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Biomedical subjects

W Xiong

Publications and source records attributed to W Xiong.

At least 73 records · Page 4Linked to original sources
PubMed1997

Role of cyclins in neuronal differentiation of immortalized hippocampal cells.

The proto-oncogene cyclin D1 and the neuron-specific cyclins p35 and p39 are expressed during brain maturation. To investigate the role of these cyclins in neuronal differentiation, we used a conditionally immortalized rat hippocampal cell line, H19-7, that expresses cyclin-dependent kinases 4 and 5 (cdk4 and -5). Cyclin D1, which activates cdk4 and binds but does not activate cdk5, was increased upon differentiation of the H19-7 cells. However, microinjection of either sense or antisense cyclin D1 cDNA or anti-cyclin D1 antibodies had no effect on morphological differentiation of the cells. On the other hand, neurite outgrowth was stimulated by expression of p35 or p39, both of which activate cdk5. A dominant-negative mutant of cdk5 blocked both p35- and p39-induced neurite extension as well as basic fibroblast growth factor (bFGF)-induced neuronal differentiation. However, of these cyclins, only antisense p39 prevented bFGF-induced neurite outgrowth. These studies indicate that cyclin D1 is neither necessary nor sufficient for morphological differentiation, that p35 is sufficient but not required, and that p39 is both necessary and sufficient for neurite outgrowth in the hippocampal cells. Taken together, these results represent the first demonstration of a specific role for p39 in neuronal differentiation, implicate the cyclin-activated kinase cdk5 in this process, and indicate that p39 is able to mediate neurite outgrowth in the presence or absence of cyclin D1.

Animals↗
PubMed1997

Cyclin D1 is required for S phase traversal in bovine tracheal myocytes.

We examined the role of cyclin D1 in cultured bovine tracheal myocyte mitogenesis. Immunoblots using a polyclonal antibody against cyclin D1 showed the appearance of this protein 4 h after treatment with platelet-derived growth factor (PDGF), a potent mitogen for these cells. Immunoblots utilizing an antibody against the 110-kDa retinoblastoma protein (Rb), a downstream phosphorylation target of the cyclin D1/cyclin-dependent kinase 4 (cdk4) complex, showed reduced electrophoretic mobility of this protein as early as 8 h after PDGF treatment, suggesting phosphorylation of Rb by the cyclin D1/cdk4 dimer in vivo. Epidermal growth factor (EGF), a nonmitogen, failed to induce either cyclin D1 synthesis or Rb phosphorylation. PDGF treatment of cells transiently transfected with the full-length cyclin D1 promoter subcloned into a luciferase reporter increased luciferase activity almost threefold, demonstrating transcriptional activation of the cyclin D1 promoter with mitogenic stimulation. Finally, microinjection of individual myocytes with an affinity-purified antibody against cyclin D1 reduced the percentage of cells traversing S phase after serum stimulation, as assessed by fractional bromodeoxyuridine labeling (isotype control antibody, 0.74 +/- 0.10; anti-cyclin D1, 0.22 +/- 0.04; P = 0.0001). We conclude that 1) mitogenic stimulation of cultured bovine tracheal myocytes by PDGF induces cyclin D1 transcriptional activation and protein expression, 2) cyclin D1 expression is accompanied by Rb phosphorylation, which is evidence of increased cyclin D1-associated kinase activity in vivo, and 3) microinjection of anti-cyclin D1 antibodies inhibits cellular DNA synthesis, which is evidence that cyclin D1 is required for airway smooth muscle S phase traversal.

Animals↗
PubMed1997

[Measures for decreasing the early mortality after valvular replacement cardiovascular surgery].

To probe the effective measure for decreasing the early mortality after artificial valvular replacement. We analyzed the cause of death among 1215 patients receiving artificial valvular replacement who were admitted in our intensive care unit during 1990-1995. All 44 deaths were serious-illed patients with preoperative cardiac functions of 3-4 degree. The factors affecting surgical survival rate include: enhancement of patient's cardiac function before operation and prevention of cardiac arrhythmia; a clear operational view and myocardial protection, especially for reoperative patients; application of membrane oxygenator; treatment of LCOS without delay, including intraveously administered agents and IABP used promptly when necessary; prevention and treatment of postoperative cardiac arrhythmia; strict, intensive care and synthesized treatment.

