Theoretical study on Cl L23 NEXAFS and UV absorption data for metal chlorides.
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Journal Article↗
Biomedical subjects
X Qian
Publications and source records attributed to X Qian.
Identification of a 140 kDa protein of rat presynaptic terminal membranes encompassing the active zones.
A polyclonal antiserum raised against the carboxy-terminal 17 amino acids of the rat p185c-neu (anct) reacted with a 140 kDa polypeptide in membranes of synaptosome fractions from neocortex and hippocampus of 11-day-old and adult rats. The same antiserum reacted with a 185 kDa polypeptide in microsome membranes from rat pheochromocytoma cells (PC12). By light microscopic immunocytochemistry, the anct antibodies against the 140 kDa protein were localized in the neuropile of brain, cerebellum and spinal cord of 11-day-old and adult rats. Especially prominent staining was obtained in the CA2-CA3 zones of the hippocampus, and in the substantia gelatinosa in the spinal cord. The finely granular and diffuse pattern of the immunostain was consistent with synaptic localizations. Interestingly, antibodies against the entire endodomain of p185c-neu (a-Bacneu) were localized in granular structures, probably representing axo-somatic and axo-dendritic synapses, on a subset of pyramidal neurons of the CA3 zone. By immunoelectron terminals in the giant mossy fiber type in the CA3 and CA4 regions. The immunolocalization of the anct antibodies was restricted in segments of the presynaptic membrane facing the synaptic cleft which include the active zone. The identify and function of the 140 kDa membrane protein of rat brain presynaptic terminals, detected by the anct antibodies, is unknown. The 140 kDa protein may be related to p185c-neu, a tyrosine kinase, or to other known or unknown kinases.
Amino Acid Sequence↗
Analysis of hepatocyte nuclear factor-3 beta protein domains required for transcriptional activation and nuclear targeting.
Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta and gamma) regulate transcription of the transthyretin (TTR) and numerous other liver-specific genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of developmental regulatory proteins including the Drosophila homeotic fork head (fkh) protein. The mammalian HNF-3/fkh family consists of at least thirty distinct members and is expressed in a variety of different cellular lineages. In addition to the winged helix motif, several HNF-3/fkh family members also share homology within transcriptional activation region II and III sequences. In the present study we further define the sequence boundaries of the HNF-3 beta N-terminal transcriptional activation domain to extend from amino acids 14 to 93 and include conserved region IV and V sequences. We also demonstrate that activity of the HNF-3 N-terminal domain was diminished by mutations which altered a putative alpha-helical structure located between amino acid residues 14 and 19. However, transcriptional activity was not affected by mutations which eliminated two conserved casein kinase I sites or increased the number of acidic amino acid residues in the N-terminal domain. Furthermore, we determined that the nuclear localization signal overlaps with the winged helix DNA-binding motif. These results suggest that conserved sequences within the winged helix motif of the HNF-3/fkh family may be involved not only in DNA recognition, but also in nuclear targeting.
Amino Acid Sequence↗
Intermolecular association and trans-phosphorylation of different neu-kinase forms permit SH2-dependent signaling and oncogenic transformation.
The neu oncogene encodes a 185 kDa receptor tyrosine kinase. A single point mutation (Val664-->Glu) within the p185neu transmembrane region results in higher efficiency of receptor dimerization, constitutive activity of tyrosine kinase and cellular transformation. The oncogenic potential of this mutated form of p185neu (termed Tneu) can be inactivated by site-directed alteration of a lysine residue in the conserved catalytic domain. In this report, we have utilized the physical and functional interaction of a full-length kinase-deficient neu protein (T757) and truncated kinase-active Tneu forms to determine critical protein domains for Tneu oligomerization and the resultant biological consequences. Analysis of various truncated Tneu mutants confirmed that the transmembrane region was crucial for p185 dimerization. Receptor association facilitates intermolecular phosphorylation of kinase-deficient mutant T757 by truncated kinase-active p185 proteins, and the trans-phosphorylated kinase-deficient T757 was able to associate in vitro with proteins containing SH2 domains. Receptor-receptor interactions resulted in enhanced signal transduction potential and transformation of cell-lines co-expressing different neu-kinase forms. These studies emphasize a novel feature of protein-protein interaction and the functional significance of p185 dimerization, intermolecular phosphorylation and signaling which may result in cellular transformation.
