[Progress in the prevention of typhoid fever in China].
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China↗
Biomedical subjects
X He
Publications and source records attributed to X He.
A Study on Organized Assemblies in the Aqueous Systems of Alkylammonium Chlorides.
Various types of organized assemblies, including micelle, planar multi-bilayer, bent multi-bilayer, fingerprint-like multi-bilayer, and multilamellar vesicles, were found in the aqueous system prepared by only one simple surfactant-alkylammonium chloride with a hydrocarbon chain length of 10 or 12. Electron microscopic images of the assemblies are provided. The properties of the system were characterized by DSC, fluorescence, and absorption probing techniques. It was found that the structure and shape of molecular assemblies in this system can be adjusted by very simple physico-chemical measures, such as pH adjustment and alcohol or salt addition. The mechanisms of formation and transformation of various molecular assemblies were discussed based on the mixing effect of the cationic amphiphile and the nonionic amphiphile which is only a hydrolysis product of the former. Copyright 1999 Academic Press.
Journal Article↗
The human acid ceramidase gene (ASAH): structure, chromosomal location, mutation analysis, and expression.
Acid ceramidase (AC) is the lysosomal enzyme that degrades ceramide into sphingosine and fatty acid. A deficiency in human AC activity leads to the lysosomal storage disorder, Farber disease (FD). The human AC gene (HGMW-approved symbol ASAH) was cloned and characterized, revealing an organization similar to that of the murine AC gene. The human gene spans about 30 kb in length and contains 14 exons ranging in size from 46 to 1201 bp. The exon/intron junctions were determined and found to follow the GT-AG rule. The putative promoter region had a GC content over 60%, lacked a TATA box, and contained several sequences matching transcription factor binding sites, including nine SP-1 sites, one AP-1 site, and three CACC boxes. The promoter activity of a 475-bp fragment from within this region was demonstrated by chloramphenicol acyltransferase assays. Northern blotting revealed variable expression of the human AC RNA; i.e., expression of the major 2.4-kb transcript was high in heart and kidney, followed by lung and placenta, but low in pancreas, liver, brain, and skeletal muscle. Two minor AC transcripts of 1.7 and 1.2 kb also were detected in heart and skeletal muscle. The human AC gene was mapped to the chromosomal region 8p21.3-p22 by in situ hybridization and FISH analyses, syntenic with the mouse chromosomal location. Finally, three new missense mutations, E138V, R254G, and P362R, were identified in the human AC gene from FD patients. Mutant AC cDNAs containing these point mutations were constructed and examined using the FLAG-tagged expression system. Although the levels of protein expression for these mutant ACs were about equivalent to that of the controls, their enzymatic activity was markedly reduced, confirming their authenticity.
3T3 Cells↗
Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.
Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion protein in COS-1 cells revealed a punctate, perinuclear distribution, although no acid ceramidase activity was detected in the transfected cells using a fluorescence-based in vitro assay system.
5' Untranslated Regions↗
Prostaglandin E2 selectively inhibits human CD4+ T cells secreting low amounts of both IL-2 and IL-4.
PGE2 is a potent inflammatory mediator with profound immune regulatory actions. The present study examined the effects of PGE2 on the activation/proliferation of CD4+ T cells using 37 cloned CD4+ T cell lines. Ten T cell clones sensitive to PGE2 and 10 T cell clones resistant to PGE2, as measured by proliferation in response to anti-CD3 Ab, were selected for comparison. It was found that the PGE2-sensitive T cells were characterized by low production (<200 pg/ml) of both IL-2 and IL-4, while PGE2-resistant T cells secreted high levels (>1000 pg/ml) of IL-2, IL-4, or both. The roles of IL-2 and IL-4 were confirmed by the finding that addition of exogenous lymphokines could restore PGE2-inhibited proliferation, and PGE2-resistant Th1-, Th2-, and Th0-like clones became PGE2 sensitive when IL-2, IL-4, or both were removed using Abs specific for the respective lymphokines. In addition, we showed that the CD45RA expression in PGE2-sensitive T cells was significantly lower than that in PGE2-resistant cells (mean intensity, 1.2 +/- 0.6 vs 7.8 +/- 5.7; p = 0.001). In contrast, CD45RO expression in PGE2-sensitive T cells was significantly higher that that in PGE2-resistant cells (mean intensity, 55.7 +/- 15.1 vs 33.4 +/- 12.9; p = 0.02). In summary, PGE2 predominantly suppressed CD45RA-RO+ CD4+ T cells with low secretion of both IL-2 and IL-4.
CD4-Positive T-Lymphocytes↗
Neuralization of the Xenopus embryo by inhibition of p300/ CREB-binding protein function.
p300/ CREB-binding protein (CBP) is a transcriptional coactivator for a plethora of transcription factors and plays critical roles in signal transduction pathways. We report that the inhibition of p300/CBP function in the Xenopus embryo abolishes non-neural tissue formation and, strikingly, initiates neural induction and primary neurogenesis in the entire embryo. The observed neuralization is achieved in the absence of anterior or posterior gene expression, suggesting that neural fate activation and anterior patterning may represent distinct molecular events. We further demonstrate that the neuralizing and anteriorizing activities of chordin and noggin are separable properties of these neural inducers. This study reveals that all embryonic cells possess intrinsic neuralizing capability and that p300/CBP function is essential for embryonic germ layer formation and neural fate suppression during vertebrate embryogenesis.
