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Biomedical subjects

W Chen

Publications and source records attributed to W Chen.

At least 757 records · Page 42Linked to original sources
PubMed1997

Differential expression of mitochondrial NADH dehydrogenase in ethanol-treated rat brain: revealed by differential display.

The technique of polymerase chain reaction (PCR) differential display was used to detect alterations in gene expression after chronic alcohol administration. Male Wistar rats were treated with ethanol vapor for 14 days. The cDNA generated from mRNA isolated from the hippocampi of ethanol-treated and control animals was compared by PCR differential display. A differentially expressed cDNA fragment was used to screen mRNA samples by Northern analysis. The level of a mRNA was significantly elevated (x 2.5) in the hippocampus, but not the cortex of alcohol-treated rats up to 48 hr after withdrawal. Sequence analysis of the cDNA fragment revealed an almost perfect homology to rat mitochondrial NADH dehydrogenase subunit 4 mRNA. The selective induction of this mRNA in alcohol-treated rat brain areas suggests altered metabolic processes and possible dysfunction of the mitochondria. The technique of PCR differential display may prove useful in further analysis of gene expression during alcohol dependence and withdrawal.

Alcoholism↗
PubMed1997

Interspousal transmission of GB virus-C/hepatitis G virus: a comparison with hepatitis C virus.

Although infection with GB virus-C/hepatitis G virus (GBV-C/HGV) by blood transfusion is well documented, little is known about the other routes of transmission. The prevalence of GBV-C/HGV infection in spouses of index patients and the related risk factors were studied. Hepatitis C virus (HCV) and GBV-C/HGV infections were studied in spouses of 100 patients with hepatitis C, of whom 12 were found to be also positive for GBV-C/HGV RNA. For couples both with GBV-C/ HGV viremia, nucleotide sequences of the divergent envelope region were analyzed by phylogenetic tree constructions. For HCV infection, anti-HCV was found in 14 (14%) of the 100 spouses. Five spouses (42%) of the 12 patients with dual infection of GBV-C/HGV and HCV had evidence of GBV-C/HGV infection, three had viral RNA, and two had antibodies to a recombinant HGV envelope protein E2. Nucleotide sequence comparison and phylogenetic tree analysis of the genome in the GBV-C/HGV infected couple revealed the isolates to be closely related. These results suggest that spouses of patients with GBV-C/HGV infection are at a higher risk of acquiring GBV-C/HGV as compared with HCV, and they should be educated to avoid GBV-C/HGV infection from their spouses, in case GBV-C/HGV is shown to be pathogenic.

Adenovirus E2 Proteins↗
PubMed1997

Lymphangioma: imaging diagnosis.

Lymphangiomas are congenital malformations of the lymphatics that are curable by extirpation. Accurate delineation of lesion extension is important for pre-operative diagnosis, surgical planning, and assessing recurrence. The radiologic findings were retrospectively evaluated to determine the imaging appearance of these benign tumours. The plain radiographs, barium meal, ultrasound, CT, and MR images of 18 patients with one or more pathologically proved lymphangiomas were reviewed. Plain radiography and barium study showed masses displacing adjacent organs. Ultrasound examination showed uni- or multilocular cystic masses with smooth, thin or irregular, thick walls. Enhancement of the cyst wall was variable on CT and MR studies. The CT density of the fluid ranged from -4 to 34 HU depending on the lipid content and the presence of haemorrhage. The cysts were isointense to muscle on T1-weighted and hyperintense to fat on T2-weighted MR images. The MR imaging delineated the tumour lesion extension more clearly than ultrasound and CT scans. Ultrasound, CT, and MR imaging are valuable for evaluating lymphangiomas. Magnetic resonance imaging allows accurate determination of lesion extension.

Adult↗
PubMed1997

Sibling aggregation of low- and high-density lipoprotein cholesterol and apolipoproteins B and A-I levels in black and white children: the Bogalusa Heart Study.

