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Biomedical subjects

X Wu

Publications and source records attributed to X Wu.

At least 19 recordsLinked to original sources
PubMed1998

cAMP response element-binding protein monomers cooperatively assemble to form dimers on DNA.

We have analyzed the properties of cAMP response element-binding protein (CREB) in solution with emphasis on dimerization and effects of phosphorylation. Using a purified CREB fusion protein, a novel dye-label technique, and sedimentation equilibrium analysis, we directly and conclusively demonstrate that, unlike Jun and Fos, CREB dimerization is DNA-dependent. CREB exists primarily as a monomer in solution and cooperatively assembles on DNA to form dimers. Sedimentation equilibrium analysis also indicates that dimerization is unaffected by cAMP-dependent protein kinase-phosphorylation or by the symmetry of the cAMP-responsive element binding site. Filter binding assays reveal that CREB binding is unaffected by phosphorylation regardless of the symmetry of the cAMP-responsive element binding site. Our results suggest that structurally similar members of the same bZIP superfamily may differ significantly in their regulation at the level of dimerization.

Animals↗
PubMed1998

Seasonal rainfall variability, the incidence of hemorrhagic fever with renal syndrome, and prediction of the disease in low-lying areas of China.

To investigate determinants of hemorrhagic fever with renal syndrome (HFRS) in low-lying areas of China, the authors studied Chuigang and Wanggang communities in Anhui Province. These adjacent farming communities have a population of about 100,000. Data were collected from the two communities in 1961-1977 and from Yingshang County in 1983-1995; information covered the incidence of HFRS, amount of precipitation, differences in the water level of the Huai River, density of Apodemus agrarius, autumn crop production, and areas of inundated farmland. Correlation and multiple linear regression analyses were used to estimate the relation between seasonal rainfall, density of mice, occupational factors, and occurrence of the disease. Associations were observed between the incidence of HFRS and the amount of precipitation, the water level of the Huai River, and the areas of inundated farmland in Chuigang community. The smaller the water-level difference, the less farmland was inundated and the higher the incidence of HFRS. In Wanggang community, the density of A. agrarius (r1=0.63, p=0.02), the water-level difference in the Huai River (r2=-0.81, p=0.007), and crop production (r3=0.96, p=0.005) were correlated with the incidence of HFRS. The regression analyses based on Wanggang community suggested that these indexes could be used as predictive variables, and the results from the model were well calibrated with the actual incidence of HFRS in that community (R2=0.88, p < 0.01) and Yingshang County (R2=0.91, p < 0.01).

Animals↗
PubMed1998

Full activity from human beta-globin locus control region transgenes requires 5'HS1, distal beta-globin promoter, and 3' beta-globin sequences.

The locus control region (LCR) activates high-level human beta-globin transgene expression. LCR cassettes composed of 5'HS2-4 linked to the 815 bp beta-globin proximal promoter do not express fully. Here, we show that LCR (5'HS2-4) beta-globin transgenes that also contain either 5'HS1 or the distal promoter fail to express fully in single- and low-copy transgenic mice. In contrast, full expression is obtained in the presence of both 5'HS1 and the distal promoter. Nine factor binding sites were identified in 5'HS1, using in vitro DNaseI footprint and gel retardation assays, and these include a strong Sp1/Sp3 site, four GATA-1 sites, and two sites that encompass an ACTAAC motif. LCR (5'HS1-4) beta-globin transgene constructs with the distal promoter deleted or replaced by spacer DNA show that specific distal promoter sequences are required for full expression. An LCR (5'HS1-4) transgene construct with truncated downstream beta-globin gene sequences indicates that 3' sequences also play an important role. These results show that full expression of the beta-globin gene directed by the LCR requires 5'HS1, the distal beta-globin promoter, and 3' sequences, and has implications for gene therapy construct design and models of LCR activation.

Animals↗
PubMed1998

Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta.

We have isolated a cDNA from rat placenta which, when expressed heterologously, mediates the transport of a wide spectrum of organic cations. The cDNA codes for a protein of 551 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is expressed most abundantly in the placenta and moderately in the intestine, heart, and brain. The expression is comparatively low in the kidney and lung and is undetectable in the liver. This transporter is distinct from the previously cloned organic cation transporters (OCT1, OCT2, NKT, NLT, RST, and OCTN1). When expressed in HeLa cells, the cDNA induces the transport of tetraethylammonium and guanidine. Competition experiments indicate that this transport process recognizes a large number of organic cations, including the neurotoxin 1-methyl-4-phenylpyridinium, as substrates. The cDNA-induced transport is markedly influenced by extracellular pH. However, when expressed in Xenopus laevis oocytes, the cDNA-induced transport is electrogenic, associated with the transfer of positive charge into the oocytes. Under voltage clamp conditions, tetraethylammonium evokes inward currents that are concentration- and potential-dependent. This potential-sensitive organic cation transporter, designated as OCT3, represents a new member of the OCT gene family.

