Biomedical subjects

W Xiong

Publications and source records attributed to W Xiong.

At least 55 records · Page 3Linked to original sources

PubMed1999

Sodium butyrate-mediated induction of the glycoprotein hormone alpha-subunit gene: requirement for continued protein synthesis, identification of a butyrate-responsive element, and inhibition of promoter activation by 2-deoxyglucose.

Production of the glycoprotein hormone alpha-subunit (GPH alpha) was enhanced by sodium butyrate (Btr) in HeLa cells. Induction of the HeLa alpha-subunit gene by Btr was inhibited by the simultaneous addition of cycloheximide (CHX), indicating a requirement for continued protein synthesis. Transient expression assays using plasmids containing the GPH alpha gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the GPH alpha promoter is inducible by Btr in HeLa cells, and this induction could be prevented by 2-deoxyglucose (dGlc). CAT production driven by the SV-40 early promoter, the cytochrome P-450-IA1 promoter, and the Rous sarcoma virus long terminal repeat was also enhanced by Btr, but the augmented synthesis was not inhibited by the addition of dGlc, demonstrating that the effect is restricted only to some promoters. CAT synthesis could be induced by Btr when the GPH alpha promoter extended upstream to position -169 (relative to the transcription start site at +1) but not when the promoter terminated at -150, classifying the DNA between these termini as a Btr-responsive element (BRE). This region overlaps the composite trophoblast-specific enhancer. Inactivation of enhancer subdomains by site-directed mutagenesis confirmed the deletion analysis and ranked their response to Btr as CRE < TSE < URE < alpha ACT. Electrophoretic mobility shift analysis failed to detect any significant difference among several enhancer binding proteins in nuclear extracts from untreated and Btr-treated cells. Together, these results suggest that Btr-mediated induction of the alpha-subunit gene in HeLa cells is manifest either through the synthesis of a new transcription factor(s), which is inhibited by CHX but required for increased transcription from the GPH alpha gene promoter, or through the activity of existing factors that may require glycosylation or phosphorylation by a modification system that is inducible by Btr and inhibited by dGlc and CHX. These results further suggest that the factor is not an enhancer-binding protein or that Btr increases its transactivation potential without altering its DNA-binding activity.

Base Sequence↗

PubMed1999

Influence of a cap site element on tissue-restricted expression of the glycoprotein hormone alpha-subunit gene.

Little is known of the transcriptional regulators important for expression of the glycoprotein hormone alpha-subunit (GPHalpha) gene in nonendocrine tumors, which secrete free alpha-subunit at an incidence of 25-80%. Consequently, attempts were made to define cis-regulatory elements and their cognate trans-acting factors that modulate promoter activity in epithelial cell types that do not normally express the glycoprotein hormones. DNA-mediated transient expression of promoter-reporter constructs was used to identify a novel negative regulatory element located at the GPHalpha gene transcription start site. Mutagenesis of this element produced a 2- to 10-fold increase in promoter activity, depending on the particular mutation and the transfected tumor cell line. Electrophoretic mobility shift analysis detected a protein that binds specifically to a DNA motif encompassing the cap site. It was present at different levels in a variety of cell types. Significantly, the degree to which activity of the wild-type promoter was suppressed relative to that of the mutant promoter was proportional to the level of cap site binding protein in the collection of cell lines examined. These results indicate that a negative regulatory element centered at the GPHalpha gene cap site and its cognate DNA-binding protein make a significant contribution to the production of alpha-subunit in a variety of tumor tissues. A detailed understanding of this cis/trans pair may further suggest a mechanism to explain, at least in part, how this gene becomes activated in nonendocrine tumors.

Binding, Competitive↗

PubMed1999

A proline- and glutamine-rich protein promotes apoptosis in neuronal cells.

During development, excess neurons are eliminated by programmed cell death. Similarly, conditionally immortalized (SV40-Tts) rat hippocampal and septal cells undergo cell death following differentiation with several factors such as fibroblast growth factor, constitutively activated Raf-1, or phorbol esters. The mechanism by which cell death occurs has not been identified. Using RNA differential display, we have identified and characterized a novel immediate early gene (denoted PQR for proline- and glutamine-rich) induced during differentiation of both rat hippocampal and septal cell lines. The 44-kDa PQR protein, rich in PQ, PH, and QQ repeats, is homologous to a murine protein (TDAG51) required for Fas-mediated apoptosis in T cells. To determine whether PQR acts as a mediator of apoptosis in neuronal cells, the hippocampal H19-7 cells were microinjected with either a plasmid expressing PQR cDNA or an antibody against PQR. Microinjection of differentiating H19-7 cells with a neutralizing antibody against PQR increased the number of surviving cells by 50%. Transient expression of PQR in both differentiating and nondifferentiating H19-7 cells decreased the number of surviving cells by 35-50%; this reduction was reversed by microinjection of PQR antibody. Finally, levels of Fas transcripts are not increased in the neuronal cells, indicating that the mechanism of action differs from that in T cells. These results demonstrate that PQR can be induced by growth factors and differentiating agents and can itself induce apoptosis in hippocampal H19-7 cells. Furthermore, these data suggest that PQR can function more generally as a mediator of apoptosis and provide a possible mechanism for induction of programmed cell death during neuronal development.