Animals↗
bFGF, neurotrophins, and the control or cortical neurogenesis.
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Animals↗
Unexpected transcriptional signals in normal and mitotically defective cells mediated through cytokine and growth factor receptors.
Polypeptide growth factors and cytokines mediate their biochemical functions through their responsive receptors. Known cytokine receptors do not possess intrinsic kinase domains whereas several polypeptide growth factor receptors do. Nevertheless, both classes of ligands are capable of activating sets of overlapping genes. In human epidermoid carcinoma cells, for example, both cytokines and epidermal growth factor (EGF) promote a common transcriptional activation signal through the tyrosine phosphorylation of stat91 (signal transducer and activator of transcription) proteins. The stat family of cytoplasmic proteins also appear to have dual functions. Tyrosine phosphorylated 'stats' are employed for signal transduction and, second, for activation of transcription of several genes. The transcription factor-SIE-DNA binding patterns are now known to be different for EGF and interferon-gamma IFN-gamma-treated cells. Nevertheless, in the active DNA-bound complex, the stat91 polypeptide is a component found in either EGF or INF-gamma-treated extracts. Other stat family members of transcription factors may also be present in the complexes. In this case, tyrosine phosphorylated stat91 polypeptides may form into homodimeric or heterodimeric assemblies with other stat-related transcription factors. We describe a novel stat-related factor, p93, that is found in EGF-treated A431 cell extracts but appears to be absent in bovine fibroblast growth factor (bFGF), IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and untreated cells. p93 appears to be antigenically related to stat91. p185c-neu+, EGFr+ (M1), and p185c-neu- kinase inactive, EGFr+ (NEN757) expressing cells undergo different mitotic responses to EGF. M1 can respond to EGF mitotically while NEN757 cannot. Both cell lines respond to 10 ng/ml of EGF and also to IFN-gamma in transducing transcriptional activation signals to the nucleus, despite the distinct growth response to EGF. Our work has analyzed the stat pathway in these types of cells and found similar patterns of usage despite the distinct EGF-responsive features. Cytoplasmic nonreceptor tyrosine kinases Jak1 and Jak2 may be involved in the activation of stat91 and other transcription factors in EGF and IFN-gamma signaling pathways. Collectively, these studies suggest that the major EGF-stimulated mitotic growth pathways may not be absolutely linked to the stat91 signaling pathways and that such transcription complexes are more complex than previously reported.
Animals↗
Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: correlation with gene suppression.
Adducts of O6-alkylguanine in DNA that are induced by cytotoxic, carcinogenic or mutagenic alkylating agents can be removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Human tumor cell lines that do not express this enzyme (Mer-) are hypersensitive to the effects of such alkylating agents, although the molecular basis of MGMT gene suppression is not yet understood. Previous studies suggested that Mer- cells deficient in this enzyme lack neither the gene nor the trans-acting factors necessary for normal transcription. Methylation of CpG dinucleotides is an attractive mechanism to account for suppression of the MGMT gene; however, there have been reports of both direct and inverse correlations between methylation and MGMT expression. We previously demonstrated an inverse correlation between methylation at a single SmaI site in the human MGMT promoter and gene expression. To substantiate this observation, we examined additional CpGs in the promoters of three Mer+ and three Mer- cell lines, using rare methylation-sensitive restriction sites, and then sought to identify the region where methylation correlated with gene expression. Six CpGs in the region from -245 bp to +225 bp (relative to the transcription start site) were completely unmethylated in all Mer+ cells, whereas in Mer- cells were at least partially methylated. The methylation status of CpGs further upstream did not correlate with MGMT expression. We conclude, therefore, that the association between CpG methylation and suppressed MGMT gene activity extends to sites other than SmaI but is limited to a core region of the promoter.
Cell Line↗
Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription.
Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta, and gamma) are known to regulate the transcription of numerous liver-specific genes. The HNF-3 proteins bind to DNA as monomers through a winged-helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic fork head (fkh) protein. We have previously characterized a strong-affinity HNF-3S site in the transthyretin (TTR) promoter region which is essential for expression in human hepatoma (HepG2) cells. In the current study, we identify an activating protein 1 (AP-1) site which partially overlaps the HNF-3S sequence in the TTR promoter. We show that in HepG2 cells the AP-1 sequence confers 12-O-tetradecanoylphorbol-13-acetate inducibility to the TTR promoter and contributes to normal TTR transcriptional activity. We also demonstrate that the HNF-3 proteins and AP-1 bind independently to the TTR AP-1-HNF-3 site, and cotransfection experiments suggest that they do not cooperate to activate an AP-1-HNF-3 reporter construct. In addition, 12-O-tetradecanoylphorbol-13-acetate exposure of HepG2 cells results in a reciprocal decrease in HNF-3 alpha and -3 gamma expression which may facilitate interaction of AP-1 with the TTR AP-1-HNF-3 site. In order to explore the role of HNF-3 in the liver, we have examined expression patterns of TTR and HNF-3 during the acute-phase response and liver regeneration. Partial hepatectomy produced minimal fluctuation in HNF-3 and TTR expression, suggesting that HNF-3 expression is not influenced by proliferative signals induced during liver regeneration. In acute-phase livers, we observed a dramatic reduction in HNF-3 alpha expression which correlates with a decrease in the expression of its target gene, the TTR gene. Furthermore, consistent with previous studies, the acute-phase livers are induced for c-jun but not c-fos expression. We propose that the reduction in TTR gene expression during the acute phase is likely due to lower HNF-3 alpha expression levels and that the induction of primarily c-jun homodimers, which are poor transcriptional activators, is insufficient to maintain normal TTR expression levels. We also discuss the role of reduced HNF-3 alpha expression in mediating decreased transcription of HNF-3 target genes which respond negatively to cytokine signalling.
Animals↗
Nitric oxide synthase in the human pituitary gland.
Nitric oxide (NO) is generated by the NO synthase family of isozymes, which is present in many mammalian cells. The constitutive NO synthase isozymes generate NO, which acts via signal transduction mechanisms in the regulation of many functions including vascular tone and blood pressure, and the inducible isozymes mediate immunological mechanisms by cytotoxic and cytostatic effects. To determine whether NO has a role in anterior pituitary cell function, immunohistochemistry and in situ hybridization analyses were used to study NO synthase expression in normal and neoplastic human pituitary tissues. Brain NO synthase was localized in the anterior pituitary in secretory and in folliculo-stellate cells and in the posterior pituitary. Pituitary adenomas had higher levels of brain NO synthase protein and mRNA compared with normal pituitaries. Endothelial NO synthase was also present in anterior and posterior pituitary cells and in endothelial cells of the pituitary. Immunoblotting studies with brain NO synthase antibodies detected a slowly migrating approximately 155-kd band and more rapidly migrating approximately 90-kd and approximately 60-kd bands. Endothelial NO synthase, but not macrophage NO synthase, was also detected in the pituitary by immunoblotting studies, confirming the immunohistochemical observations. These findings indicate that NO synthase is expressed in normal and neoplastic human pituitary tissues with increased levels of brain NO synthase protein and mRNA in adenomas compared with non-neoplastic pituitary cells and suggest that NO may play a regulatory role in hormone secretion in anterior pituitary cells.
Adenoma↗
[Surgical management of severe hemorrhage and retroperitoneal perforation after endoscopic sphincterotomy].
From 1990 to 1993, 5 patients with severe hemorrhage and retroperitoneal perforation following endoscopic sphincterotomy (EST) were surgically treated. Two patients with hemorrhage and one with perforation survived. The remaining two died of hemorrhage plus perforation. Early diagnosis and surgical intervention was very important to lower the mortality, especially in those with common bile duct stones retention, acute pancreatitis, or cholangitis. For patients with late stage perforation or recurrent hemorrhage, palliative procedure aimed at isolating the EST incision from the digestion of bile and pancreatic juice should be performed.
Duodenal Diseases↗
Analysis of the chromogranin A post-translational cleavage product pancreastatin and the prohormone convertases PC2 and PC3 in normal and neoplastic human pituitaries.