Amino Acid Sequence↗
PubMed1998

Regulation of brain G-protein go by Alzheimer's disease gene presenilin-1.

To investigate a possible association between G-proteins and presenilin-1 (PS-1), a series of glutathione S-transferase-fusion proteins containing portions of PS-1 were prepared and used in vitro in binding experiments with tissue and recombinant G-proteins. The results demonstrate that the 39 C-terminal amino acids of PS-1 selectively bind the brain G-protein, Go. Addition of guanosine 5'-3-O-(thio)triphosphate promoted Go dissociation from PS-1, indicating that this domain mimics the function of G-protein-coupling domains found in receptors. The 39-amino acid synthetic polypeptide activated Go in a magnesium ion-dependent manner. Physical interaction of full-length PS-1 and Go was also demonstrated. Following transfection of Goalpha and N-terminally FLAG-tagged PS-1 in COS-7 cells, Go was immunoprecipitated by FLAG antibodies. In addition, endogenous PS-1 and Goalpha were colocalized immunocytochemically in human glioma cell lines. The results indicate that PS-1 regulates Go activities in living cells.

Alzheimer Disease↗
PubMed1998

cDNA sequence, transport function, and genomic organization of human OCTN2, a new member of the organic cation transporter family.

We have cloned OCTN2, a new member of the organic cation transporter family, from a human placental trophoblast cell line. The hOCTN2 cDNA codes for a protein of 557 amino acids with twelve putative transmembrane domains. The octn2 gene, located on human chromosome 5q31, consists of ten exons. The OCTN2-specific transcript, 3.5 kb in size, is expressed widely in human tissues and in cell lines of human origin. At the level of amino acid sequence, OCTN2 is more closely related to OCTN1 than to OCT1, OCT2 and OCT3. When expressed heterologously in HeLa cells, OCTN2 mediates the transport of tetraethylammonium, a prototypical organic cation, in a pH-dependent manner. Several organic cations, including the neurotoxins 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and methamphetamine, compete for the OCTN2-mediated transport process.

Amines↗
PubMed1998

Interaction of secretory leukocyte protease inhibitor with heparin inhibits proteases involved in asthma.

Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfated polysaccharides. The nature of the SLPI-polysaccharide interaction, explored with affinity chromatography, indicated that this interaction was sensitive to the charge and type of polysaccharide. Dextran and chondroitin had the lowest affinity for SLPI, followed by dermatan, heparan, and dextran sulfates. While heparin bound SLPI tightly, the highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin oligosaccharides, prepared using heparin lyase I were SLPI-affinity fractionated. Surprisingly, undersulfated heparin oligosaccharides bound SLPI with the highest affinity, suggesting the importance of free hydroxyl groups for high affinity interaction. Isothermal titration calorimetry was used to determine the thermodynamics of SLPI interaction with a low molecular weight heparin, an undersulfated decasaccharide and a tetrasaccharide. The studies showed 12-14 saccharide units, corresponding to molecular weight of approximately 4,800, were required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore, an undersulfated decasaccharide was able to bind SLPI tightly (Kd approximately 13 nM), resulting in its activation and the inhibition of neutrophil elastase and pancreatic chymotrypsin. The in vitro assessment of heparin and the decasaccharide and tetrasaccharide using stopped-flow kinetics suggested that heparin was the optimal choice to study SLPI-based in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.

Animals↗
PubMed1998

Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin. Synergism with cyclic nucleotide-activated kinase.

Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.

Animals↗
PubMed1998

CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor.

DFF45 is a subunit of the DNA fragmentation factor (DFF) that is cleaved by caspase-3 during apoptosis. However, the mechanism by which DFF45 regulates apoptotic cell death remains poorly understood. Here we report the identification and characterization of two mammalian genes, CIDE-A and CIDE-B, encoding highly related proteins with homology to the N-terminal region of DFF45. CIDE-A and CIDE-B were found to activate apoptosis in mammalian cells, which was inhibited by DFF45 but not by caspase inhibitors. Expression of CIDE-A induced DNA fragmentation in 293T cells, which was inhibited by DFF45, further suggesting that DFF45 inhibits the apoptotic activities of CIDEs. In addition to mammalian CIDE-A and CIDE-B, we identified DREP-1, a Drosophila melanogaster homolog of DFF45 that could inhibit CIDE-A-mediated apoptosis. Mutant analysis revealed that the C-terminal region of CIDE-A was necessary and sufficient for killing whereas the region with homology to DFF45 located in the N-terminus was required for DFF45 to inhibit CIDE-A-induced apoptosis. CD95/Fas-mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. These studies suggest that DFF45 is evolutionarily conserved and implicate CIDEs as DFF45-inhibitable effectors that promote cell death and DNA fragmentation.

