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Biomedical subjects

W Xie

Publications and source records attributed to W Xie.

At least 145 records · Page 8Linked to original sources
PubMed1996

Transcriptional regulation of prostaglandin synthase 2 gene expression by platelet-derived growth factor and serum.

Prostaglandin synthase 2 (PGS2) is an immediate-early gene induced in a variety of cellular contexts. We investigate here the transcriptional activation of the murine PGS2 gene in NIH 3T3 cells, in response to the mitogens platelet-derived growth factor (PDGF) or serum. Site-directed mutagenesis experiments demonstrate that a consensus cyclic AMP response element (CRE) in the murine PGS2 promoter is essential for optimal PGS2 gene expression in response to PDGF or to serum. Overexpression of c-Jun potentiates PDGF- or serum-induced luciferase expression from a reporter construct containing the first 371 nucleotides of the PGS2 promoter. In contrast, overexpression of other transcription factors binding to the CRE element of the PGS2 gene inhibits induction by PDGF or serum. Moreover, positioning the c-Jun activation domain next to the minimal PGS2 promoter via a GAL4 DNA binding site rather than the CRE is sufficient to permit serum or PDGF stimulation of luciferase expression from this modified reporter construct. PDGF or serum treatment both activate c-Jun N-terminal kinase (JNK), the mitogen-activated protein kinase responsible for phosphorylation and activation of c-Jun. Cotransfection of plasmids expressing dominant-negative Ras, Rac1, MEKK-1, or JNK along with the [PGS2][luciferase] reporter prevents induction by PDGF or serum, demonstrating that serum and PDGF induction of the PGS2 gene in NIH 3T3 cells requires activation of a Ras/Rac1/MEKK-1/JNK kinase/JNK signal transduction leading to phosphorylation of c-Jun. Additional cotransfection experiments with plasmids expressing dominant-negative Raf1 and ERK demonstrate that induction of PGS2 gene expression by PDGF and serum also requires activation of a Ras/Raf1/mitogen-activated protein kinase kinase (MAPKK)/ERK signal transduction pathway.

3T3 Cells↗
PubMed1996

Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.

The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.

Alleles↗
PubMed1996

Characterization of PCP-2, a novel receptor protein tyrosine phosphatase of the MAM domain family.

DNA sequences encoding a novel member of the receptor protein tyrosine phosphatase (R-PTP) family, termed PCP-2, were identified in a human pancreatic adenocarcinoma cDNA library. Human PCP-2 cDNA predicts a protein of 1430 amino acids with a calculated Mr of 160 kDa. The predicted PCP-2 enzyme consists of a 740 amino acid extracellular region, a single transmembrane domain, and a 666 amino acid intracellular portion. The extracellular sequence contains a MAM (meprin/A5/PTPmu) domain, an immunoglobulin-like domain and four fibronectin type III-like repeats, suggesting that it is a member of the PTPkappa and PTPmu subfamily. The intracellular region contains two tandemly-repeated protein tyrosine phosphatase domains. Northern blot analyses revealed a single transcript of 5.5 kilobases, which is expressed at different levels in many human tissues except spleen and placenta. Upon transfection of PCP-2 cDNA into human embryonic kidney fibroblast 293 cells, a protein with an apparent Mr of 180 000 was detected by immunoblot analysis. This size was reduced to the predicted Mr upon treatment with endoglycosidase F, indicating that PCP-2 is glycosylated and, hence, expressed at the cell surface. A potential role of PCP-2 in cell-cell recognition and adhesion is supported by its co-localization with cell adhesion molecules, such as catenin and E-cadherin, at sites of cell-cell contact.

Adenocarcinoma↗
PubMed1996

Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84 colonic epithelial cells.

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

Calcimycin↗
PubMed1996

Biosynthesis of fumonisin and aal derivatives by Alternaria and Fusarium in laboratory culture.

Cultures of Fusarium moniliforme and Alternaria alternata f. sp. lycopersici were grown in the laboratory and analyzed for various fumonisin derivatives. Analyses were made by continuous flow fast atom bombardment and ionspray mass spectrometry interfaced to microcapillary HPLC. Besides FB1, FB2 and FB3 derivatives, two isomers of the one-armed FB1 (protonated molecular ion at m/z 564) and two isomers of the one-armed FB2 (m/z 548) were found. Two different isolates of A. alternata when grown in culture yielded FB1, FB2 and FB3. One of them also yielded the one-armed FB1 which was identical to that metabolite found in Fusarium and naturally infected corn. FB1, FB2 and FB3 have been found in 2 different isolates of Alternaria alternata, both obtained from tomato.

Alternaria↗
PubMed1996

In vivo and in vitro expression of a non-mammalian cyclooxygenase-1.

