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Biomedical subjects

W Xu

Publications and source records attributed to W Xu.

At least 19 recordsLinked to original sources
PubMed1999

Vitamin D receptor interacts with DnaK/heat shock protein 70: identification of DnaK interaction site on vitamin D receptor.

Vitamin D receptor (VDR) regulates the expression of vitamin D-dependent genes upon binding to its cognate ligand, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). This process represents a complex interaction of ligand-bound VDR with nuclear proteins like retinoid X receptor, nuclear accessory factors, and regulatory elements of the target gene. Expression of full-length VDR in Escherichia coli revealed that VDR binds DnaK, a member of heat-shock protein (Hsp) family, with high affinity. By systematic N-terminal truncation of VDR, the interaction site of DnaK on VDR was localized within a 17-amino-acid segment (105-122) representing the "hinge region" between the DNA-binding and hormone-binding domains of VDR. The putative DnaK-binding site was further localized between residues 105 to 109 of VDR by using binding-energy-minimization studies. The interaction of DnaK with VDR did not influence the binding of 1,25(OH)2D3 or nuclear accessory factor(s) to VDR. Furthermore, bovine brain Hsp 70, similar to DnaK, interacted with VDR-ligand-binding domain (105-427). These results suggest that DnaK/Hsp 70 may interact with VDR prior to the activation of the latter by 1,25(OH)2D3-binding.

Amino Acid Sequence↗
PubMed1999

(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: implications for congenital myotonic dystrophy.

Although the mutation for myotonic dystrophy has been identified as a (CTG)n repeat expansion located in the 3'-untranslated region of a gene located on chromosome 19, the mechanism of disease pathogenesis is not understood. The objective of this study was to assess the effect of (CTG)n repeats on the differentiation of myoblasts in cell culture. We report here that C2C12 myoblast cell lines permanently transfected with plasmid expressing 500 bases long CTG repeat sequences, exhibited a drastic reduction in their ability to fuse and differentiate into myotubes. The percentage of cells fused into myotubes in C2 C12 cells (53.4+/-4.4%) was strikingly different from those in the two CTG repeat carrying clones (1.8+/-0.4% and 3.3+/-0. 7%). Control C2C12 cells permanently transfected with vector alone did not show such an effect. This finding may have important implications in understanding the pathogenesis of congenital myotonic dystrophy.

Animals↗
PubMed1999

Neuroleukin mediated differentiation induction of myelogenous leukemia cells.

Leukemia cells enriched from peripheral blood of a patient with myelogenous leukemia were induced to differentiate with a purified T cell lymphokine neuroleukin. With sufficient neuroleukin concentrations, cells with macrophage-like morphology were identified among the developing adherent cells. After 2-5 days, approximately 38-50% of the suspension cells became macrophage-like and acquired CD21, alpha-naphthyl acetate reactivity and immune adherence capability. The amount of these nonproliferating cells increased along with cells containing fragmented DNA. Induction with insufficient neuroleukin quantity or with patient plasma alone developed few or no mature cells, indicating differentiation to mature cells is dose-dependent. The possibility of insufficient quantity of neuroleukin in regulation of patient plasma for differentiation was discussed.

Cell Differentiation↗
PubMed1999

Genome scan for human obesity and linkage to markers in 20q13.

Obesity is a highly prevalent, multigenic trait that predicts increased morbidity and mortality. Here we report results from a genome scan based on 354 markers in 513 members of 92 nuclear families ascertained through extreme obesity and normal body weight. The average marker interval was approximately 10 cM. We examined four correlated obesity phenotypes, including the body-mass index (BMI) (both as a quantitative trait and as a discrete trait with a threshold of BMI > or /=30 kg/m2) and percentage of fat (both as a quantitative trait and as a discrete trait with a threshold of 40%) as assessed by bioelectrical impedance. In the initial stage of the genome scan, four markers in 20q gave positive evidence for linkage, which was consistent across most obesity phenotypes and analytic methods. After saturating 20q with additional markers (25 markers total) in an augmented sample of 713 members from 124 families, we found linkage to several markers in a region, 20q13, previously implicated in both human and animal studies. Three markers (D20S107, D20S211, and D20S149) in 20q13 had empirical P values (based on Monte Carlo simulations, which controlled for multiple testing) < or /=. 01 for single-point analysis. In addition, the parametric, affecteds-only analysis for D20S476 yielded a LOD score of 3.06 (P=. 00009), and the affected-sib-pair test yielded a LOD score of 3.17 (P=.000067). Multipoint analyses further strengthened and localized these findings. This region includes several plausible candidate genes for obesity. Our results suggest that one or more genes affecting obesity are located in 20q13.

