization and sequence heterogeneity of the iceA locus of Helicobacter pylori was studied, and the existence of two distinct gene families, iceA1 and iceA2, at this locus was confirmed. iceA1 has significant sequence homology to nlaIIIR, encoding an endonuclease in Neisseria lactamica, but the similarity at the protein level is limited, due to frameshift mutations of iceA1 in most H. pylori strains. In only five of the 19 iceA1 strains studied, a full-length open reading frame (ORF), capable of encoding a 228aa protein, with 52% homology to NlaIII was observed. The region upstream of iceA2 is highly variable in length, containing up to 15 copies of 8bp tandem repeats. iceA2 can encode proteins of 24, 59, 94, or 129 amino acids, consisting of 14 and 10aa domains, conserved in all iceA2 strains, flanking 0, 1, 2, or 3 copies of a 35aa cassette. This 35aa cassette consists of domains of 13, 16 and 6aa, respectively. The 13aa and 6aa domains are highly conserved, but the 16aa domain exists in two variants. In total, five distinct iceA2 subtypes were defined. Database searches did not reveal any homologous sequences. Recombinant IceA1 and IceA2 proteins were expressed in Escherichia coli, confirming the predicted ORFs. Genotype-specific PCR primers permitted iceA genotyping in 318 (99. 1%) of a worldwide collection of 321 H. pylori strains. The conserved sizes of the amplification products confirmed the worldwide distribution of discrete variants of iceA1 and iceA2.

Amino Acid Sequence↗
PubMed2000

BAR: An apoptosis regulator at the intersection of caspases and Bcl-2 family proteins.

Two major pathways for induction of apoptosis have been identified-intrinsic and extrinsic. The extrinsic pathway is represented by tumor necrosis factor family receptors, which utilize protein interaction modules known as death domains and death effector domains (DEDs) to assemble receptor signaling complexes that recruit and activate certain caspase-family cell death proteases, namely procaspases-8 and -10. The intrinsic pathway for apoptosis involves the participation of mitochondria, which release caspase-activating proteins. Bcl-2 family proteins govern this mitochondria-dependent apoptosis pathway, with proteins such as Bax functioning as inducers and proteins such as Bcl-2 and Bcl-X(L) serving as suppressors of cell death. An apoptosis regulator, BAR, was identified by using a yeast-based screen for inhibitors of Bax-induced cell death. The BAR protein contains a SAM domain, which is required for its interactions with Bcl-2 and Bcl-X(L) and for suppression of Bax-induced cell death in both mammalian cells and yeast. In addition, BAR contains a DED-like domain responsible for its interaction with DED-containing procaspases and suppression of Fas-induced apoptosis. Furthermore, BAR can bridge procaspase-8 and Bcl-2 into a protein complex. The BAR protein is anchored in intracellular membranes where Bcl-2 resides. BAR therefore may represent a scaffold protein capable of bridging two major apoptosis pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Reduced neointima hyperplasia of vein bypass grafts in intercellular adhesion molecule-1-deficient mice.

Recently, we established a new mouse model of vein graft arteriosclerosis through the grafting of vena cava to carotid arteries. In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft disease. With this model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of vein graft arteriosclerosis in ICAM-1-deficient mice. Neointimal hyperplasia of vein grafts in ICAM-1 -/- mice was reduced 30% to 50% compared with that of wild-type control animals. Immmunofluorescent analysis revealed that increased ICAM-1 expression was observed on the endothelium and smooth muscle cells (SMCs) of the grafted veins in wild-type, but not ICAM-1 -/-, mice. MAC-1 (CD11b/18)-positive cells that adhered to the surface of vein grafts in ICAM-1 -/- mice were significantly less as identified with en face immunofluorescence, and these positive cells were more abundant in the intimal lesions of vein grafts in wild-type mice. Furthermore, aortic SMCs cultivated from wild-type mice exhibited high ICAM-1 expression in response to tumor necrosis factor-alpha. When tumor necrosis factor-alpha-stimulated SMCs were incubated with mouse spleen leukocytes, the number of cells that adhered to ICAM-1 -/- SMCs was significantly lower than the number that adhered to ICAM-1 +/+ SMCs, which was markedly blocked through pretreatment of leukocytes with the anti-MAC-1 antibody. Taken together, our findings demonstrate that ICAM-1 is critical in the development of venous bypass graft arteriosclerosis, which provides essential information for therapeutic intervention for vein graft disease in patients undergoing bypass surgery.

Animals↗
PubMed2000

Studies on the fluorescence reaction between nucleic acid and the complex of cobalt(II) with 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline and its applications.

Based on the enhancement of the fluorescence intensity of the complex of 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline (FCPBSQ) with Co(2+) by nucleic acid in the presence of Tween 80 and in the weakly basic medium, a fluorescence method for the determination of nucleic acid was proposed. The calibration graphs for the determination of denatured calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA (yt RNA) were obtained in concentration ranges 0.050-4.0, 0.10-3.0, and 0.050-3.0 microg/ml with limits of detection of 0.010, 0.030, and 0.020 microg/ml, respectively. The method has been satisfactorily used for the determination of DNA in wheat cell extraction and ct DNA, fs DNA, and yt RNA in synthetic samples. Investigations on the binding mode by the Scatchard plots method suggested that both intercalation and electrostatic binding modes existed in this system.