Unlike cyclooxygenase 2 (COX-2), COX-1 has never been identified, purified or cloned in a non-mammalian species. Here we report the RT-PCR cloning of a chicken cDNA that encodes the amphipathic membrane binding region and parts of the dimerization and catalytic domains of COX-1-like enzyme. Sequence comparison showed this putative COX-1 to be evolutionarily less conserved than COX-2. Furthermore, whereas COX-1 in mammals is broadly expressed in tissues as a constitutive enzyme, the mRNA detected by our clone in chicken was almost absent in tissues and embryo fibroblasts (CEF). Highest expression was in brain and seminal vesicle. This transcript was not detectable during chick embryogenesis and, as is the case for mammalian COX-1, was not induced above background by mitogen stimulation. The identification of an avian COX-1 shows that COX-1 and COX-2 existed as separate catalysts for prostaglandin synthesis before the divergence of birds and mammals.

Amino Acid Sequence↗
PubMed1996

Expression of cecropin CMIV fusion protein in E. coli under T7 promoter.

A synthetic gene coding for cecropin CMIV from Bombyx mori was cloned into plasmid pEZZ318 and fused to a DNA segment encoding the signal peptide of Staphylococcal protein A and IgG binding domain. The fusion gene was then subcloned into expression vector pET11c under the control of T7 promoter and expressed in E. coli. The fusion protein did not exhibit any antibacterial activity either in cell lysate or in medium. After cleavage from the fusion protein with CNBr the biological activity of recombinant cecropin CMIV was obtained.

Amino Acid Sequence↗
PubMed1996

Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Amino Acid Sequence↗
PubMed1996

The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.

Amino Acid Sequence↗
PubMed1996

Shrinkage activates a nonselective conductance: involvement of a Walker-motif protein and PKC.

The ability of all cells to maintain their volume during an osmotic challenge is dependent on the regulated movement of salt and water across the plasma membrane. We demonstrate the phosphorylation-dependent gating of a nonselective conductance in Caco-2 cells during cellular shrinkage. Intracellular application of exogenous purified rat brain protein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na(+)-to-Cl- permeability ratio of approximately 1.7:1. To prevent possible PKC- and/or shrinkage-dependent activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR505-511, was introduced into the cells via the patch pipette. Anti-CFTR505-511, which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC/shrink-age current activation. The peptide CFTR505-511 also induced current inhibition, suggesting the possible involvement of a regulatory element in close proximity to the channel that shares sequence homology with the first nucleotide binding fold of CFTR and whose binding to the channel is required for channel gating.

Animals↗
PubMed1996

[Effects of selenium and germanium on lipid peroxidation in rats fed with low-selenium grain].

Rats were fed with grain produced in the area prevalent of Keshan disease for 12 weeks, by adding certain amount of sodium selenite (Na2SeO3) and carboxylethylgermanium (Ge-132) to study the combined effects of selenium and germanium on lipid peroxidation and metabolism of free radicals in their bodies. Results showed selenium and germanium added to the grain could reduce the content of free radicals in rats' liver and kidney tissues, and that of lipid peroxide in their heart, liver and kidney, and increase the activities of glutathione peroxidase (GSH-Px) in their blood, in a synergic way.

Animals↗
PubMed1996

Rat liver catalase is sorted to peroxisomes by its C-terminal tripeptide Ala-Asn-Leu, not by the internal Ser-Lys-Leu motif.

The molecular signal for targeting catalases to peroxisomes has not been defined. In this study, a plant in vivo import system (tobacco BY-2 suspension culture cells) was used to test the current postulate that the peroxisome targeting signal (PTS) for mammalian catalases is the internal Ser-Lys-Leu (SKL) motif found approximately eight amino acid residues from the C-terminus. Elucidation of the catalase PTS has been hampered previously by the ubiquitous presence of catalase in peroxisomes. The current study was possible because antibodies to mammalian catalases did not recognize endogenous, tobacco peroxisome catalase. Rat and mouse liver catalases (Rcat and Mcat), with an internal Ser-His-Ile (SHI) and Ser-His-Met (SHM), respectively, and both with a C-terminal Ala-Asn-Leu (ANL), were expressed transiently in BY-2 cells and targeted to the peroxisomes. Sorting was demonstrated by double-label immunofluorescence colocalization of these catalases with tobacco catalase. Peroxisome targeting of Rcat was abolished as expected when the internal SHI residues were removed by deletion of three C-terminal portions (28, 16, or 11 residues). Surprisingly, peroxisome targeting was still abolished when SHI (or SHL produced by site-directed mutagenesis) were at the extreme C-terminus as a consequence of deleting eight residues. However, when SHL was at the C-terminus in full-sized Rcat via a mutation of ANL-COOH, the enzyme sorted to peroxisomes indicating that the position of the PTS is significant in Rcat. The importance of the internal context of the SHI (or SHL) was examined further by changing ANL-COOH to a non-SKL motif, AGS-COOH. This Rcat did not sort to the peroxisomes, nor did Rcat with its ANL-COOH deleted; these data indicated the necessity of the C-terminal tripeptide. Sufficiency of ANL was demonstrated when chloramphenicol acetyltransferase with an appended ANL-COOH was redirected from the cytosol to peroxisomes. Collectively, these results do not support the internal PTS hypothesis, but indicate that a type 1 PTS slightly divergent from the typical SKL motif serves as the necessary and sufficient PTS for rat liver and probably other eukaryotic catalases.