Adolescent↗
PubMed1996

1-O-octadecyl-2-O-methyl-glycerophosphocholine inhibits the transduction of growth signals via the MAPK cascade in cultured MCF-7 cells.

1-O-Octadecyl-2-O-methyl-glycerophosphocholine (ET18-OCH3) is an ether lipid with selective antiproliferative properties whose mechanism of action is still unresolved. We hypothesized that since ET18-OCH3 affects a wide variety of cells, its mechanism of action was likely to involve the inhibition of a common widely used pathway for transducing growth signals such as the mitogen-activated protein kinase (MAPK) cascade. To test this, we established conditions whereby quiescent MCF-7 cells took up ET18-OCH3 in sufficient quantities that inhibited cell proliferation subsequent to the addition of growth medium and examined the activation of components of the MAPK cascade under these conditions. ET18-OCH3 inhibited the sustained phosphorylation of MAPK resulting in a decrease in the magnitude and duration of activation of MAPK in cells stimulated with serum or EGF. ET18-OCH3 had no effect on the binding of EGF to its receptors, their activation, or p21ras activation. However, an interference in the association of Raf-1 with membranes and a resultant decrease in Raf-1 kinase activity in membranes of ET18-OCH3-treated cells was observed. ET18-OCH3 had no direct effect on MAPK or Raf-1 kinase activity. A direct correlation between ET18-OCH3 accumulation, inhibition of cell proliferation, Raf association with the membrane, and MAPK activation was also established. These results suggest that inhibition of the MAPK cascade by ET18-OCH3 as a result of its effect on Raf-1 activation may be an important mechanism by which ET18-OCH3 inhibits cell proliferation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Nck inhibits NGF and basic FGF induced PC12 cell differentiation via mitogen-activated protein kinase-independent pathway.

Proto-oncogene Nck, an adapter molecule containing three SH3 and one SH2 domains, binds to cell surface receptors and mediates mitogenic effects in the cells. Overexpression of Nck caused cell transformation in vitro and tumor formation in the nude mice. The mechanism of this action by Nck, however, remained unclear. Rat adrenal pheochromocytoma cell line PC12 provides a useful system for studying growth factor-regulated cell proliferation and differentiation. Serum and epidermal growth factor (EGF) stimulate proliferation, whereas nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) cause growth arrest and sympathetic neurite outgrowth in these cells. To study the function of Nck, we generated stable clones of PC12 cells overexpressing the human Nck. We report here that the overexpressed Nck caused continued proliferation of PC12 cells even in the presence of NGF and blocked both the NGF- and bFGF-induced neurite outgrowth. Anti-sense but not sense oligonucleotides to the human Nck resumed the NGF-induced differentiation, indicating the specific inhibitory effect of Nck. Interestingly, Nck did not interfere with the kinetics of NGF- and EGF-stimulated protein tyrosine phosphorylation and the mitogen-activated protein kinase (MAPK) activation, suggesting that Nck inhibited the induced PC12 cell differentiation via a MAPK-independent mechanism. This study has provided a useful system for further understanding the function of Nck.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Wild-type and transactivation-defective mutants of human immunodeficiency virus type 1 Tat protein bind human TATA-binding protein in vitro.