Animals↗
PubMed2000

[{Pt]

Prolonged evacuation of [{Pt(CO)(3)}(2)](2+) (1), the first homoleptic, dinuclear, cationic platinum(I) carbonyl complex, results in reversible disproportionation. Complex 1 was formed by dissolution of PtO(2) in concentrated H(2)SO(4) under an atmosphere of CO [Eq. (a)], and completely characterized by NMR ((13)C, (195)Pt), IR, and Raman spectroscopy.

Journal Article↗
PubMed2000

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection.

Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 microm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal-to-noise ratio. The limit of detection is 1 x 10(-7) mol/L or 26 amol and the linear dynamic range is 2 x 10(-7) to 2 X 10(-5) mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser-induced fluorescence detection.

Adsorption↗
PubMed2000

Quantitative assay of metronidazole by capillary zone electrophoresis with amperometric detection at a gold microelectrode.

Capillary zone electrophoresis was employed for the determination of metronidazole using end-column amperometric detection with a gold microelectrode at a constant potential of -0.52V vs. saturated calomel electrode. To overcome interference of oxygen in the solution, a deaeration injector and a deaeration protector at the detection cell were used. The optimum conditions of separation and detection are 1.0 x 10(-3) mol/L potassium dihydrogen citrate (KH2C6H5O7) for the buffer solution, 20 kV for the separation voltage, and 5 kV and 10 S for injection voltage and injection time, respectively. The limit of detection is 6.0 x 10(7) mol/L or 0.78 fmole (S/N = 3). The relative standard deviation is 3.9% for the electrophoretic peak current. The method was applied to the determination of metronidazole in human urine.

Electrophoresis, Capillary↗
PubMed2000

Determination of clozapine by capillary zone electrophoresis following end-column amperometric detection with simplified capillary/electrode alignment.

Capillary zone electrophoresis was employed for the determination of clozapine using an end-column amperometric detection at a carbon fiber array microdisk electrode with simplified capillary/electrode alignment. The optimum conditions of separation and detection are: Britton-Robinson buffer, pH 2.0 (1.3 x 10(-2) mol/L total concentration of acids, 3.2 x 10(-3) mol/L NaOH), 15 kV for separation voltage, 5 kV and 10 s for injection voltage and injection time, respectively. The limit of detection is 4.2 x 10(-7) mol/L or 1.2 fmole (signal to noise, S/N = 2). The relative standard deviation is 1.4% for the migration time and 2.5% for the electrophoretic peak current. The method was applied to the determination of clozapine in human blood. The recovery of the method is between 94-104%.

Clozapine↗
PubMed2000

A new capillary electrophoresis end-column amperometric detection system without the need for capillary/electrode alignment.

A self-aligning end-column amperometric detection system for capillary electrophoresis was constructed. In this system, the electrode and capillary were exchanged easily and the capillary/electrode alignment procedure is not required. Gold, gold/mercury amalgam, copper and carbon fiber could be used as the working electrode. The principle is in the use of two disk holders with the capillary and the electrode in the center, so that by inserting the disk holders into a groove in the working electrode port, the capillary and the electrode are automatically aligned and the distance between the capillary and the electrode is assured at 0.24 mm. The relative standard deviation obtained using five different gold/mercury amalgam microdisk electrodes for determination of cysteine was 1.5% for the migration time and 3.3% for the electrophoretic peak current. The simple and convenient system was attractive for the routine analysis by capillary electrophoresis with electrochemical detection. The system was applied to the determination of promethazine hydrochloride in human serum.

Electrodes↗
PubMed2000

Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis.

OBJECTIVE: To investigate whether stress- and mitogen-activated protein kinases (SAPK/MAPK), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, are significantly activated in rheumatoid arthritis (RA) synovial tissue compared with their activation in degenerative joint disease; to assess the localization of SAPK/MAPK activation in rheumatoid synovial tissue; and to search for the factors leading to stress kinase activation in human synovial cells. METHODS: Immunoblotting and immunohistology by antibodies specific for the activated forms of SAPK/MAPK were performed on synovial tissue samples from patients with RA and osteoarthritis (OA). In addition, untreated and cytokine-treated human synovial cells were assessed for SAPK/MAPK activation and downstream signaling by various techniques. RESULTS: ERK, JNK, and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not OA, patients. ERK activation was localized around synovial microvessels, JNK activation was localized around and within mononuclear cell infiltrates, and p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells. Tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6 were the major inducers of ERK, JNK, and p38 MAPK activation in cultured human synovial cells. CONCLUSION: Signaling through SAPK/MAPK pathways is a typical feature of chronic synovitis in RA, but not in degenerative joint disease. SAPK/MAPK signaling is found at distinct sites in the synovial tissue, is induced by proinflammatory cytokines, and could lead to the design of highly targeted therapies.

Arthritis, Rheumatoid↗
PubMed2000

Endotoxin exacerbates immunologically induced liver injury in cooperation with interferon-gamma.