OBJECTIVE: To examine sibling aggregation of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein (apo) B and apo A-I in white versus black school-aged children. DESIGN: Study subjects included 790 full sibships representing 1305 sibpairs aged 5 to 17 years from a biracial community. METHODS: Intraclass correlation coefficients estimated by analyses of covariance were used to examine sibling aggregation of LDL-C, HDL-C, apo B and apo A-I. The influences of obesity and other lifestyle variables on sibpair differences in LDL-C, HDL-C, apo B and apo A-I were evaluated in black and white children by stepwise multiple regression analyses. RESULTS: Intraclass correlation coefficients for LDL-C and apo B in black children were lower than those in white children (0.17 and 0.11 versus 0.32 and 0.33, respectively, P < 0.05-0.01); no racial difference was found for HDL-C and apo A-I. Intraclass correlation coefficients for LDL-C and HDL-C were similar to that of apo B and apo A-I, respectively and the sibpair differences in LDL-C and HDL-C were correlated with those of apo B and apo A-I, respectively (P < 0.01). Obesity exerted a greater effect on sibpair differences in LDL-C, HDL-C and apo B in black children than in white children. CONCLUSIONS: These results suggest that the hereditary influence on LDL-C and apo B is more important in white children than in black children, especially for apo B. Further, genetic influence on LDL-C versus apo B, or HDL-C versus apo A-I may be similar.

Adolescent↗
PubMed1997

Change of level and expression of endothelin-1 in the lungs of rats with hypoxic pulmonary hypertension.

OBJECTIVE: To determine whether mRNA for endothelin-1 was present and changed in the lungs of rats exposed to chronic hypoxia. METHODS: cRNA probes for rat ET-1 and c-fos were labelled by in vitro transcription with digoxigenin-UTP for in situ hybridization. Endothelin-1 like immunoreactivity (ET-1-LI) in plasma as well as in lung homogenate was measured by radioimmunoassay. RESULTS: The vascular endothelial cells of pulmonary arteries and the bronchiolar epithelial cells were strongly positive for the ET-1 probe. The perivascular and peribronchiolar smooth muscle were positive for the c-fos probe, primarily. There was little expression of ET-1 and c-fos in the rat lungs in controls. There was significant increase of ET-1-LI levels in rats exposed to hypoxia for 3 weeks, the concentration ET-1-LI in venous samples was 3.85 +/- 1.52 ng/L (P < 0.05 as compared with the control groups), in arterial sample was 4.72 +/- 1.66 ng/L (P < 0.05) and in lung was 2.06 +/- 0.68 ng/g wet lung weight (P < 0.05). CONCLUSIONS: Chronic hypoxia elevated the mean pulmonary arterial pressure, caused vessel remodelling and the right ventricular hypertrophy. These changes were accompanied by an increase of ET-1 in plasma and lung homogenate. The expression and production of ET-1 were localized to endothelium and airway epithelium in the lungs.

Animals↗
PubMed1997

Evaluation of transforming growth factor beta and bone morphogenetic protein composite on healing of bone defects.

OBJECTIVE: To evaluate the effect of transforming growth factor-beta/bone morphogenetic protein (TGF-beta/BMP) composite on the healing of large segmental bone defects and to discuss the interaction between TGF-beta and BMP during bone repair. METHODS: A 1.5 cm segmental defect was made in the mid-upper part of the radial shaft of 48 adult rabbits. The defects were filled with implants of TGF-beta/carrier, BMP/carrier or TGF-beta/BMP/carrier separately. 120 micrograms of purified bovine TGF-beta and 12 mg of BMP were used in the composite. The defects were examined radiographically and histologically at 4, 8, 12 and 16 weeks post-operation. RESULTS: In the group filled with TGF-beta/BMP composite, the defect areas were bridged at 4 weeks, with callus of uniform radiodensity. Cortices of the cut ends were obscured by new bone. By 16 weeks post-operation, the defects were bridged by uniform new bone and the cut ends of the cortex could not be seen in all groups. In group of BMP/carrier, the defects were filled with more irregular woven bone callus than in other two groups. TGF-beta/BMP implanted defects in animals killed at 16 weeks showed histologically new lamella and woven bone that was formed in continuity with the cut ends of the cortex. Medullar canal was recanalized and contained marrow elements with normal appearance. CONCLUSIONS: These data demonstrate not only the synergistic action between TGF-beta and BMP in the process of bone healing, but also a better effect of TGF-beta/BMP composite than single TGF-beta or BMP on bone repair, especially in the early stage of bone repair process.