SUMMARY: Tat regulates human immunodeficiency virus type 1 (HIV-1) gene expression by increasing both the rate of transcription initiation and the efficiency of transcription elongation. The ability of Tat to facilitate HIV-1 transcription preinitiation complex formation suggests that components of the basal transcriptional machinery may be targeted by Tat. Previous studies have demonstrated that Tat interacts directly with the human TATA-binding protein (TBP) and specific TBP-associated factors (TAFS) that comprise the TFIID complex. Here, in vitro glutathione S-transferase protein binding assays containing fully functional or transactivation-defective mutant Tat proteins have been used to investigate the functional significance of the direct interaction between Tat and TBP relative to Tat transactivation. Results demonstrate that full-length Tat, as well as the activation domain of Tat alone, binds human TBP in vitro. Site-directed mutations within the activation domain of Tat (C22G and P18IS) that abrogate transactivation by Tat in vivo fail to inhibit Tat-TBP binding. Full-length Tat, the activation domain of Tat alone, and a transactivation-defective mutant of Tat that lacks N-terminal amino acid residues 2-36 bind with equal efficiencies to TBP provided that the H1 alpha helical domain that maps to amino acids 167-220 within the highly conserved carboxyl terminus of TBP is maintained. These data indicate that an activity mapped within the activation domain of Tat, which is distinct from Tat-TBP binding. is required for transactivation by Tat.

Binding Sites↗
PubMed1996

Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain.

Nucleoside transport may be involved in the regulation of extracellular levels of adenosine, an inhibitory neuromodulator in the central nervous system. Previous reports have provided functional evidence for Na+-dependent nucleoside transport in rat brain. We isolated total RNA from various regions of rat brain and tested for the presence of mRNA for two recently cloned Na+/nucleoside cotransporters using reverse transcriptase PCR (RT-PCR). Messenger RNA for a pyrimidine-selective Na+/nucleoside cotransporter mRNA (rCNT1) was detected in samples from each brain region tested by RT-PCR amplification of a 309-bp DNA product. Southern blot and sequence analysis confirmed that this product was derived from rCNT1 mRNA. A purine-selective Na+/nucleoside cotransporter mRNA (rCNT2, also termed SPNT) was detected throughout brain by amplifying a 235-bp DNA product, the sequence of which was identical to that published. These experiments demonstrate the presence of both rCNT1 and rCNT2 mRNA in rat brain.

Animals↗
PubMed1995

Muscle delivery of human kallikrein gene reduces blood pressure in hypertensive rats.

We recently found that transgenic mice expressing human tissue kallikrein develop sustained hypotension. The result suggests that a continuous supply of human tissue kallikrein could have a prolonged effect on blood pressure reduction. In the present study, we investigated the potential of using human tissue kallikrein for gene therapy by injecting a kallikrein gene construct into the skeletal muscle of spontaneously hypertensive rats. Expression of the human tissue kallikrein messenger RNA in spontaneously hypertensive rats was identified by reverse transcription-polymerase chain reaction with Southern blot. Human tissue kallikrein was detected in the injected animals by an enzyme-linked immunosorbent assay. Injection of the human kallikrein gene into spontaneously hypertensive rats caused a significant reduction of systemic blood pressure, ranging from 15 to 26 mm Hg, compared with the control group. The differences were significant 1 week after the injection and continued for more than 2 months. Blood pressure reduction could be reversed after the administration of the bradykinin antagonist Hoe 140. The results indicate that somatic delivery of the human tissue kallikrein gene induces a sustained reduction of systemic blood pressure in spontaneously hypertensive rats. The present study raises the possibility of applying kallikrein gene therapy to the treatment of human hypertensive diseases.

Animals↗
PubMed1995

[Effect of calmodulin antagonist on chemosensitivity of primary cultured lung cancer cells].

The effect of calmodulin antagonist trifluoperazine (TFP) on in vitro drug sensitivity of primary cultured lung cancer cells was studied in 28 cases with MTT assay. TFP was found to enhance significantly the anticancer activities of VCR, ADR and VP16 (P < 0.01 or < 0.05). But TFP could not sensitize tumor cells to DDP. TFP may therefore be useful as an adjunct in chemotherapy of lung cancer.

Adenocarcinoma↗
PubMed1994

Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells.