OBJECTIVE AND DESIGN: To investigate the role of endotoxin in the development of immunologically induced liver injury. MATERIALS AND METHODS: A new model of liver injury induced in BALB/c mice by delayed-type hypersensitivity to picryl chloride and its in vitro assay for the interaction between liver nonparenchymal and parenchymal cells were used. RESULTS: Plasma endotoxin in the injured liver correlated well with serum alanine transaminase (ALT) activity (r = 0.601). Tolerance to lipopolysaccharide led to a significant inhibition of serum ALT elevation. However, lipopolysaccharide in vitro increased the ALT release from hepatocytes caused by nonparenchymal cells only in the presence of IFN-gamma. When nonparenchymal cells were separated into Kupffer and non-Kupffer cell populations, the synergistic hepatotoxicity of lipopolysaccharide and IFN-gamma was still observed in the former but not in the latter cell type. Lipopolysaccharide with IFN-gamma also enhanced TNF-alpha levels. Anti-TNF-alpha almost completely inhibited the ALT release. Combined use of TNF-alpha and IFN-gamma caused marked hepatocyte damage, while these cytokines alone did not. CONCLUSIONS: We suggest that elevated endotoxin levels accompanying the development of liver injury may activate Kupffer cells to release TNF-alpha leading to exacerbation of hepatocyte damage in cooperation with IFN-gamma produced during liver injury.

Alanine Transaminase↗
PubMed2000

Protective involvement of nitric oxide in the liver injury induced by delayed-type hypersensitivity to picryl chloride.

OBJECTIVE AND DESIGN: To investigate the role of nitric oxide (NO) in the liver injury induced by delayed-type hypersensitivity to picryl chloride (PCl-DTH). MATERIALS AND METHODS: Liver injury was induced in mice by PCl-DTH. NO production was examined using the Griess reagent. Isolated hepatocytes (HC) and nonparenchymal cells (NPC) were used. RESULTS: NO production in both serum and liver tissue reached a peak at 36 h after the elicitation of liver injury. The in vitro NO production was only observed by NPC or HC isolated at 24 h after the injury. Co-stimulation of IFN-gamma and TNF-alpha significantly triggered the HC and NPC isolated at 0 h to produce NO. As NO synthase inhibitors, Nomega-Nitro-L-Arginine exacerbated the liver injury in mice and NG-Monomethyl-L-Arginine enhanced the hepatotoxicity of IFN-gamma and TNF-alpha in vitro. In contrast, NO producer, S-nitroso-N-acetylpenicillamine dose-dependently inhibited the hepatotoxicity of NPC. CONCLUSIONS: NO may be produced by HC and NPC under the co-stimulation of IFN-gamma and TNF-alpha, and may play an important role for alleviating the liver injury.

Animals↗
PubMed2000

Amperometric detection studies of Nafion/indium hexacyanoferrate film for the determination of electroinactive cations in ion chromatography.

An amperometric detector based on the chemical modification of Nafion and indium (III) hexacyanoferrate (II, III) thin film (Nafion/In-CN-Fe) onto a glassy carbon (GC) electrode, was first successfully used for the determination of electroinactive cations (Li+, Na+, K+, Rb+, Cs+, NH4+) in single column ion chromatography (IC). A set of well-defined peaks of electroinactive cations was obtained. The detection limits of the cations are 8.9 x 10(-6) mol/L for Li+, 2.3 x 10(-6) mol/L for Na+, 5.2 x 10(-6) mol/L for K+, 4.8 x 10(-6) mol/L for Rb+, 4.0 x 10(-6) mol/L for Cs+ and 5.3 x 10(-6) mol/L for NH4+ at a single-to-noise ratio of 3. The proposed method was quick, sensitive and simple. The cations in rainwater and mineral water were successfully analyzed by this method.

Acid Rain↗
PubMed2000

Polydiphenylamine-dodecyl sulfate films for the simultaneous amperometric determination of electroinactive anions and cations in ion-exclusion cation-exchange chromatography.

An amperometric detector with two working electrodes both modified with polydiphenylamine-dodecyl sulfate (PDPA-DS) was successfully used for the simultaneous determination of electroinactive anions (SO42-, Cl-, NO3-) and cations (Na+, NH4+ and K+) in single-column ion-exclusion cation-exchange chromatography (IEC-CEC). The PDPA-DS chemical modified electrode (CME) was based on the incorporation of dodecyl sulfate (DS) into PDPA by electropolymerization of diphenylamine in the presence of sodium dodecyl sulfate. The electrochemical responses against the anions and cations at the PDPA-DS CME in differential pulse voltammetry were studied. A set of well-defined peaks of electroinactive anions and cations were obtained. The anions and cations were detected conveniently and reproducibly in a linear concentration range 0.01-5.0 mmol/L and their detection limits were in the range 5-9 micromol/L at a signal-to-noise ratio of 3 (S/N = 3). The proposed method was quick, sensitive and simple and was successfully applied to the analysis of lake water samples. The working electrode was stable over one week period of operation with no evidence of chemical and mechanical deterioration.

Journal Article↗