Amino Acid Sequence↗
PubMed1998

Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression.

The recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. We examined PTEN/MMAC1 in advanced prostate cancer for gene mutations or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1-specific antibody. Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and reverse transcription-PCR of mRNA. PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by in vitro treatment with the demethylating agent 5-azadeoxycytidine. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblot analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. These results demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer.

Azacitidine↗
PubMed1998

Sensitive method for the quantification of beta-glucuronidase activity in human urine using capillary electrophoresis with fluorescence detection.

Capillary electrophoresis (CE) with fluorescence detection was used to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-beta-D-glucuronide by beta-glucuronidase. Enzyme substrate saturation kinetics were studied in buffer and the pH range for the enzyme reaction was optimized. A linear relationship of initial enzyme reaction velocity as a function of peak area of enzyme product was obtained for enzyme activity ranging from 1 to 100 units. The beta-glucuronidase activity in urine was next determined. Freshly collected urine samples were dialyzed, the retentate was incubated with 4-methylumbelliferyl-beta-D-glucuronide, boiled and centrifuged. The supernatant was separated by CE in an uncoated capillary with 0.1 M sodium acetate buffer by applying a voltage of 12 kV. The product of the enzymatic reaction, 4-methylumbelliferone, was detected by fluorescence, facilitating the determination of as little as one unit of beta-glucuronidase activity in a 0.5-h incubation time, with an error of less than +/-5%.

Adult↗
PubMed1998

Benzo[a]pyrene diol epoxide-induced 3p21.3 aberrations and genetic predisposition to lung cancer.

3p deletion, a common chromosome defect in lung cancer, occurs more frequently in the lung tumor tissues of smoking patients than it does in those of nonsmoking patients. This pilot study evaluated whether 3p aberrations induced by benzo[a]pyrene diol epoxide (BPDE), the metabolic product of benzo[a]pyrene, a constituent of tobacco smoke, were more common in the peripheral blood lymphocytes of 40 lung cancer patients than they were in those of 54 matched controls. Our hypothesis was that 3p sensitivity to BPDE reflects the susceptibility of a specific locus to damage from carcinogens in tobacco smoke. BPDE-induced chromosome 3p21.3 aberrations were significantly more frequent in cases (34.1 per 1000) than they were in controls (22.1 per 1000; P < 0.0001). However, no such difference was observed for 6q27, a control locus. Using the median value in the controls (20 per 1000) as a cutoff point to classify BPDE-induced sensitivity at 3p21.3 and after adjustment by age, sex, ethnicity, and smoking status, 3p BPDE sensitivity was associated with an elevated risk of 14.1 (95% confidence interval: 3.5, 56.2) for lung cancer. There was also a dose-response relationship between the degree of BPDE sensitivity at 3p21.3 and increased risk for lung cancer. Therefore, 3p may be a molecular target for BPDE damage in lung cancer cases.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

[Sequencing analysis of human K-ras oncogene exon 1 in Chinese people].

OBJECTIVE: To investigate the mutation of human K-ras oncogene in the blood of patients with lung cancer. METHODS: PCR and sequencing methods were used to analyse the exon 1. Twelve patients'blood samples and 31 normals' blood samples were detected. RESULTS: In all 43 samples, the K-ras codon 11 was CCT (proline), and the opposite strand sequenced was AGG. It was different from the standard GCT(Alanine) sequenced by foreign authors. CONCLUSION: The 11th codon of the human K-ras oncogene in Chinese is CCT (Proline). As the K-ras codon 12 is a hot spot, this finding may be of importance to clinical genetic diagnosis.

Asian People↗
PubMed1998

Effects of 2,3-butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes.