Interferons (IFNs) exert antiproliferative effects on many types of cells. The underlying molecular mechanism, however, is unclear. One possibility is that IFNs block growth factor-induced mitogenic signaling, which involves activation of Ras/Raf-1/MEK/mitogen-activated protein kinase. We have tested this hypothesis by using HER14 cells (NIH 3T3 cell expressing both platelet-derived growth factor [PDGF] and epidermal growth factor [EGF] receptors) as a model system. Our studies showed that IFNs (alpha/beta and gamma) blocked PDGF-and phorbol ester- but not EGF-stimulated DNA synthesis and cell proliferation. While the ligand-stimulated receptor tyrosine phosphorylation and interaction with downstream signaling molecules, such as GRB2, were not affected, IFNs specifically blocked PDGF- and phorbol ester- but not EGF-stimulated activation of Raf-1, mitogen-activated protein kinases, and tyrosine phosphorylation of an unidentified 34-kDa protein. This inhibition could be detected as early as 5 min after IFN treatments and was insensitive to cycloheximide, indicating that de novo protein synthesis is not required. The IFN-induced inhibition acted upstream of Raf-1 kinase and downstream of diacyl glycerol/phorbol ester, suggesting that protein kinase C (PKC) is the potential primary target. Consistently, downregulation of PKC by chronic phorbol myristate acetate treatment or inhibition of PKC by H7 and staurosporine blocked PDGF- and phorbol myristate acetate- but not EGF-induced signaling and DNA synthesis. Moreover, incubating cells with antisense oligodeoxyribonucleotides of PKC delta eliminated production of PKC delta protein and specifically blocked PDGF- but not EGF-stimulated mitogenesis in these cells. Thus, these studies have elucidated a major difference in the early events of EGF-and PDGF-stimulated signal transduction and, more importantly, revealed a novel mechanism by which IFNs may execute their antiproliferative function.

3T3 Cells↗
PubMed1994

Human tissue kallikrein induces hypotension in transgenic mice.

We investigated the role of the kallikrein-kinin system in blood pressure control by developing transgenic mice overexpressing human tissue kallikrein. Two lines of transgenic mice carrying the human tissue kallikrein gene under the control of the mouse metallothionein metal-responsive promoter were established. Human tissue kallikrein was identified in pancreas, salivary gland, kidney, liver, and spleen of the transgenic mice by a specific radioimmunoassay for human tissue kallikrein. The immunoreactive human tissue kallikrein reached high levels in the circulation. The linear displacement curves for the transgenic product were parallel with the human tissue kallikrein standard curve, indicating their immunologic identity. The expression of human tissue kallikrein transcript in the transgenic mice was further confirmed by Northern blot analysis and by reverse transcription-polymerase chain reaction followed by Southern blot. Both lines of transgenic mice had significantly lowered blood pressure (86.4 +/- 13.5 mm Hg [mean +/- SD], n = 8 and 78.9 +/- 12.4 mm Hg, n = 8) compared with control mice (100.9 +/- 5.0 mm Hg, n = 8). Induction with zinc did not lower the blood pressure further despite elevated expression of the transgene. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored the blood pressure of the transgenic mice but had no significant effect on control littermates. Our findings raise the possibility of tissue kallikrein being a powerful modulator of blood pressure and provide a new animal model for the study of blood pressure regulation.

Animals↗
PubMed1994

Family-based intervention for schizophrenic patients in China. A randomised controlled trial.

BACKGROUND: We developed and evaluated a comprehensive, ongoing intervention for families of schizophrenic patients appropriate for China's complex family relationships and unique social environment. METHOD: Sixty-three DSM-III-R schizophrenic patients living with family members were enrolled when admitted to hospital and randomly assigned to receive standard care or a family-based intervention that included monthly 45-minute counselling sessions focused on the management of social and occupational problems, medication management, family education, family group meetings, and crisis intervention. RESULTS: At 6, 12, and 18-month follow-ups by blind evaluators, the proportion of subjects rehospitalised was lower, the duration of rehospitalisation was shorter, and the duration of employment was longer in the experimental group than in the control group; these differences were statistically significant at the 12 and 18-month follow-ups and were not explained by differences in drug compliance. Family intervention was associated with significantly lower levels of family burden. CONCLUSIONS: This intervention is less costly than standard treatment, is suitable for urban families of schizophrenic patients in China and feasible given the constraints of the Chinese mental health system.