Animals↗

PubMed1999

Distribution of equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporters (ENT1) in brain.

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [3H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35S-labeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra- and extracellular levels of adenosine in brain.

Adenosine↗

PubMed1999

Recent developments in gene therapy: applications for the treatment of pituitary tumours.

Pituitary tumours are normally benign, highly differentiated and slow growing neoplasms. Nevertheless, as many as half of them will show evidence of local invasion into the surrounding structures. Despite their benign growth characteristics and slow clinical progression, pituitary tumours commonly cause serious morbidity. The mass effects of large tumours, including headache and visual failure from optic chiasm compression, may cause lifelong disability. Hormone hypersecretion or deficiency causes major clinical problems that often require expensive and long-term medical therapy. Major advances have been made in the therapy of pituitary tumours over the past 20-30 years, but despite this, their treatment often remains an unsatisfactory compromise in practice. There is, therefore, a place for improvements in therapy, and to this end, gene therapy may come to hold a significant place in the future treatment of human pituitary tumours. With the development of new gene delivery vehicles, this concept can now be explored with a view to treating specific types of pituitary tumours.

Animals↗

PubMed1999

Role of voltage-sensitive release mechanism in depression of cardiac contraction in myopathic hamsters.

We investigated excitation-contraction (EC) coupling in isolated ventricular myocytes from prehypertrophic cardiomyopathic (CM) hamster hearts. Conventional and voltage-clamp recordings were made with high-resistance microelectrodes, and cell shortening was measured with a video-edge detector at 37 degrees C. Contractions were depressed in myocytes from CM hearts, whether they were initiated by action potentials or voltage-clamp steps. As in guinea pig and rat, contraction in hamster myocytes could be triggered by a voltage-sensitive release mechanism (VSRM) or Ca(2+)-induced Ca(2+) release (CICR). Selective activation of these mechanisms demonstrated that the defect in EC coupling was primarily caused by a defect in the VSRM. However, activation and inactivation properties of the VSRM were not altered. When the VSRM was inhibited, the remaining contractions induced by CICR exhibited identical bell-shaped contraction voltage relations in normal and CM myocytes. Inward Ca(2+) current was unchanged. Thus a defect in the VSRM component of EC coupling precedes the development of hypertrophy and failure in CM hamster heart.

Animals↗

PubMed1999

The roles of receptor abnormalities in the pathogenesis and chronic complications of type 2 diabetes mellitus.

Type 2 diabetes mellitus is a polygenic disorder with complicated biochemical alterations. Numerous investigators have examined the implication of receptor abnormalities in the pathogenesis of the disorder. The authors review the potential roles of some important receptors, such as the insulin receptor, beta 3-adrenergic receptor, leptin receptor and peroxisome proliferator-activated receptor gamma, in the pathogenesis of human type 2 diabetes. They emphasize the significance of effective glycemic control by examining the evidence that strongly suggests the association of chronic complications of type 2 diabetes with abnormalities of receptors for the advanced glycation end products, transforming growth factor-beta and platelet-derived growth factor. The molecular understanding of receptor abnormalities and alterations in postreceptor signalling pathways may not only clarify the pathogenesis of human type 2 diabetes and the development of chronic complications in the disorder but also provide insight into more efficacious drug regimens that target these receptors.

Carrier Proteins↗

PubMed1999

Cloning and expression of truncated PTHrP receptor in Escherichia coli.

To evaluate the effects of recombinant soluble parathyroid hormone related protein receptor(sPTHrPR), the RNA prepared from renal cell carcinoma was reverse-transcribed and amplified by a polymerase chain reaction(PCR). A 543 base pair fragment corresponding to extracellular domain of PTHrPR was obtained and cloned. Sequencing was performed by the dideoxy chain-termination method. Comparison of the nucleic acid sequence with the published data revealed that only one nucleotide had changed. A high level expression vector referred to as pET-3a/sPTHrPR was constructed. The binding specificity of the expression products was proved by indirect ELISA. Our preliminary experiment also showed that the expression products can block the biological activity of PTHrP.

