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X Qian

Publications and source records attributed to X Qian.

At least 73 records · Page 4Linked to original sources
PubMed1999

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

Animals↗
PubMed1999

Preparation of an IMI dye (imidazole functional group) containing a 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole fluorophore for labeling of phosphomonoesters.

We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.

Electrophoresis↗
PubMed1999

Domain-specific interactions between the p185(neu) and epidermal growth factor receptor kinases determine differential signaling outcomes.

We expressed the epidermal growth factor receptor (EGFR) along with mutant p185(neu) proteins containing the rat transmembrane point mutation. The work concerned the study of the contributions made by various p185(neu) subdomains to signaling induced by a heterodimeric ErbB complex. Co-expression of full-length EGFR and oncogenic p185(neu) receptors resulted in an increased EGF-induced phosphotyrosine content of p185(neu), increased cell proliferation to limiting concentrations of EGF, and increases in both EGF-induced MAPK and phosphatidylinositol 3-kinase (PI 3-kinase) activation. Intracellular domain-deleted p185(neu) receptors (T691stop neu) were able to associate with full-length EGFR, but induced antagonistic effects on EGF-dependent EGF receptor down-regulation, cell proliferation, and activation of MAPK and PI 3-kinase pathways. Ectodomain-deleted p185(neu) proteins (TDelta5) were unable to physically associate with EGFR, and extracellular domain-deleted p185(neu) forms failed to augment activation of MAPK and PI 3-kinase in response to EGF. Association of EGFR with a carboxyl-terminally truncated p185(neu) mutant (TAPstop) form did not increase transforming efficiency and phosphotyrosine content of the TAPstop species, and proliferation of EGFR.TAPstop-co-expressing cells in response to EGF was similar to cells containing EGFR only. Thus, neither cooperative nor inhibitory effects were observed in cell lines co-expressing either TDelta5 or TAPstop mutant proteins. Unlike the formation of potent homodimer assemblies composed of oncogenic p185(neu), the induction of signaling from p185(neu).EGFR heteroreceptor assemblies requires the ectodomain for ligand-dependent physical association and intracellular domain contacts for efficient intermolecular kinase activation.

Animals↗
PubMed1999

Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes.

The protein sequence encoded by a creatine transporter cDNA cloned from a human heart library was identical to that cloned from a human kidney library (Nash et al., Receptors Channels 2, 165-174, 1994), except that at position 285 the former contained an Ala residue and the latter contained a Pro residue. Expression of this human heart cDNA clone in Xenopus laevis oocytes induced a Na+- and Cl--dependent creatine uptake activity that saturated with a Km of approximately 20 microM for creatine. The induced uptake was inhibited by beta-guanidinopropionic acid (IC50 approximately 44.4 microM), 2-amino-1-imidazolidineacetic acid (cyclocreatine; IC50 approximately 369.8 microM), gamma-guanidinobutyric acid (IC50 approximately 697.9 microM), gamma-aminobutyric acid (IC50 approximately 6.47 mM), and amiloride (IC50 approximately 2.46 mM). The inhibitors beta-guanidinopropionic acid, cyclocreatine, and gamma-guanidinobutyric acid also inhibited the uptake activity of the Ala285 to Pro285 (A285P) mutant as effectively as that of the wild type. In contrast, guanidinoethane sulfonic acid, a potent inhibitor of taurine transport, inhibited the uptake activity of the A285P mutant approx. two times more effectively than that of the wild type. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not its inactive analog, 4alpha-phorbol 12, 13-didecanoate, inhibited the creatine uptake, and the inhibitory effect of PMA was both time and concentration dependent. The protein kinase A activator 8-bromo-cyclic AMP, however, had no effect on the creatine uptake. The rate of uptake increased hyperbolically with the increasing concentration of the external Cl- (equilibrium constant KCl- approximately 5 mM) and sigmoidally with the increasing concentration of the external Na+ (equilibrium constant KNa+ approximately 56 mM). Further analyses of the Na+ and Cl- concentration dependence data suggested that at least two Na+ and one Cl- were required to transport one creatine molecule via the creatine transporter.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers.