Black People↗
PubMed1999

Impact of clozapine on negative symptoms and on the deficit syndrome in refractory schizophrenia. Department of Veterans Affairs Cooperative Study Group on Clozapine in Refractory Schizophrenia.

OBJECTIVE: This study compared the effect of clozapine and haloperidol on positive and negative symptoms of schizophrenia and in patients with high levels of negative symptoms or the deficit syndrome. METHOD: Patients were participants in a 15-site double-blind, random-assignment Veterans Administration trial comparing clozapine (N=205) and haloperidol (N=217) in hospitalized patients with refractory schizophrenia. Analysis of covariance examining change at 6 weeks, 3 months, and 1 year evaluated 1) clozapine's effect on positive and negative syndromes; 2) clozapine's effect on each syndrome, statistically controlling for the other; and 3) the interaction of clozapine treatment and the presence or absence of high levels of negative symptoms at baseline and the deficit syndrome. RESULTS: Patients treated with clozapine showed significantly greater improvement than control subjects on positive symptoms at all time points and on negative symptoms at 3 months. Clozapine had no independent effect on negative symptoms at any time after control for positive symptoms, but its effects on positive symptoms persisted after control for negative symptoms at 6 weeks only. There were no significant differences in response to clozapine between patients with high and low levels of negative symptoms at baseline or between patients with and without the deficit syndrome. CONCLUSIONS: The greater effectiveness of clozapine as compared to conventional medications in refractory schizophrenia is not specific to either negative clinical symptoms or clinical subtypes defined by prominent negative symptoms or evidence of the deficit syndrome.

Analysis of Variance↗
PubMed1998

Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations.

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized protein coats 24% or less of the sorbent surface. It was found that columns on which proteins were immobilized still behaved as a cation-exchange chromatography sorbents, but their selectivity was different from the parent weak cation-exchange column. This was interpreted to mean that in addition to the normal electrostatic interactions expected in ion- exchange chromatography, protein analytes interact with immobilized protein on the sorbent surface. Anionic proteins were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It is concluded that these protein-protein interactions occur after proteins are captured by the primary interaction mechanism of the column, in this case, electrostatic interaction. Protein-protein interaction is a secondary, lateral process. These lateral interactions were observed between 4% and 24% surface saturation. The significance of this observation is that in preparative chromatography and the case of "fouled" columns, strongly adsorbed proteins could alter the elution characteristics of sample proteins being target for analysis or purification.

Cation Exchange Resins↗
PubMed1998

Palmitoylation of the rat mu opioid receptor.

We examined whether the mu opioid receptor was palmitoylated and attempted to determine sites of palmitoylation. Following metabolic labeling with [3H]palmitic acid and immunoaffinity purification of the mu opioid receptor, SDS-PAGE and fluorography revealed a broad labeled band with Mr of approximately 80 kDa in CHO cells stably expressing the rat mu receptor, but not in CHO cells transfected with the vector alone, indicating that the mu receptor is palmitoylated. Activation of the receptor with morphine did not affect the extent of palmitoylation. Hydroxylamine or dithiothreitol treatment removed most of the radioactivity, demonstrating that [3H]palmitic acid is incorporated into Cys residue(s) via thioester bond(s). Surprisingly, mutations of the only two Cys residues in the C-terminal domain did not reduce [3H]palmitic acid incorporation significantly. Thus, unlike many G-protein coupled receptors, the palmitoylation site(s) of the rat mu opioid receptor do(es) not reside in the C-terminal domain.

Amino Acid Sequence↗
PubMed1998

Congenital erythropoietic porphyria successfully treated by allogeneic bone marrow transplantation.