Several members of the chromogranin/secretogranin (Cg/Sg) family are post-translationally processed in neuroendocrine cells and tumors to smaller peptides, some of which are biologically active. For example, CgA is processed to pancreastatin, parastatin, and other peptides. We analyzed the distribution of pancreastatin and CgA proteins in normal and neoplastic pituitaries as well as the prohormone convertases PC2 and PC3/1 (PC3), the putative processing enzymes for the Cg/Sg family, in 35 pituitary adenomas and 4 non-neoplastic pituitaries by immunohistochemistry and immunoblotting with highly specific antisera. CgA and CgB mRNAs were also examined. Pancreastatin was present in all subtypes of pituitary tumors, although prolactin-secreting adenomas expressed this peptide less frequently than did other tumor types. CgA protein and CgA mRNA expression were also restricted in prolactin adenomas and in normal prolactin cells, as shown by combined in situ hybridization and immunostaining. The prohormone convertases PC2 and PC3 were present in pituitary tumors and in non-neoplastic pituitaries. Immunoblot analysis and immunostaining showed a principal approximately 69-kd PC3 band and a approximately 68-kd PC2 band. Adrenocorticotrophic hormone-secreting adenomas expressed mainly PC3 as determined by immunoblotting and immunohistochemistry, whereas all other adenoma groups expressed predominantly PC2. These results indicate that the enzymes capable of processing CgA and other members of the Cg/Sg family to peptides with biological activity such as pancreastatin are widely expressed in human pituitary adenomas and in non-neoplastic pituitaries, with adrenocorticotrophic hormone tumors expressing predominantly PC3 and other adenomas expressing mainly PC2. The infrequent expression of CgA protein and pancreastatin peptides in normal and neoplastic prolactin cells suggests a unique role of CgA in these tumors.
Adult↗
[Clinical significance of fibrinolytic system defectiveness in exercise induced myocardial ischemia and its mechanism].
Plasma levels of tissue type plasminogen activator (tPA), plasminogen activator inhibitor (PAI), fibrinogen (Fg), thromboxane B2 (TXB2), 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), renin activity (PRA) and angiotensin II (ANGII) were assayed in 18 patients with coronary heart disease and 11 healthy subjects before and after submaximal treadmill exercise test according to "Bruce programme". All patients showed significant (> or = 50%) stenosis of at least one branch of the coronary arteries in coronary angiography and normal levels of serum trinitroglycerin. Drugs of dipyridamole, heparin, warfarin and aspirin were not administered to the patients two weeks before the test. The results were as follows: (1) Plasma PAI activity and Fg levels in 12 patients with positive test were remarkablly higher than in the remaining 6 subjects with negative test in the CHD group and in the control before and after exercise. Plasma tPA antigen changed in a reverse way. (2) Plasma levels of TXB2, PRA and ANGII in the positive test subgroup of CHD were higher than in the negative test subgroup and in the control after exercise, but differences of the parameters among the three groups were insignificant before exercise. The study suggests that defectiveness of fibrinolytic system in CHD patients was shown mainly as disorder of tPA-PAI equilibrium and that decreased fibrinolytic activity and increased PAI in exercise-induced myocardial ischemia have relations with activation of platelets and renin-angiotensin system.
Angiotensin II↗
Epidermal growth factor in acute promyelocytic leukemia treated with retinoic acid.
We studied 18 patients with acute promyelocytic leukaemia and 13 with relapsed APL. We found a significantly elevated EGF in acute leukaemia, especially in APL, being 418.59 +/- 19.2 micrograms in the 24-h urine that was much higher than that of the normal controls. When eight APL patients achieved complete remission by RA treatment, the EGF value decreased to 149.9 +/- 27.3 micrograms in the 24-h urine near to normal. In 13 patients with relapsed APL, EGF rose to 446.9 +/- 82.6 micrograms in the 24-h urine. Most interestingly, this elevated EGF could be detected before the relapse by 5 +/- 0.84 months in seven out of eight APL with relapse. We suggest that the unaccountably elevated EGF during remission period may be an indicator of the occurrence of relapse.
Acute Disease↗
[Hemodynamic and renal effects of strong diuretic atrial natriuretic peptide in patients with acute myocardial infarction].
The hemodynamic and renal effects of strong diuretic atrial natriuretic peptide (ANP) were investigated in 12 patients with acute myocardial infarction. Strong diuretic ANP was administered as a bolus injection of 100 micrograms and followed by a continuous infusion of 2 micrograms/kg within 10 minutes into the right atrium via a catheter. We found that strong diuretic ANP induced an increase in cardiac index and stroke volume index. Right atrial pressure, pulmonary arterial pressure, pulmonary capillary wedge pressure, systemic vascular resistance and pulmonary vascular resistance were decreased after ANP infusion. The maximal responses were observed immediately after the infusion was completed. No significant change of arterial blood pressure and heart rate was observed. Furthermore, strong diuretic ANP also increased the urine volume, urinary sodium, urinary chloride and urinary potassium. These results suggest that strong diuretic ANP infusion may induce significant changes of hemodynamics and may be used to prevent the development of severe circulatory volume overload in patients with AMI.