p27kip1 (p27) is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. p27 expression is regulated by cell contact inhibition and by specific growth factors, such as transforming growth factor (TGF)-beta. Since the cloning of the p27 gene in 1994, a host of other functions have been associated with this cell cycle protein. In addition to its role as a CDKI, p27 is a putative tumor suppressor gene, regulator of drug resistance in solid tumors, and promoter of apoptosis; acts as a safeguard against inflammatory injury; and has a role in cell differentiation. The level of p27 protein expression decreases during tumor development and progression in some epithelial, lymphoid, and endocrine tissues. This decrease occurs mainly at the post-translational level with protein degradation by the ubiquitin-proteasome pathway. A large number of studies have characterized p27 as an independent prognostic factor in various human cancers, including breast, colon, and prostate adenocarcinomas. Here we review the role of p27 in the regulation of the cell cycle and other cell functions and as a diagnostic and prognostic marker in human neoplasms. We also review studies indicating the increasingly important roles of p27, other CDKIs, and cyclins in endocrine cell hyperplasia and tumor development.

Cell Cycle↗
PubMed1999

Apoptosis in nontumorous and neoplastic human pituitaries: expression of the Bcl-2 family of proteins.

Analyses of apoptosis and of the apoptosis regulatory proteins Bcl-2, Bax, Bcl-X, and Bad were done in 95 nontumorous and neoplastic pituitary tissues by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), immunohistochemistry, and Western blotting. The apoptotic index was relatively low in all groups but was at least fourfold higher in pituitary carcinomas compared with any other groups. Pituitaries from pregnant and postpartum women had a fivefold higher apoptotic index compared with matched controls from nonpregnant females. Preoperative treatment of adenomas with octreotide or dopamine agonists did not change the apoptotic index significantly. The lowest levels of Bcl-2, Bax, and Bcl-X expression were in pituitary carcinomas as detected by immunostaining. An immortalized human pituitary adenoma cell line, HP75, developed in our laboratory using a replication-defective recombinant human adenovirus with an early large T-antigen, had a much higher level of apoptosis than nontumorous and neoplastic pituitaries. Treatment with transforming growth factor (TGF)-beta1 and protein kinase C (PKC) inhibitors increased apoptosis in this cell line. Analysis of the Bcl-2 family of proteins after treatment with TGF-beta1 and PKC inhibitors showed a 20% to 30% decrease in Bcl-X in the treated groups compared with controls. These results, which represent the first study of apoptosis in pituitaries from pregnant and postpartum cases and in pituitary carcinomas, indicate that 1) the apoptotic rate is low in nontumorous and neoplastic pituitary tissues but is relatively higher in pituitary carcinomas, 2) there are alterations in the expression of the Bcl-2 family of proteins in pituitary neoplasms with a decrease in Bcl-2 expression in pituitary carcinomas that may contribute to pituitary tumor pathogenesis and/or proliferation, and 3) cultured pituitary tumor cells respond to TGF-beta1 and PKC inhibitors by undergoing apoptotic cell death.

Adult↗
PubMed1999

Inflammatory modulation of calcium-activated potassium channels in canine colonic circular smooth muscle cells.

BACKGROUND & AIMS: The characteristics of colonic circular smooth muscle slow waves are altered during inflammation. The aim of this study was to examine whether inflammation modulates the open-state probability of Ca2+-activated K+ (KCa) channels in these cells to contribute to these alterations. METHODS: The experiments were performed on freshly dissociated single smooth muscle cells from the canine colon using standard patch clamp methods. Inflammation was induced by mucosal exposure to ethanol and acetic acid. RESULTS: Inflammation decreased the open-state probability of large-conductance KCa (BK) channels in the cell-attached and excised inside-out configurations. The voltage sensitivity of the channels was also reduced during inflammation. Inflammation had no significant effect on the large, medium, and small conductances or the unitary current levels of channel openings. However, it decreased the maximum number of simultaneous channel openings. The channels were Ca2+-dependent and were blocked by tetraethylammonium and charybdotoxin in normal and inflamed cells. CONCLUSIONS: Inflammation decreases the open-state probability of BK channels. This may partially reverse the decrease in duration and amplitude of slow waves and depolarization of membrane potential seen in inflammation.

Animals↗
PubMed1999

Quantitative studies on structure--activity relationship of sulfonylurea and benzoylphenylurea type pesticides and their substituents' bioisosterism using Synthons' activity contribution.