The long-term biochemical and clinical effectiveness of allogenic bone marrow transplantation (BMT) was shown in a severely affected, transfusion-dependent 18-month-old female with congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis resulting from mutations in the uroporphyrinogen III synthase (URO-synthase) gene. Three years post-BMT, the recipient had normal hemoglobin, markedly reduced urinary porphyrin excretion, and no cutaneous lesions with unlimited exposure to sunlight. The patient was homoallelic for a novel URO-synthase missense mutation, G188R, that expressed less than 5% of mean normal activity in Escherichia coli, consistent with her transfusion dependency. Because the clinical severity of CEP is highly variable, ranging from nonimmune hydrops fetalis to milder, later onset forms with only cutaneous lesions, the importance of genotyping newly diagnosed infants to select severely affected patients for BMT is emphasized. In addition, the long-term effectiveness of BMT in this patient provides the rationale for future hematopoietic stem cell gene therapy in severely affected patients with CEP.

Bone Marrow Transplantation↗
PubMed1998

Senescent BALB/c mice exhibit decreased expression of lambda5 surrogate light chains and reduced development within the pre-B cell compartment.

Although senescent BALB/c mice (approximately 2 years old) have reduced numbers of small pre-B cells, early pre-B cells (CD43+CD25+B220+) are present in comparable numbers within the bone marrow of both young (3-6-month-old) and senescent BALB/c mice. The transition of CD43+ pre-B cells to the CD43- pre-B cell compartments is dependent on proliferation and clonal maturation dictated by the pre-B cell receptor (mu/lambda5/VpreB). In vivo, senescent CD43+B220+ pro-B/early pre-B cells demonstrated reduction of lambda5 mRNA, by RT-PCR analysis, and of both surface and cytoplasmic lambda5 protein. Decreased lambda5 protein expression was also seen among pro-B/pre-B cells derived from senescent bone marrow after stimulation in vitro with IL-7. We propose that diminished expression of the lambda5 surrogate light chain results in decreased pre-B cell receptor formation and contributes to reduced recruitment of nascent CD43+ pre-B cells into the CD43- large and small pre-B cell compartments.

Aging↗
PubMed1998

Assembly of a tailed bacterial virus and its genome release studied in three dimensions.

We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Bacillus Phages↗
PubMed1998

Molecular cloning of human ABPL, an actin-binding protein homologue.

Based on two partial cDNA sequences, a full-length cDNA sequence for an actin-binding like protein previously named ABPL has been isolated and characterized. ABPL is homologous to the human actin-binding proteins ABP-280 and ABP-278. The predicted sequence for ABPL is 2,705 amino acids in length with a calculated molecular mass of 289 kDa. It contains an amino terminal actin-binding domain followed by 24 tandem repeats of approximately 96 amino acids. Two hinge regions, Hinge I and Hinge II, were located prior to repeats 16 and 24, respectively. An isoform of ABPL lacking Hinge I, with a calculated molecular mass of 286 kDa, was also identified by the reverse transcriptase PCR (RT-PCR) method. A comparison with genomic sequences indicated the isoform resulted from alternative RNA splicing. ABPL has a unique insertion sequence of 82 amino acids in repeat 20 that was not present in the other two homologues and has a tissue distribution that was also different from the other two homologues.

Actins↗
PubMed1998

Predictors of differential response to clozapine and haloperidol. Veterans Affairs Cooperative Study Group on Clozapine in Refractory Schizophrenia.

BACKGROUND: We sought to identify baseline predictors of response to clozapine. METHODS: Data were from a 15-site randomized clinical trial comparing clozapine and haloperidol in hospitalized patients with refractory schizophrenia (n = 423). Three-month outcomes were analyzed with the full sample (n = 368 due to attrition). Because of crossovers, analyses of 12-month outcomes were conducted with crossovers excluded (n = 291). Clinical predictors included age, race, diagnosis (current substance abuse, paranoid subtype of schizophrenia, or depressive syndrome), severity of symptoms, quality of life, age at onset of schizophrenia, extrapyramidal symptoms, and VA compensation payment. Multiple regression analysis was used to examine the interaction of treatment condition and each of these variables in predicting outcomes for symptoms, quality of life, side effects, and days hospitalized. RESULTS: Patients with higher quality of life at baseline (p = .04) and higher symptoms (p = .02) had relatively smaller declines in hospital days at 6 months. In the 12-month sample patients with higher levels of symptoms had greater symptom reductions at 12 months (p = .03) and greater improvement in quality of life (p = .004). CONCLUSIONS: Although high levels of symptoms were associated with greater improvement on clozapine, these findings are not robust enough to suggest that any specific, clinically defined subgroup of refractory patients should be preferentially targeted for clozapine treatment.