Animals↗
PubMed1996

[Internalization of tumor associated antigen on human lung adenocarcinoma cell line SPC-A-1 by McAb LC-1].

Antigenic modulation of tumor cells is a kind of immunophenomenon that the antigenicity of tumor cell surface antigen could be lessened, weakened or completely lost. It is a potential route for tumor cells to escape the immunosurveillance and immunoattack of the host. One of the means to cause the antigenic modulation is by means of the antibody. In present research, gold labeled monoclonal antibody LC-1, which is raised against human lung cancer in our lab, as a molecular tracer to study the internalization and the subsequent fate of the membrane tumor associated antigen-LC-1 complex on the SPC-A-1 cell surface has been used. We found that this complex was internalized via the receptor-mediated endocytic pathway and concentrated in the multivesicular bodies and transported to lysosomes for proteolysis in the end. Strikingly, we noted that LC-1 had induced the autophagocytosis of ribosomes in SPC-A-1 cells in the median time it induced the internalization of the cell surface antigen cause by internalization and the restoration after antigenic modulation were also analyzed by the fluorescence activated cell sorter (FACS).

Adenocarcinoma↗
PubMed1995

v-src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor.

A consensus cyclic AMP response element (CRE) in the murine prostaglandin synthase-2 (PGS2) promoter is essential for pgs2 gene expression induced by pp60v-src, the v-src oncogene product. In this study, we investigate (i) the transcription factors active at the PGS2 "CRE site" in response to v-src activation and (ii) the signal transduction pathways by which pp60v-src activates these transcription factors. Transient transfection assays with pgs2 promoter/luciferase reporter chimeric genes suggest that c-Jun mediates v-src-induced pgs2 gene expression. Antibody supershift experiments demonstrate that c-Jun can participate in a complex with the pgs2 promoter CRE site. Moreover, in vitro immuno-complex assays demonstrate that pp60v-src expression strongly activates c-Jun N-terminal kinase (JNK1) enzyme activity. Serines 63 and 73, the sites of c-Jun phosphorylation by JNK, are essential for v-src-induced, pgs2 promoter-mediated luciferase expression. Cotransfection studies with plasmids expressing wild-type JNK, dominant-negative JNK, and dominant-negative MEKK-1 confirm that activation of the Ras/MEKK-1/JNK/c-Jun pathway is required for v-src-induced pgs2 gene expression. Overexpression of either wild-type ERK-1 or ERK-2 proteins also potentiate v-src-mediated luciferase expression driven by the pgs2 promoter, and expression of dominant-negative mutants of ERK-1, ERK-2, or Raf-1 attenuate this response. Thus, in response to v-src expression, a Ras/MEKK-1/JNK signal transduction pathway activating c-Jun and a Ras/Raf-1/ERK pathway converge to mediate pgs2 gene expression via the CRE site in the pgs2 promoter.

3T3 Cells↗
PubMed1995

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.

Animals↗
PubMed1995

Inflammation, reproduction, cancer and all that.... The regulation and role of the inducible prostaglandin synthase.

Discovery of a second, inducible prostaglandin synthase provides explanations for many previously puzzling observations, but also raises new questions about prostanoid synthesis. A cis-acting sequence closely related to the cyclic AMP response element has been shown to play a role in both basal and induced prostaglandin synthase 2 gene expression. Aspirin and other currently available non-steroidal anti-inflammatory drugs that inhibit prostaglandin synthase activity do not effectively discriminate between the inducible prostaglandin synthase 2 and constitutive prostaglandin synthase 1 enzymes. Identification of a second prostaglandin synthase, induced by inflammatory stimuli, initiated a search for isoform-specific inhibitors. Use of prostaglandin synthase 2 specific inhibitors and antisense oligonucleotides has led to the suggestion that specific ligands activate alternative pathways of prostanoid production, using one of the prostaglandin synthase isoforms preferentially. The coupling mechanisms by which these pathways are activated in response to alternative stimuli should provide additional routes of intervention in prostanoid production.

Animals↗