With the Free-Wilson mathematical model method in its Fujita-Ban variant, the quantitative structure-activity relationships of some famous pesticides, their substituents' quantitative bioisosterism sequence, and their physicochemical meaning analysis are studied. In the case of sulfonylurea herbicides, the heterocycle connected with the amino group of the urea part was found to play an important role in inhibition of rape rooting, which showed positive activity contribution accounted for by the highest occupied orbital energy E(HOMO) and the lowest unoccupied orbital energy E(LUMO) compared with the negative contribution of the substituents on the sulfonylphenyl ring. In the case of benzoylphenylurea type insect-growth regulators, the substituent X(1), at the benzoyl moiety, was found to play an important role in the inhibition of insects' chitin synthesis, which gave strong positive activity contribution mainly accounted for by electronic sigma and steric Es parameters, whereas the substituent X(2) gave a negative to weakly positive activity contribution mainly resulting from hydrophobic pi.

Animals↗
PubMed1999

Distribution and regulation of proconvertases PC1 and PC2 in human pituitary adenomas.

Pituitary adenomas are members of the family of neuroendocrine cells and tumors which have secretory granules containing chromogranins/secretogranins and other proteins. Pituitary adenomas express the neuroendocrine specific proconvertases PC1 (also known as PC3) and PC2, which are important for the proteolytic processing of chromogranins/secretogranins molecules. We examined the distribution of PC1 and PC2 in primary cultures of 20 pituitary adenomas and analyzed the regulation of the proconvertase mRNAs and proteins by various secretagogues including hypothalamic hormones and phorbol ester to determine the role of PC1 and PC2 in CgA processing in pituitary adenomas. Although PC2 was present in all adenomas, there was a differential distribution of PC1 with PRL adenomas expressing lower levels of PC1 compared to other adenoma types by RT-PCR analysis, in situ hybridization and immunostaining. Treatment of primary cultures of pituitary adenomas with phorbol 12-myristrate 13-acetate (PMA) resulted in an increase in pancreastatin (PST) secretion in most pituitary adenomas and increased PC1 mRNA and protein expression in gonadotroph adenomas, but not in other types of adenomas. Analysis of a human pituitary adenoma cell line, immortalized by recombinant defective adenovirus (HP75), which expressed chromogranin A, FSH, PC1 and PC2 showed that PST was secreted by these immortalized cells. Treatment with TGF beta 1 resulted in an increase in PST secretion and in PC1 mRNA and protein. These results indicate that a) there is a differential distribution of PC1 in human pituitary adenomas with PRL adenomas expressing very little PC1 mRNA and protein and b) that PC1 expression in gonadotropin hormone-producing adenomas is regulated by PMA and TGF beta 1. These findings support the observation that chromogranin A is a substrate for the endoproteinase PC1 in human pituitary adenoma cells.

Adenoma↗
PubMed1999

Sustained mitogen-activated protein kinase activation is induced by transforming erbB receptor complexes.

We used a genetic approach to characterize features of mitogen-activated protein kinase (MAPK) activation occurring as a consequence of expression of distinct erbB receptor combinations in transformed human cells. Kinase-deficient erbB proteins reduced epidermal growth factor (EGF)-induced tyrosine phosphorylation of endogenous Shc proteins and also reduced immediate and sustained EGF-induced ERK MAPK activities in human glioblastoma cells, although basal ERK MAPK activities were unaffected. Basal and EGF-induced JNK and p38 MAPK kinase activities were equivalent in parental cancer cells and EGFR-inhibited subclones. When ectopically overexpressed in murine fibroblasts and human glioblastoma cells, a constitutively activated human EGF receptor oncoprotein (deltaEGFR) induced EGF-independent elevation of basal ERK MAPK activity. Basal JNK MAPK kinase activity was also specifically induced by deltaEGFR, which correlated with increased phosphorylation of a 54-kDa JNK2 protein observed in deltaEGFR-containing cells. The JNK activities in response to DNA damage were comparably increased in cells containing wildtype EGFR or deltaEGFR. Consistent with the notion that transforming erbB complexes induce sustained and unregulated MAPK activities, coexpression of p185(neu) and EGFR proteins to levels sufficient to transform murine fibroblasts also resulted in prolonged EGF-induced ERK in vitro kinase activation. Transforming erbB complexes, including EGFR homodimers, deltaEGFR homodimers, and p185(neu)/EGFR heterodimers, appear to induce sustained, unattenuated activation of MAPK activities that may contribute to increased transformation and resistance to apoptosis in primary human glioblastoma cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Ras-specific exchange factor GRF: oligomerization through its Dbl homology domain and calcium-dependent activation of Raf.