Amino Acid Sequence↗

PubMed1999

[The effect of human cytomegalovirus on human marrow granulocyte/macrophage progenitor].

OBJECTIVE: To investigate whether the suppression of CFU-GM is caused directly by human cytomegalovirus(HCMV). METHODS: The effect of HCMV AD 169 strain on CFU-GM growth was assayed in vitro and HCMV AD 169 DNA in CFU-GM was detected by in situ PCR(ISPCR). RESULTS: Significant inhibition of CFU-GM was observed when HCMV AD 169 containing fluid was diluted to 2 x 10(5) pfu/ml and 2 x 10(6) pfu/ml (P < 0.01). The suppression was in a dose-dependent fashion. HCMV AD 169-DNA was detected by ISPCR on the nuclear of CFU-GM. CONCLUSION: HCMV might cause suppression of CFU-GM growth by direct infection.

Adolescent↗

PubMed1998

Regulation of interleukin 1 signalling through integrin binding and actin reorganization: disparate effects on NF-kappaB and stress kinase pathways.

Interleukin 1 (IL-1)-mediated gene regulation is dependent on cell-matrix interactions. Both IL-1-activated pathways, nuclear factor kappaB (NF-kappaB) and the stress-activated protein kinase (SAPK), can be regulated by cell adhesion and changes in the cytoskeleton, suggesting that cell-matrix effects on IL-1 responses are initiated in part though effects on signal transduction. Here we show that IL-1-induced transient alterations in cell shape and in the cytoskeleton in fibronectin attached cells are correlated with effects on peak activity of NF-kappaB and SAPK. Cells on fibronectin showed a 1.5-2-fold enhancement in IL-1-induced NF-kappaB activity compared with levels in cells on poly(l-lysine) or bare tissue culture plates. The effect was increased with increasing concentrations of fibronectin and was most prominent at lower concentrations of IL-1. In contrast, fibronectin attachment caused an approx. 50% decrease in the IL-1 activation of SAPK, eliminating the peak activity after 15 min of stimulation with IL-1. IL-1-induced NF-kappaB activity showed a successive, substratum-independent increase during 4 h of attachment and spreading, whereas the inhibitory effect of fibronectin on the SAPK pathway was induced at the initial stages of attachment. Further, the addition of a peptide containing the motif RGD resulted in a 40% decrease in NF-kappaB activity in cells on fibronectin, largely accounted for by an effect on the p50/p65 heterodimer. Similarly, blocking of integrin aggregation by RGD-containing peptide resulted in a total abrogation of the fibronectin effect on IL-1-induced SAPK activity. The results demonstrate disparate effects of cell adhesion on the activation by IL-1 of the NF-kappaB and SAPK pathways. Thus fibronectin attachment causes an up-regulation of NF-kappaB activity in the presence of IL-1, whereas in contrast it results in a pronounced decrease in IL-1-induced SAPK activity.

Actins↗

PubMed1998

Expression of anti-CD4 human/murine chimeric antibody and their killer tumor activity.

From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup II (A) and kappa chain subgroup III, respectively. The VH and VL genes were subcloned into p gamma 1-Expr and p kappa-Expr respectively, then transfected into XL2-Blue. The VH- p gamma 1 and VL- p kappa were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specifically in vitro.

Animals↗

PubMed1998

cDNA sequence analysis of monoclonal antibodies against the human placental acidic isoferritin.

By using human placental acidic isoferritin (PAF) as antigen to immunize BALB/c mice and conventional cell fusion, we have established three mouse hybridoma cell lines that secrete IgG monoclonal antibodies (MAbs) to PAF, termed as Z-2-3, Z-2-5, and Z-3-6, respectively. In ELISA, the MAbs were shown to react specifically with human PAF. We then applied the polymerase chain reaction (PCR) technique to clone variable region genes of the heavy (V(H)) and light (V(L)) chains of these MAbs, and appropriate full-length cDNA clones were obtained and characterized by nucleotide sequence analysis. V(H) and V(L) segments of anti-human PAF MAbs belong to the J558 and Vkappa19 family, respectively. The nucleotide sequence of Z-3-6 in the V(H) segment is highly homologous to that of MAb 18.1.16 except for their diversity minigenes. The light chain sequences of these MAbs show high homology with that of MAb cc92. It is implied that the D segment and the nucleotides inserted at the V(H)-D and D-J splice junctions are mostly responsible for the specificity of Z-3-6, and that the differences between the V(H) and V(L) sequences of these MAbs may determine their different affinity or recognition of different antigenic determinants.

Animals↗

PubMed1998

Effects of age on the posttranscriptional regulation of CCAAT/enhancer binding protein alpha and CCAAT/enhancer binding protein beta isoform synthesis in control and LPS-treated livers.