Adolescent↗
PubMed1992

Transgenic mice expressing full-length human apolipoprotein B-100. Full-length human apolipoprotein B mRNA is essentially not edited in mouse intestine or liver.

Apolipoprotein (apo) B-100 mRNA is edited in the small intestine (in all mammals examined) and the liver (in mice and rats only) to produce apoB-48 mRNA. ApoB mRNA editing involves a C-->U conversion of the first base of the codon CAA for Gln-2153 in apoB-100, changing it to an in-frame stop codon (UAA). The edited mRNA encodes apoB-48, which is colinear with the N-terminal 48% of apoB-100. ApoB mRNA editing can be reproduced in vitro using cellular extracts from one species to edit synthetic apoB mRNA sequences from a different species. Editing of transcripts from transfected genes also appears not to be species-specific. We have produced transgenic mice that express full-length human apoB-100 mRNA at high levels in the liver and small intestine. Human apoB-100 (a 550-kDa protein) but not apoB-48 (a 260-kDa protein) is detected in total plasma (at approximately 22 mg/dl) and in very low density and low density lipoproteins. The endogenous mouse plasma apoB concentration is reduced by approximately 45% in the transgenic animals. Thus, the transgenic mice form an animal model for familial hyperapolipoprotein B, an inherited form of hyperlipidemia. To our surprise, we found that the full-length human apoB mRNA consists of > 99% apoB-100 mRNA in both the liver and small intestine; < 1% of edited (apoB-48) mRNA was detected. The proportions of endogenous mouse apoB-48 (edited) mRNA (60 and 90% in the liver and small intestine, respectively) were identical in transgenic mice and their nontransgenic littermates. Therefore, full-length human apoB mRNA is resistant to editing by the mouse editing enzyme in vivo; the unchanged proportion of endogenous mouse apoB-48 mRNA in the transgenic mice suggests that the human mRNA competes poorly with the mouse sequence for interacting with the editing enzyme. This observation has implications for the sequence specificity and mechanism of RNA editing. Furthermore, we should exercise caution in the interpretation of in vitro RNA-editing experiments.

Animals↗
PubMed1992

Substrate specificities of tissue kallikrein and T-kininogenase: their possible role in kininogen processing.

The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the Km values of T-kininogenase while Val at P'2 increases its kcat values.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

In vivo catabolism of human kallikrein-binding protein and its complex with tissue kallikrein.

We recently identified and purified a novel human kallikrein-binding protein (HKBP) from human plasma. The HKBP forms a 92 kd sodium dodecyl sulfate-stable and heat-stable complex with tissue kallikrein. This study was undertaken to characterize the plasma clearance and tissue distribution of exogenously administered HKBP and its complex with tissue kallikrein. Human tissue kallikrein was first incubated with purified HKBP, and the high-molecular-weight complex was separated from unbound proteins on a high-pressure liquid chromatography gel filtration column. Tissue kallikrein, kallikrein-binding protein, and their complex were labeled with iodine-125 and then injected intravenously into Sprague-Dawley rats. The disappearance rates of trichloracetic acid-precipitable radioactivity from the circulation were determined. The clearance profile of HKBP shows a nonlinear pattern with an apparent half-life of 65 minutes (n = 4). The plasma clearance of HKBP complexed with kallikrein shows a similar profile but a shorter half-life of 33 minutes (n = 3). HKBP and its complex with kallikrein were mainly taken up by the liver but to a lesser degree by the kidney, lung, and other tissues. Labeled human kallikrein has an apparent half-life of 8 minutes (n = 4), and its clearance consists of a fast and a slow component. The data indicate that kallikrein-HKBP complex is cleared from the circulation two times faster than that of the binding protein alone and that it persists in the circulation four times longer than kallikrein alone. The results support the notion that more than one pathway exists for the metabolism of tissue kallikrein and that HKBP plays a role in modulating tissue kallikrein's bioavailability.

Animals↗