Adult↗
PubMed1998

Direct Observation of Polymer Network Structure in Macroporous N-Isopropylacrylamide Gel by Raman Microscopy.

The Raman microscopy technique is used to characterize the temperature-induced evolution of the pore structures of a macroporous N-isopropylacrylamide (NIPA). The gel is synthesized using a suspension of 45 wt % toluene and 55 wt % pregel NIPA solution. The intensity of the band due to the CH2 bending vibration, centered at 1445 cm-1, is used to monitor the distribution of the polymer chain density in the lateral plane. It is found that the macroporous gel consists of water-rich areas, which can be interpreted as the pores, and polymer-rich areas. At room temperature, the average sizes of the pores and the width of polymer-rich areas are 75 µm and 20 µm, respectively. Both the pores and their surrounding polymer-rich areas have random geometry, as demonstrated by the Raman microimaging. With increased temperature the size of the pores decreases. This process is accompanied by a narrowing of the polymer-rich areas. At higher temperatures polymer chains bunch together and this process accelerates rapidly near the volume phase transition temperature (34 degreesC). Above 36 degreesC, the pore sizes become too small to be resolved using Raman microscopy.

Journal Article↗
PubMed1998

Isolation and characterization of the mouse beta 2/neuroD gene promoter.

Beta2/neuroD is a basic helix-loop-helix protein involved in differentiation of the endocrine pancreas and the central nervous system. A 2-kb fragment containing the 5' upstream region of the mouse beta2/neuroD gene was cloned and sequenced. The cloned fragment was tested for promoter activity in six cell lines. Strong promoter activity was apparent in the three pancreatic beta cell lines, beta-HC3, beta-HC9, and NIT-1, whereas weak activity was seen in NIH 3T3, Rat-1, and MCF-7 cell lines. Analysis of promoter activity of deletion mutants in beta-HC3 cells indicated that while basal promoter activity was observed with a fragment which extended -109 bp upstream of the transcription start site, maximal activity required the fragment -2091 to -297 bp.

Animals↗
PubMed1998

Neuronal expression of the glutamate transporter GLT-1 in hippocampal microcultures.

To address the question of the relative contributions of glial and neuronal glutamate transport in the vertebrate CNS, we studied the distribution of forebrain glutamate transporters in rat hippocampal microcultures, a preparation in which physiological functions of glutamate transporters have been well characterized. Two of the three transporters, GLAST (EAAT1) and EAAC1 (EAAT3), are localized to microculture glia and neurons, respectively, as expected. However, we find strong immunoreactivity for the third glutamate transporter GLT-1 (EAAT2), a putatively glial transporter, in microculture neurons and in a small subset of microculture glia. Indistinguishable immunohistochemical staining patterns for GLT-1 were obtained with antibodies directed against both the N terminal and C terminal of the GLT-1 protein. Double-labeling experiments suggest that neuronal GLT-1 protein is primarily localized to the dendrites of excitatory neurons. Neuronal electrogenic transport currents in response to D-aspartate applications were occluded by the selective GLT-1 inhibitor dihydrokainate. In contrast, glia exhibited a larger transporter current density than did neurons, and the glial transport current was less sensitive to dihydrokainate. Neuronal transport currents were potentiated less than were glial currents when the chaotropic anion thiocyanate was substituted for gluconate in the whole-cell recording pipette, consistent with the previously reported lower anion permeability of EAAC1 and GLT-1 compared with that of GLAST. After microculture glia were rendered nonviable, excitatory autaptic currents (EACs) were prolonged in the presence of dihydrokainate, suggesting that neuronal GLT-1 is capable of participating in the clearance of synaptically released glutamate. Our results suggest that the initially proposed characterization of GLT-1 as a purely glial transporter is too simplistic and that under certain conditions functional GLT-1 protein can be expressed in brain neurons. The study suggests that changes in GLT-1 levels that occur with pathology or experimental manipulations cannot be assumed to be glial.

ATP-Binding Cassette Transporters↗