The CCAAT/enhancer binding protein alpha (C/EBPalpha) and CCAAT/enhancer binding protein beta (C/EBPbeta) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa and C/EBPbetas of 35, 20, and approximately 8.5 kDa. The DNA-binding activities and pool levels of p42(C/EBPalpha) and p30(C/EBPalpha) in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPalpha and C/EBPbeta isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42(C/EBPalpha) levels and binding activity, whereas those of p20(C/EBPalpha) and p20(C/EBPbeta) are increased. However, translation of 42-kDa C/EBPalpha is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPalpha- and C/EBPbeta-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPalpha and C/EBPbeta isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

Aging↗

PubMed1998

Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line.

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 Tts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-XL or inhibition of Jun kinase activity.

Androstadienes↗

PubMed1997

Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase.

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase beta (CAKbeta) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFalpha and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH2-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.

Animals↗

PubMed1997

Tissue-specific expression and promoter analyses of the human tissue kallikrein gene in transgenic mice.

The expression of the tissue kallikrein gene is tissue-specific and exhibits a complex pattern of transcriptional and post-translational regulation. Information concerning the mechanism of its tissue-specific expression has been limited owing to the lack of suitable cell lines for the expression study. We approached this problem by introducing human tissue kallikrein gene constructs into mouse embryos, creating transgenic lines carrying its coding sequence with varying lengths of the promoter region. One construct (PHK) contained 801 bp in the 5'-flanking region and two deletion constructs contained either 302 bp (D300) or 202 bp (D200) of the promoter region. The expression of human tissue kallikrein in these transgenic mice was monitored by Northern blot, reverse transcriptase-PCR followed by Southern blot, and radioimmunoassay. In all three lines, human tissue kallikrein was expressed predominantly in the pancreas and at lower levels in other tissues, including salivary gland, kidney and spleen. This pattern was similar to that of tissue kallikrein expression in human tissues. The D300 line has higher levels of transgene expression than the D200 and PHK lines. The results indicate that the 202 bp segment immediately upstream of the translation starting site is sufficient to direct a tissue-specific expression pattern of the human tissue kallikrein gene, and that regulatory elements might exist between -801 and -202.

Animals↗

PubMed1997

Expression and localization of human kallistatin in rat submandibular gland after intracapsular gene injection.

Gene delivery into rat submandibular gland in vivo by direct intracapsular injection has been studied. After the administration of adenovirus constructs, Ad-RSV-LacZ and Ad-CMV-LacZ, beta-galactosidase expression was localized in the granular convoluted tubular and striated duct cells of rat submandibular gland by in situ enzyme histochemistry. Adenovirus-mediated delivery of the human kallistatin gene (Ad-RSV-HKBP) into rat submandibular gland results in the expression of human kallistatin in a time-dependent manner. The expression of immunoreactive kallistatin in submandibular gland was detected 1 day after the Ad-RSV-HKBP injection and it reached a plateau (1-2 ng/mg protein) 2 days after gene delivery. Higher levels of human kallistatin were found in the submandibular gland of 6-month-old rats than in one-month-old rats. After direct gene injection, human kallistatin was localized mainly in cells of the granular convoluted tubules and striated ducts of rat submandibular gland using a specific monoclonal antibody to human kallistatin. The results indicate that direct intracapsular gene delivery into the submandibular gland provides a simple and reliable method for introducing foreign genes into the gland. This method can be used for studying gene regulation in vivo and may have potential for gene therapy in oral diseases.

Animals↗

PubMed1997

Protein kinase C activity and light sensitivity of single amphibian rods.

Biochemical experiments by others have indicated that protein kinase C activity is present in the rod outer segment, with potential or demonstrated targets including rhodopsin, transducin, cGMP-phosphodiesterase (PDE), guanylate cyclase, and arrestin, all of which are components of the phototransduction cascade. In particular, PKC phosphorylations of rhodopsin and the inhibitory subunit of PDE (PDE ) have been studied in some detail, and suggested to have roles in downregulating the sensitivity of rod photoreceptors to light during illumination. We have examined this question under physiological conditions by recording from a single, dissociated salamander rod with a suction pipette while exposing its outer segment to the PKC activators phorbol-12-myristate,13-acetate (PMA) or phorbol-12,13-dibutyrate (PDBu), or to the PKC-inhibitor GF109203X. No significant effect of any of these agents on rod sensitivity was detected, whether in the absence or presence of a background light, or after a low bleach. These results suggest that PKC probably does not produce any acute downregulation of rod sensitivity as a mechanism of light adaptation, at least for isolated amphibian rods.

Ambystoma↗