The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other organs, possess an ionomycin-dependent activation of Erk mitogen-activated protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature 376:524-527, 1995; N. P. Fam et al., Mol. Cell. Biol. 17:1396-1406, 1996). Each GRF protein contains a Dbl homology (DH) domain. A yeast two-hybrid screen was used to identify polypeptides that associate with the DH domain of GRF1. In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacent ilimaquinone domain. Deletion analysis verified that the two-hybrid interaction required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interaction, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction. Oligomers between GRF1 and GRF2 were detected in a rat brain extract, and forced expression of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers. Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in oligomer formation, while GRF1 containing the DH cluster mutations formed homo-oligomers with an efficiency similar to that of wild type. Compared to wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH cluster mutant was reduced, while the L263Q mutant was inactive. Both mutants were impaired in their ability to mediate ionomycin-dependent Erk activity in 293T cells. In the absence of ionomycin, 293T cells expressing wild-type GRF1 contained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further increase in the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cells GRF1 mediates at least two pathways for Raf activation: one a constitutive signal that is mainly Ras-dependent, and one an ionomycin-induced signal that cooperates with the constitutive signal without further augmenting the level of GTP-Ras.

3T3 Cells↗
PubMed1999

Estrogen downregulates neuronal nitric oxide synthase in rat anterior pituitary cells and GH3 tumors.

The anterior pituitary gland produces neuronal nitric oxide synthase (nNOS) and nitric oxide regulates secretion of various anterior pituitary hormones. Estrogen has many functions in anterior pituitary cells including stimulation of prolactin (PRL) cell proliferation and secretion of various anterior pituitary hormones. However, the role of estradiol-17beta (E2) in regulating pituitary nNOS expression has not been previously examined. We studied the regulation of nNOS in normal pituitaries, and neoplastic GH3 pituitary tumors in order to analyze the effects of E2 on nNOS in pituitary cells. GH3 tumors expressed higher levels of nNOS proteins compared to normal pituitaries. Estrogen downregulated nNOS mRNA and protein in both estrogen-treated pituitaries with PRL cell hyperplasia and in GH3 tumors implanted into the flank of rats treated with E2 in silastic tubing. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated three alternatively spliced nNOS transcript isoforms--nNOSa, nNOSb, and nNOSc mRNAs--with distinct 5' untranslated first exons that arose from alternative splicing to a common second exon. All three spliced isoforms were found in the normal rat pituitary, whereas nNOSa and nNOSb, but not nNOSc, were expressed in GH3 tumors implanted into Wistar-Furth rats. E2 also downregulated the nNOSa alternative mRNA transcript isoform in vivo. These results indicate that the biological activity of nNOS in the normal rat anterior pituitary and in pituitary tumors is regulated by a complex pattern of alternative splicing and that some of these mRNA isoforms as well as nNOS protein are regulated by estrogen. Our results also indicate that the levels of nNOS and the alternatively spliced nNOS transcript between normal and GH3 pituitary tumors are different.

Animals↗
PubMed1999

[Capillary free zone electrophoresis of globin chains].

OBJECTIVE: To introduce a new technique for rapid separation of globin chains from clinical samples. METHODS: The authors used the uncoated capillary and the electrophoresis buffer which contained 100mmol/L NaH2PO4 +7mmol/L urea +1% Triton X-100 +1% 2-mercaptoethanol +0.25% PEG-600. Capillary free zone electrophoresis was employed in analyzing the hemoglobin-water solutions which were made from HbA control and 74 clinical blood samples. The globin chains were separated from the samples. RESULTS: IN 40 minutes, five globin chains: alpha, beta, Ggamma, delta, Agamma were separated clearly by capillary free zone electrophoresis using nanolitres of sample. In electrophoresis figure the baselines were stable and the peaks were clear. The time coefficient of variation (CV) was under 3%, and the area CV below 4%. Also, The authors measured the relative concentration of globin chains from 60 normal cases and 14 thalassemia patients. CONCLUSION: The capillary free zone electrophoresis for the separation of globin chains from clinical samples has been successfully achieved. It is a rapid, simple and sensitive technique suitable for rapid diagnosis of hemoglobinopathies.

