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Biomedical subjects

W Chen

Publications and source records attributed to W Chen.

At least 883 records · Page 49Linked to original sources
PubMed1995

Suppression of potato spindle tuber viroid replication and symptom expression by mutations which stabilize the pathogenicity domain.

Nucleotides within the pathogenicity domain of potato spindle tuber viroid (PSTVd) are known to play an important role in regulating symptom expression, but the underlying molecular mechanism is unknown. In order to determine more precisely how structural features within the pathogenicity domain regulate symptom expression, we have characterized a series of mutations that progressively stabilize premelting region 1 and the rest of the "virulence modulating" region. The structural effects of these mutations were monitored by temperature gradient gel electrophoresis of circularized RNA transcripts, and their biological effects were assessed by quantitative bioassays in tomato. Closure of a 4-nucleotide loop within the premelting region 1 virtually abolished PSTVd infectivity, especially when a nearby 2-nucleotide loop was also closed. Although RNA transcripts containing less stabilizing mutations were readily infectious, none of the four single and one double substitutions examined were stably maintained in vivo. The pattern of spontaneous, apparently compensatory sequence changes observed in the progeny suggests that PSTVd variants with less stable secondary structures enjoy a selective advantage. Mutations which stabilize the pathogenicity domain of PSTVd in vitro also suppressed symptom expression, but at least one other mutation having no obvious structural effects was associated with a similar phenotype. Conformational stability appears to be only one of several factors regulating PSTVd replication and pathogenicity.

Base Sequence↗
PubMed1995

A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways.

Current assays for functional activation of Gs-coupled receptors usually involve quantitation of adenylyl cyclase or measurement of cAMP concentration by radioimmunoassay. The activation of Gq-coupled receptors is commonly assayed by measurement of the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-bisphosphate or of changes in intracellular calcium. These assays generally require large numbers of cells (10(5)-10(6)) and/or the use of radioactive materials. We have developed a rapid nonradioactive colorimetric assay that utilizes a beta-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein that results from an increase in intracellular cAMP or calcium. This assay can be performed using as few as 30,000 cells in a 96-well format with the end products measured simultaneously in a microplate reader. Consequently, a single individual can readily assay 1000 samples a day. Using this assay, the fold increase in beta-galactosidase activity was similar in magnitude to increases in cAMP or adenylyl cyclase activity and was approximately linear from 0.01 to 0.27 fmol/cell of intracellular cAMP. Furthermore, pharmacological characterization of one of the melanocortin receptors, mMC5-R, using this assay resulted in a similar order of potency for several melanocortin peptides to that obtained with a commonly used adenylyl cyclase enzyme assay. This assay is also useful for the characterization of Gq-coupled receptors as is demonstrated here using cells transfected with the mouse bombesin receptor. The large-scale capacity of this assay makes it an excellent method for screening molecules of interest acting on Gs- and Gq-coupled receptors.

Base Sequence↗
PubMed1995

Characterization of a 1.8-kb DNA segment located upstream from the human Ha-ras protooncogene and possibly regulating its function.

We have cloned a 1.8-kb segment of DNA just adjacent to the 5' end of the 6.4-kb BamHI fragment which contains the Ha-ras oncogene (ras1; GenBank notation HUMRASH). Electrophoretic mobility shift assays (EMSA) indicate the presence of multiple nuclear protein-binding sites within the 1.8-kb segment. At least three putative regulatory protein-binding sites have been identified in a 206-bp subfragment by DNase I footprint analysis. Within the subfragment containing the DNase-protected regions, there is also a segment containing a number of consensus sequences for DNA-binding proteins and two sub-sequences that exhibit strong sequence homology to at least two previously characterized enhancers. These data suggest that a novel set of regulatory elements may lie as far as 2 kb upstream from the normal Ha-ras transcription start points.

Amino Acid Sequence↗
PubMed1995

The conserved box 1 motif of cytokine receptors is required for association with JAK kinases.

The JAK2 tyrosine kinase is known to associate with the receptors for growth hormone (GH) and erythropoietin (EPO) and with the interleukin-6 receptor signal transducing protein, gp130. Here we demonstrate that chimeric cytokine receptors which contain the cytoplasmic domain of the receptors for GH and EPO or for gp130 can form complexes with JAK2 when transiently co-expressed in HeLa cells. Mutational analyses of chimeras for the the GH and EPO receptors and gp130 demonstrated that box 1, a motif critical for cytokine receptor signal transduction, was required for the association of JAK2. Although JAK2 was capable of associating with all three of the chimeras, JAK1 co-precipitated only with the gp130 chimera. Association of JAK1 and JAK2 with cytokine receptor proteins, therefore, requires the highly conserved box 1 domain, but other sequences within the receptor proteins may influence the specificity of JAK binding. Mutational analysis of JAK2 revealed that multiple or complex protein sequences within JAK2 are required for association with cytokine receptors.