Animals↗
PubMed1997

[Experimental study of the effect of motion on repairing defect of articular cartilage following autogenous periosteal graft].

In order to investigate the effect of motion on repairing articular cartilage defect following autogenous periosteal graft, sixty adult rabbits were divided randomly into three groups: out-cage motion (OCM), in-cage motion (ICM) and immobilization (IMM). A defect of the articular cartilage, 1 cm x 0.5 cm in size, was made in the patellar-groove of femur of each hind limb. Free autogenous periosteal graft from the proximal tibia was sutured on the base of the left defect, while the right limb was served as control. The animals were sacrificed at 4, 8 and 12 weeks, respectively, after operation. The regeneration of the cartilage implanted was observed through gross, histology, histochemical assay and electronic microscope. The influence of different amount of motion on the chondrogenesis from the periosteal implant was also compared. The result showed that the hyaline cartilage produced from periosteal implant could be capable to repair full-thickness of articular cartilage. From statistical study, there was significant difference between OCM and ICM groups (P < 0.05), ICM and IMM (P < 0.05) as well as OCM and IMM (P < 0.01). It was suggested that the periosteal graft was effective in repair of defect of articular cartilage and the amount of motion was important for chondrogenesis.

Animals↗
PubMed1996

Isolation of the gene coding for Caenorhabditis elegans Rac2 homologue, a Ras-related small GTP-binding protein.

When screening a Caenorhabditis elegans genomic library using the human Rac1 cDNA as probe, a hybridizing fragment of 2.7 kb was isolated which contained four exons with high sequence similarity to CeRac1, coding for the nematode homologue of the Ras-related small GTP-binding protein Rac1. The putative translational product of 195 amino acids (aa) from the exons displayed 88% identity to the sequence of CeRac1. Interestingly, three alterations were found in the N-terminal "effector domain' (residues 22-45) which hitherto was identical among all known Rac p21s, suggesting that CeRac2 might have different targets/functions for nematode development. Additionally, an insertion of 4 aa was found in the hypervariable region at the C terminus of CeRac2.

Amino Acid Sequence↗
PubMed1996

Genetic analysis of a Japanese cerebrotendinous xanthomatosis family: identification of a novel mutation in the adrenodoxin binding region of the CYP 27 gene.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive lipid-storage hereditary disorder, is caused by mutations in the sterol 27-hydroxylase gene (CYP 27). A 24-year-old female Japanese CTX patient and her parents were studied for a CYP 27 mutation. Multiple xanthomas were the main complaint of the patient and plasma cholestanol level was markedly elevated. Sterol analysis of a xanthoma biopsy confirmed cholesterol and cholestanol deposition, and the cholestanol accounted for 8.1% of the total sterols. Sterol 27-hydroxylase activity in fibroblasts derived from the patient was undetectable, while the activities in fibroblasts from her mother and father were 54% and 41% of the normal level, respectively. Direct sequence analysis showed a missense mutation of A for G substitution in the CYP 27 gene at codon 362 (CGT 362Arg to CAT 362His) with a homozygous pattern in the patient, and a heterozygous pattern in the parents. The mutation, which eliminates a normal HgaI endonuclease site at position 1195 of the cDNA and is located at the adrenodoxin binding region of the gene, is most probably responsible for the decreased sterol 27-hydroxylase activity in this Japanese CTX family. The combined data strongly support that the primary enzymatic defect in CTX is the disruption of sterol 27-hydroxylase and that the disease is inherited in an autosomal recessive trait.

Adrenodoxin↗
PubMed1996

Expression of glutamyl-tRNA reductase in Escherichia coli.

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNAglu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.

Aldehyde Oxidoreductases↗
PubMed1996

Baculovirus expression and purification of human and rat cytochrome P450 2E1.