Animals↗
Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma.
BACKGROUND: Focal adhesion kinase (FAK) is a tyrosine kinase that is important to such key functions such as cell adhesion, motility, and invasion. A MEDLINE search of the years 1980-1998 found no previous reports of FAK expression in human ovarian carcinoma. The authors performed experiments to determine whether FAK expression is elevated in this disease. METHODS: Ten normal human ovarian tissue samples and 26 cancer samples from patients with Stage I-IV ovarian carcinoma were obtained. Two ovarian carcinoma cell lines were also analyzed. FAK expression was determined by Western blot analysis with the V39 anti-human FAK polyclonal antibody. The level of FAK protein expression was determined using densitometric scanning of the 125 kD band on autoradiographs of Western immunoblots. RESULTS: Serous cancers expressed fourfold-increased values of FAK relative to normal ovarian tissue (P < 0.0001), and nonserous adenocarcinomas expressed threefold- to fourfold-increased values of FAK (P < 0. 0006). Ovarian carcinoma cell lines also expressed increased values of FAK. With a cutoff of 40, an elevated FAK level was associated with a sensitivity of 93% and specificity of 100%. There was no significant difference in FAK expression with regard to grade or stage of tumor. CONCLUSIONS: FAK is significantly overexpressed in ovarian carcinoma, implying that FAK may play an important role in ovarian carcinogenesis. FAK expression may be useful as a screening tool to identify newly developed disease or as a tumor marker in confirmed cases of epithelial ovarian carcinoma. FAK may also serve as a potential target for therapeutic disruption of ovarian carcinoma progression.
Blotting, Western↗
A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.
Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.
Acid Ceramidase↗
[Toxic effects of cooking oil fumes on testis of rats].
To investigate the toxic effects of inhaling cooking oil fumes (COF), 30 male SD rats were submitted to inhaling COF at a constant concentration and duration (35-53 mg/m3, 30 min/d) for different length of time (20, 40 and 60 days), and examined for change in body weight, spermtogenesis and content of trace elements in their testes. The results indicated that the body weight and the ratio of gonad to body weight in rats inhaling COF for 40 and 60 days were lower than those in the controls (P < 0.05). Fewer alive sperms and more malformed or dead sperms were fond in the epididymis of rats exposed to COF for 40 and 60 days. Among the four trace elements(Zn, Mn, Cu, Fe) studied, the content of Zn in the testis was found to be significantly decreased and that of Mn significantly increased in the rats exposed to COF for longer periods. There existed a time-effect relationship in all of the above findings. The results suggested that inhaling COF was significantly toxic to male gonads, and the imbalance of Zn, Mn might be contributive to the damage of testis.
Animals↗
Protease M/neurosin mRNA is expressed in mature oligodendrocytes.
No proteases have been identified to be oligodendrocyte-specific though there are phenomena in which proteases may be committed. We have cloned a cDNA for mouse homologue of protease M/neurosin, serine protease, and localized its mRNA in the mouse brain. The cDNA and the deduced amino acid sequences were 66% identical to those of human protease M/neurosin. In situ hybridization histochemistry revealed that the mRNA was localized in the mature oligodendrocytes which expressed proteolipid protein mRNA. Developmentally, protease M/neurosin mRNA was expressed by oligodendrocytes 2 to 7 days after the maturation. Thus, protease M/neurosin may be important to the processes occurring after the maturation of oligodendrocytes such as myelination or turnover of the proteins in the myelin.
Amino Acid Sequence↗
Method of medians for lifetime data with Weibull models.
The Weibull family of distributions is frequently used in failure time models. The maximum likelihood estimator is very sensitive to occurrence of upper and lower outliers, especially when the hazard function is increasing. We consider the method of medians estimator for the two-parameter Weibull model. As an M-estimator, it has a bounded influence function and is highly robust against outliers. It is easy to compute as it requires solving only one equation instead of a pair of equations as for most other M-estimators. Furthermore, no assumptions or adjustments are needed for the estimator when there are some possibly censored observations at either end of the sample. About 16 per cent of the largest observations and 34 per cent of the smallest observations may be censored without affecting the calculations. We also present a simple criterion to choose between the maximum likelihood estimator and the method of medians estimator to improve on the finite-sample efficiency of the Weibull model. Robust inference on the shape parameter is also considered. The usefulness with contaminated or censored samples is illustrated by examples on three lifetime data sets. A simulation study was carried out to assess the performance of the proposed estimator and the confidence intervals of a variety of contaminated Weibull models.
Aged↗
Physiological concentrations of gangliosides GM1, GM2 and GM3 differentially modify basic-fibroblast-growth-factor-induced mitogenesis and the associated signalling pathway in endothelial cells.