Animals↗
PubMed1995

Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection.

Treatment of infected patients with ABT-538, an inhibitor of the protease of human immunodeficiency virus type 1 (HIV-1), causes plasma HIV-1 levels to decrease exponentially (mean half-life, 2.1 +/- 0.4 days) and CD4 lymphocyte counts to rise substantially. Minimum estimates of HIV-1 production and clearance and of CD4 lymphocyte turnover indicate that replication of HIV-1 in vivo is continuous and highly productive, driving the rapid turnover of CD4 lymphocytes.

Antiviral Agents↗
PubMed1995

Cloning and complete coding sequence of a novel human cathepsin expressed in giant cells of osteoclastomas.

A gene encoding a possible novel human cathepsin, a cysteine proteinase that is distinct from previously characterized enzymes, has been identified by differential screening of a human osteoclastoma cDNA library. This molecule, termed cathepsin X, appears to represent the human homolog of the osteoclast-expressed rabbit cathepsin OC-2. Cathepsin X (GenBank accession number U20280) is 93.9% identical to OC-2 at the amino acid level, and is 92% identical at the nucleotide level within the coding region. Cathepsin X is 52.2 and 46.9% identical to cathepsins S and L, respectively, and is therefore clearly distinct from these enzymes. Cathepsin X mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. These data strongly support the hypothesis that cathepsin X represents a novel cysteine proteinase which is expressed at high levels in osteoclasts.

Amino Acid Sequence↗
PubMed1995

Phylogenetic ordinal placement based on rDNA sequences of the freshwater genera Ophioceras and Pseudohalonectria.

The ordinal placement of two closely related freshwater genera, Ophioceras and Pseudohalonectria, was assessed by using phylogenetic analysis of morphological characters, partial sequences of the large subunit ribosomal DNA and restriction site variations in the internal transcribed spacer (ITS). The two genera have some morphological features that are used to define taxa in both the Sordariales and Diaporthales, and, hence, their phylogenetic relationships are unclear. Equally weighted analyses of thirty-eight morphological characters produced unresolved phylogenetic trees and unequivocal conclusions could not be drawn based on the morphological data. The polymerase chain reaction-amplified ITS region was variable in length between the two genera and restriction sites in the ITS region were determined. Analysis of variation in restriction sites in the ITS region placed Ophioceras and Pseudohalonectria in one clade with taxa sampled from Sordariales. About 350 basepairs of DNA sequence from the 5' end of the large subunit rDNA were also determined. In phylogenetic analysis of the sequence data with Hypocrea lutea and Nectria cinnabarina as outgroups, Ophioceras and Pseudohalonectria showed a closer relationship to Neurospora crassa, Schizothecium sp., and Sordaria fimicola of the Sordariales than to Cryphonectria parasitica and Endothia gyrosa of the Diaporthales.

Ascomycota↗
PubMed1995

Evolution and persistence mechanisms of mouse hepatitis virus.

We established and characterized persistently-infected DBT cells with mouse hepatitis virus to study the molecular mechanisms of MHV persistence and evolution in vitro. Following infection, viral mRNA and RF RNA were coordinately reduced by about 70% as compared to acute infection suggesting that the reduction in mRNA synthesis was due to reduced levels of transcriptionally active full length and subgenomic length negative-stranded RNAs. Although the rates of mRNA synthesis were also reduced, the relative percent molar ratio of the mRNAs and RF RNAs were similar to those detected during acute infection. In contrast to the finding during BCV persistence, analysis of the MHV leader RNA indicated that the leader RNA and leader/body junction sequences were extremely stable. These data suggested that polymorphism and mutations resulting in intraleader ORFs was not required for MHV persistence. Conversely MHV persistence was significantly associated with a A to G mutation at nt 77 in the 5' end untranslated region (UTR) of the genomic RNA.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of a hop stunt viroid variant isolated from citrus growing in Taiwan.

The 303 nucleotide sequence of HSVd-citrus(T), a hop stunt viroid (HSVd) variant present in Etrog citron growing in Taiwan, was determined from cDNAs amplified by the polymerase chain reaction. HSVd-citrus(T) is very similar to several HSVd isolates previously recovered from citrus or cucumber, and exhibits microsequence heterogeneity at positions 154 and 181. Phylogenetic analysis using maximum parsimony grouped HSVd-citrus(T) with seven other isolates from citrus and cucumber in a large cluster of "citrus-type" isolates. A similar analysis revealed marked differences in both the extent and distribution of sequence variation among naturally occurring isolates of potato spindle tuber viroid.

Base Sequence↗