High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) was achieved using a baculovirus expression system. A full length cDNA encoding human CYP2E1 was cloned from a human liver cDNA library and sequenced using the dideoxy sequencing method. Insect cells were infected with the homologous recombinant baculoviruses containing the human and rat CYP2E1 cDNAs, respectively. The infected cells were harvested at a time when 450-nm peak intensities were at a maximal level and there was no 420-nm peak observed in the reduced CO difference spectrum. Both human and rat CYP2E1 were then purified to electrophoretic homogeneity by a relatively rapid and efficient procedure. The specific contents of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg protein, respectively. The lambda(max) of the reduced CO difference spectra of both purified rat and human CYP2E1 was found to be 451.5 nm. When the purified rat and human CYP2E1 were reconstituted with purified rat NADPH-P450 reductase and human cytochrome b5, they were able to metabolize several known CYP2E1 substrates: chlorzoxazone, p-nitrophenol, acetaminophen, and carbon tetrachloride. Interestingly, cytochrome b5 markedly stimulated the CYP2E1-mediated two-electron oxidation of the first three substrates, while it had almost no effect on the presumed one-electron reduction of carbon tetrachloride.

Animals↗
PubMed1996

Crystal structure of p38 mitogen-activated protein kinase.

p38 mitogen-activated protein kinase is activated by environmental stress and cytokines and plays a role in transcriptional regulation and inflammatory responses. The crystal structure of the apo, unphosphorylated form of p38 kinase has been solved at 2.3 A resolution. The fold and topology of p38 is similar to ERK2 (Zhang, F., Strand, A., Robbins, D., Cobb, M. H., and Goldsmith, E. J. (1994) Nature 367, 704-711). The relative orientation of the two domains of p38 kinase is different from that observed in the active form of cAMP-dependent protein kinase. The twist results in a misalignment of the active site of p38, suggesting that the orientation of the domains would have to change before catalysis could proceed. The residues that are phosphorylated upon activation of p38 are located on a surface loop that occupies the peptide binding channel. Occlusion of the active site by the loop, and misalignment of catalytic residues, may account for the low enzymatic activity of unphosphorylated p38 kinase.

Amino Acid Sequence↗
PubMed1996

The Caenorhabditis elegans p21-activated kinase (CePAK) colocalizes with CeRac1 and CDC42Ce at hypodermal cell boundaries during embryo elongation.

The p21-activated kinase (PAK) is a downstream target of Rac and CDC42, members of the Ras-related Rho subfamily, that mediates signaling pathway leading to cytoskeletal reorganization. To investigate its function in Caenorhabditis elegans development, we have isolated the cDNA coding for the p21-activated kinase homologue (CePAK) from a C. elegans embryonic cDNA library. This 2.35-kilobase pair cDNA encodes a polypeptide of 572 amino acid residues, with the highly conserved N-terminal p21-binding and the C-terminal kinase domains. Similar to its mammalian and Drosophila counterparts, the CePAK protein expressed in E. coli exhibits binding activity toward GTP-bound CeRac1 and CDC42Ce. Polyclonal antibodies raised against the recombinant CePAK recognize a specific 70-kDa protein from embryonic extracts that displays CeRac1/CDC42Ce-binding and kinase activities. Immunofluorescence analysis indicates that CePAK is specifically expressed at the hypodermal cell boundaries during embryonic body elongation, which involves dramatic cytoskeletal reorganization. Interestingly, CeRac1 and CDC42Ce are found at the same location, which might point to their common involvement in hypodermal cell fusion, a crucial morphogenetic event for nematode development.

Amino Acid Sequence↗
PubMed1996

Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter capsulatus.

A synthetic oligodeoxynucleotide probe based on a highly conserved region of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R. capsulatus. The deduced polypeptide contains 674 amino acids and has a predicted molecular mass of 75,942 Da. The deduced amino acid sequence of R. capsulatus RpoD protein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E. coli, respectively. Unsuccessful attempts to inactivate the single chromosomal rpoD gene of R. capsulatus by gene replacement technique indicate that this gene is essential for cell survival, as expected for the primary sigma factor. The rpoD transcript 5'-end was mapped by primer extension analysis, 74 bp upstream of the initiation codon and DNA sequence analysis has identified a motif resembling the delta 70 E. coli consensus promoter sequences at the expected distance from this proposed transcription start site. rpoD gene expression, as measured by the activity of the phi (rpoD'-lacZ+) translational fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stationary phase.

Amino Acid Sequence↗