1. We examine the actions of a chemical phosphatase, 2,3-butanedione monoxime (BDM), on endogenous and expressed Ca2+ channel currents in Xenopus oocytes. In previous studies on L-type Ca2+ channel currents in cardiomyocytes and dorsal root ganglia, the inhibitory effects of BDM were attenuated by activation of protein kinase A. 2. Ba2+ currents (IBa) through a human wild-type L-type Ca2+ channel complex (i.e. halpha1C, alpha2-deltaa and hbeta1b) are inhibited by BDM with an IC50 of 16 mM, with 10 mM producing a 36.1 +/- 2.2 % inhibition. IBa through endogenous oocyte N-type Ca2+ channels, upregulated by exogenous alpha2-deltaa and hbeta1b subunits, are inhibited to a similar degree by BDM. 3. To examine whether the action of BDM is dependent on PKA-dependent phosphorylation, a clone of halpha1C deficient in all five serine PKA consensus sites (halpha1C-SA5) was co-expressed with alpha2-deltaa and the human cardiac hbeta3 subunit, which naturally lacks PKA consensus sites. This complex exhibited a sensitivity to BDM that was similar to the wild-type complex, with 10 mM BDM producing 31.6 +/- 1.5 % inhibition. 4. As limited proteolysis upregulates Ca2+ channels in cardiomyocytes and renders them less sensitive to BDM, experiments were performed with a carboxyl terminus deletion mutant, halpha1C-Delta1633. IBa through this subunit showed a sensitivity to BDM that was similar to the wild-type complex, with 10 mM BDM producing 31.3 +/- 1.4 % inhibition. However, co-expression with alpha2-deltaa and hbeta3 subunits reduced potency, and is reflected by an increased IC50 of 22.7 mM. 5. The actions of BDM were examined on a rat brain rbA-1 Ca2+ channel clone, alpha1A, co-expressed with alpha2-deltab and beta1b subunit homologues from rat brain. BDM inhibited the current through this channel complex to a similar degree to that seen for cardiac wild-type channels, with 10 mM BDM causing a 33.1 +/- 3.5 % inhibition. 6. The effects of BDM were compared at two holding potentials, -80 and -30 mV, using the halpha1C-Delta1633, alpha2-deltaa and hbeta3 subunit combination. At -30 mV BDM is more potent with 10 mM BDM reducing IBa by 39.8 +/- 2.7 %, compared with 20.8 +/- 2.2 % at -80 mV. 7. The data suggest that BDM may not exert its inhibitory action by means of a chemical phosphatase effect, but by channel block. The similar potency observed between alpha1C, alpha1A and endogenous (N-type) channels may help point towards a possible site of action; differences with the carboxyl deletion mutant may help further to define a locus of interaction.

Animals↗
PubMed1998

DNA bending is induced by binding of the peroxisome proliferator-activated receptor gamma 2 heterodimer to its response element in the murine lipoprotein lipase promoter.

The peroxisome proliferator activated receptor gamma 2 (PPAR gamma 2) is a critical transcriptional regulator of adipogenesis. Lipoprotein lipase is one of the earliest genes induced following exposure of pre-adipocytes to PPAR gamma 2 ligands such as the thiazolidinediones. A unique PPAR gamma 2 DNA recognition element was mapped to the region between -171 to -149 bp of the murine LPL promoter, based on transfection analysis of deletion constructs and gel retention assays using bacterially expressed, affinity purified recombinant proteins. Circular permutation analysis determined that binding of the PPAR gamma 2/retinoic acid X receptor (RXR) heterodimer to its LPL promoter recognition element induced DNA bending at an angle of approximately 46 degrees. Parallel studies using an optimal PPAR recognition element obtained a comparable bending angle of 56 degrees. This is the first demonstration that binding of a PPAR protein to its recognition element causes a distortion of the DNA configuration. It indicates that PPAR gamma 2 utilizes a common mechanism shared by other nuclear hormone receptor proteins reported to induce bending at their DNA binding sites.

Animals↗
PubMed1998

Case-control study of the D2 dopamine receptor gene and smoking status in lung cancer patients.

BACKGROUND: Interindividual differences in the structure and expression of the dopamine receptor genes affect dopamine availability and may be the genetic basis for variation in vulnerability to tobacco smoking. In this study, prevalences of polymorphisms in the TaqIA allele (A1 and A2) and the TaqIB allele (B1 and B2) of the D2 dopamine receptor gene in 157 lung cancer case patients and 126 control subjects were determined to assess whether individuals homozygous or heterozygous for the less common A1 and B1 alleles are more vulnerable to nicotine addiction. METHODS: Case and control subjects were accrued from an ongoing epidemiologic study. Blood samples were collected from them and subjected to molecular genetic analyses. Subjects were interviewed to obtain relevant information. Current and former smokers were administered a questionnaire to quantify their addiction to nicotine. RESULTS: The combined B1B2 genotypes appeared to be more prevalent in ever smokers than in never smokers among case patients (30.3% versus 13.3%; two-sided P = .233) and among control subjects (30.9% and 0%; two-sided P = .02); statistically significant differences were not observed among those with A1 genotypes. Statistically significant correlations between the presence of the A1 and B1 alleles were observed (r = .73 for case subjects and r = .76 for control subjects; two-sided P<.001). Individuals with rarer genotypes reported having been substantially younger at the time of smoking initiation (statistically significant for both A1 and B1) and having attempted to quit smoking fewer times (statistically significant for only A1). CONCLUSION: Variant alleles of the D2 dopamine receptor gene may play a role in determining nicotine addiction, although the associations between the at-risk genotypes and measures of nicotine addiction were not entirely consistent.

Aged↗