Adult↗
PubMed1999

Non-invasive assessment of myocardial risk and infarct area in canine model of myocardial reperfusion by intravenous contrast echocardiography.

OBJECTIVE: To evaluate the newly developed perfluoropropene-filled echo-contrast agent (FCT-188) in non-invasive assessment of risk areas (RA) and infarct areas (IA) with intravenous myocardial contrast echocardiography (MCE) in canine model of ischemia followed by reperfusion. METHODS: Eight chest-opened Beagle dogs with a 90-minute ischemia followed by a 240-minute reperfusion were studied. MCE was performed after a bolus injection of FCT-188 (0.025 ml/kg, i.v.) into a superficial vein of the forelimb at baseline, 20 minutes after occlusion, and 4 h after reperfusion to non-invasively assess the left ventricular myocardium area (LVMA), myocardial ischemic risk area (RA), and infarct area (IA) in a short-axis view of left ventricle. The accuracy of detecting myocardial perfusion with intravenous MCE was further assessed by in vitro myocardial staining of the matched cross-sections. Both RA and IA were expressed as percent of LVMA. RESULTS: LVMA, RA, IA, and IA/RA ratio were accurately assessed by MCE (LVMA: 6.60 cm2 +/- 0.76 cm2; RA: 35.7% +/- 6.68%; IA: 21.0% +/- 13.2%; IA/RA: 60.3% +/- 31.4%; n = 7) as compared with those of the matched cross-section (LVMA: 6.81 cm2 +/- 0.73 cm2, P = 0.062; RA: 35.3% +/- 9.9%, P = 0.84; IA: 25.10% +/- 14.5%, P = 0.07; IA/RA: 68.0% +/- 22.2%, P = 0.28, respectively). There was a significant correlation of MCE-assessed IA/RA ratio and its corresponding pathologically determined finding in vitro (Y = 1.21 x -21.6, r = 0.73, P = 0.015). No significant changes of electrocardiogram (ECG), mean artery pressures (MAP), pulmonary artery pressures (PAP), and pulmonary artery wedge pressures (PAWP) were found between pre- and post-intravenous injection of FCT-188 at each time point. CONCLUSION: These indicate that FCT-188 can be used to assess risk areas and infarct areas accurately and non-invasively with intravenous MCE in the canine model of a 90-minute ischemia followed by a 240-minute reperfusion and might have potential significance for non-invasive assessment of myocardial reperfusion clinically.

Animals↗
PubMed1999

[The application of Apo-1/Fas to evaluate apoptosis of myocardial cells in patients with congestive heart failure].

OBJECTIVE: To study the changes of serum level of Apo-1/Fas in patients with congestive heart failure (CHF) and evaluate apoptosis of failing myocardial cells. METHODS: Strepavidin-Biotin ELISA was used to determine serum level of Apo-1/Fas, interleukin-6 (IL-6) and tumor necrosis factor (TNF alpha) in 60 patients with CHF. Cardiac ejection fraction (EF) of the patients were measured by acusson 128XP/10 echocardiograph. RESULTS: Serum levels of Apo-1/Fas and TNF alpha in class III and IV patients with CHF were significantly higher than those in class I and II (P < 0.01). Serum levels of IL-6 in all the patients were obviously higher than those in controls (P < 0.05 and 0.01) and the levels in class III and IV patients were significantly higher than those in class I and II (P < 0.05). Serum levels of Apo-1/Fas in patients with EF < 55 percent were higher than in those with EF > 55 percent (P < 0.05). CONCLUSION: Serum level of Apo-1/Fas in patients with CHF reflects a state of apoptosis in failing myocardial cells. IL-6 and TNF alpha have important effects on immune regulation of myocardial cell apoptosis.

Adult↗
PubMed1999

[Theory and application of affinity capillary electrophoresis].

Affinity capillary electrophoresis (ACE) is a technique for studying receptor-ligand interactions. ACE experiments are based on the different values of electrophoretic mobility of free and bound receptor. ACE can be introduced to many capillary electrophoresis models, such as CZE, MECC, CIEF, CGE etc. It's applications include immunoassays, determining the binding constants of formed complexes, screening libraries for lead compounds, sequence-specific and base composition-specific recognition of oligodeoxy-nucleotide etc.

Animals↗