It has been suggested that gangliosides can influence the growth of cells by modulation of growth-factor-receptor signalling. The activation of endothelial cells (EC) during angiogenesis is crucial for tumour growth and for metastasis, also for numerous other physiological and pathological situations. Pre-treatment of bovine aortic endothelial cells (BAEC) with GM1 or GM2 (5-20 microM) inhibited basic-fibroblast-growth-factor (bFGF)-induced mitogenesis, but GM3 (0.1-20 microM) acted synergistically, increasing proliferation above that of bFGF alone (p < 0.05). The mitogenic effect of all 3 gangliosides was markedly reduced if the cells were washed to remove excess gangliosides from the medium before addition of bFGF. We further show that GM1 and to a lesser extent GM2 modify bFGF binding to its receptor and inhibit the associated mitogenic signal-transduction pathway of protein-tyrosine phosphorylation of 40 to 120 kDa, PLCgamma1, MAP kinase and protein-kinase-C activation. In contrast, GM3 increased tyrosine phosphorylation and MAP kinase activity, as compared with bFGF alone. The observed differential modulation of bFGF-induced mitogenesis by GM1, GM2 and GM3 was at concentrations routinely occurring in the serum of cancer patients. The results suggest that circulating gangliosides may have a role in regulating solid-tumour growth by modulating angiogenesis.
Animals↗
Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells.
A rapid purification method was developed to isolate milligram quantities of human acid sphingomyelinase from the media of overexpressing Chinese hamster ovary cells. The purified, recombinant enzyme (rhASM) had physical and kinetic characteristics that were consistent with those reported for the non-recombinant enzyme, including an acidic pH optimum and sensitivity to sulfhydryl reducing reagents and the zinc specific chelator, 1, 10-phenanthroline. A novel assay using fluorescently conjugated sphingomyelin was developed to explore the substrate binding properties of rhASM. Substrate binding required a fatty acid chain length of at least six carbons and the presence of the phosphocholine headgroup on sphingomyelin. Substrate binding also required an acidic pH, and was inhibited by pretreatment of the enzyme with sulfhydral reducing reagents or 1,10-phenanthroline. rhASM was rapidly internalized by cultured skin fibroblasts from Niemann-Pick disease (NPD) patients, and approximately 50% of this uptake was dependent on the mannose 6-phosphate receptor system. Studies using FITC-labeled rhASM revealed that by 1 h the internalized enzyme was localized to acidic compartments and could degrade sphingomyelin, the first demonstration that a lysosomal sphingolipid hydrolase can be fluorescently labeled and retain its biological activity. Intravenous injection of rhASM into ASM knock-out mice showed that the t(1/2) in the plasma was less than 5 min, and that the majority of the injected enzyme was taken up by the liver, followed by the spleen. Thus, these studies lay the foundation for future structure/function investigations of ASM, further investigations into this enzyme's role in ceramide mediated signal transduction, and the evaluation of enzyme replacement therapy for NPD using the mouse model.
Animals↗
Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence.
Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and we have reported that protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homologue of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. To our knowledge, these results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression.
Amino Acid Sequence↗
[Effects of high manganese on cerebral development of the offspring of rats].
Pregnant rats were divided into three groups. Two groups are exposed to manganese from drinking water contaminated with manganese in 10 and 2 g/L respectively. Cerebral development of the offspring of rats was studied. The results are as follows. (1) The thickness of frontal and parental cortex of high dose Mn-exposed groups was significantly reduced, but glial fibrillary acid protein (GFAP) and the densities of its products were increased. (2) Cingular cortex was thickened, GFAP and the density of its products of cingular cortex was increased in both Mn-exposed groups compared with the control group. (3) The area, GFAP and the densits of its products of corpus callosium in the high Mn-exposed group were significantly increased compared with those in both low Mn-exposed and control groups.
Animals↗
beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation.
Regulation of beta-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of beta-catenin by the Axin-glycogen synthase kinase (GSK)-3beta complex targets beta-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize beta-catenin and cause human cancers. How beta-catenin phosphorylation results in its degradation is undefined. Here we show that phosphorylated beta-catenin is specifically recognized by beta-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. beta-catenin harboring mutations at the critical phosphoserine residues escapes recognition by beta-Trcp, thus providing a molecular explanation for why these mutations cause beta-catenin accumulation that leads to cancer. Inhibition of endogenous beta-Trcp function by a dominant negative mutant stabilizes beta-catenin, activates Wnt/beta-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, beta-Trcp plays a central role in recruiting phosphorylated beta-catenin for degradation and in dorsoventral patterning of the Xenopus embryo.
Animals↗
Injury induces neuropsin mRNA in the central nervous system.
We have shown that neuropsin is expressed in the neurons of the limbic system in the adult mouse. After the central nervous system was injured by incision or intraperitoneal kainate injection, neuropsin mRNA was induced in the peri-lesioned region. The cells in which neuropsin mRNA was induced were localized mainly in axon fiber pathways and closely associated to proteolipid protein (PLP) mRNA expressing oligodendrocytes.
Animals↗
Identification and characterization of the sodium-binding site of activated protein C.
Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.
Allosteric Regulation↗