Adult↗
Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.
Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.
Base Sequence↗
Retinoic acid-mediated activation of HNF-3 alpha during EC stem cell differentiation.
We present evidence demonstrating that the liver-enriched transcription factor HNF-3 alpha is activated upon retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells. We have detected increases in the DNA binding activity and mRNA level of HNF-3 alpha. Both are reflections of the actual activation mechanism at the level of transcriptional initiation, which we showed with the help of HNF-3 alpha promoter constructs. Time course studies clearly show that HNF-3 alpha activation is a transient event. Employing Northern blots, HNF-3 alpha mRNA can be detected between 16 and 24 hours post-differentiation, reaches its zenith at approximately 1 day, and then declines to virtually undetectable levels. F9 cells can give rise to three distinct differentiated cell types; visceral endoderm, parietal endoderm, and primitive endoderm. We have clearly shown that HNF-3 alpha stimulation occurs upon primitive endoderm formation. In addition, the transcription factor is also activated during the induction of cell lineages that give rise to parietal and visceral endoderm. HNF-3 alpha stimulation upon visceral endoderm differentiation is accompanied by the activation of HNF-3 target genes such as transthyretin, suggesting that HNF-3 alpha is involved in the developmental activation of this gene. In contrast, HNF-3 alpha target genes in parietal and primitive endoderm have yet to be identified. However, the stimulation of HNF-3 alpha during primitive endoderm formation, which is an extremely early event during murine embryogenesis, points towards a role for the factor in crucial determination processes that occur early during development.
Animals↗
Newly discovered stereochemical requirements in the side-chain conformation of delta opioid agonists for recognizing opioid delta receptors.
Topographic design of peptide ligands using specialized topographically constrained amino acids can provide new insights into the stereochemical requirements for delta opioid receptors. A highly constrained tyrosine derivative, (2S,3S)-beta-methyl-2',6'-dimethyltyrosine [(2S,3S)-TMT], was prepared by asymmetric synthesis and incorporated in [D-Pen2,D-Pen5] enkephalin (delta 1) and Deltorphin I (delta 2). The results of binding assays and bioassays showed that the two analogues (3 and 4) acted very differently at delta opioid receptors. Further pharmacological evaluations suggested that they actually interact primarily with the delta 1 and delta 2 receptor subtypes, respectively. These results, and conformational studies using NMR and computer-assisted modeling, provided insights into the different stereochemical requirements for these two delta opioid ligands to recognize the delta opioid receptor and its subtypes.
Amino Acid Sequence↗
Ligand and p185c-neu density govern receptor interactions and tyrosine kinase activation.
The neu protooncogene (also known as c-erbB2, NGL, and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity that resembles the receptor for epidermal growth factor. The p185 gene and protein were originally identified in the brain and are thought to play a critical role in neurogenesis. Aberrant c-erbB2 protein overexpression also occurs in several human adenocarcinomas. A ligand for p185, neu-activating factor (NAF), specifically binds to neu receptor and increases the p185c-neu tyrosine phosphorylation in vitro and in vivo in a dose-dependent manner. We now show that NAF specifically binds to purified p185 expressed in baculovirus. Direct binding analysis showed that NAF binds with high affinity (Kd = 1.3 nM). We have investigated changes in the structure and association state of baculovirus-produced neu holoreceptor that are induced by ligand binding. In this study, we used sucrose gradients to show that purified p185c-neu exists mainly in the monomeric form at low concentrations, whereas at higher concentrations p185c-neu exists as dimers or multimers. At low concentrations, but in the presence of ligand, p185c-neu sediments as a dimeric or multimeric form. Monomer-oligomer interconversion is absolutely ligand dependent at low receptor concentrations. The high molecular weight form of the receptor is enzymatically more active, as a consequence of ligand-driven activation of the receptor kinase. Oncogenic p185neu receptors sediment predominantly as high molecular weight forms and have constitutively active kinases.
Animals↗