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S Chen

Publications and source records attributed to S Chen.

At least 271 records · Page 15Linked to original sources
PubMed2001

Assembly of tapasin-associated MHC class I in the absence of the transporter associated with antigen processing (TAP).

The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and beta(2)-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Distinct differences in association of MHC class I with endoplasmic reticulum proteins in wild-type, and beta 2-microglobulin- and TAP-deficient cell lines.

In this study we have compared the interaction of human MHC class I molecules with IgG heavy chain (HC) binding protein (BiP), calnexin, calreticulin, tapasin and TAP in beta(2)-microglobulin (beta(2)m)- or TAP-deficient cells, as well as in wild-type B-LCL cells. Distinct differences between the association of HC and these endoplasmic reticulum (ER) proteins were found in the three cell lines. In the absence of beta(2)m (Daudi cells), HC associated with both BiP and calnexin. A prominent portion of HC was complexed simultaneously to both chaperones, as indicated by co-precipitation with either anti-calnexin or anti-class I antisera. In the presence of beta(2)m, but absence of TAP (T2 cells), HC could be co-precipitated with calnexin, whereas no detectable interaction with BiP could be demonstrated. This suggests that calnexin interacts with HC at a later stage than BiP. In B-LCL cells, HC-beta(2)m associated with calreticulin and tapasin, whereas no interaction with calnexin and BiP was observed. In the absence of beta(2)m, HC were rapidly degraded in the ER, while the ER retained HC were stabilized in the presence of beta(2)m, even in the absence of TAP. The dissociation of class I molecules from TAP in B-LCL cells correlated with the kinetics of appearance of class I molecules on the cell surface, suggesting that TAP retains peptide-free class I molecules in the ER. Taken together, our results suggest the model that BiP and calnexin sequentially control the folding of MHC class I, before MHC class I molecules associate with the loading complex.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

White button mushroom phytochemicals inhibit aromatase activity and breast cancer cell proliferation.

Estrogen is a major factor in the development of breast cancer. In situ estrogen production by aromatase/estrogen synthetase in breast cancer plays a dominant role in tumor proliferation. Because natural compounds such as flavones and isoflavones have been shown to be inhibitors of aromatase, it is thought that vegetables that contain these phytochemicals can inhibit aromatase activity and suppress breast cancer cell proliferation. Heat-stable extracts were prepared from vegetables and screened for their ability to inhibit aromatase activity in a human placental microsome assay. The white button mushroom (species Agaricus bisporus) suppressed aromatase activity dose dependently. Enzyme kinetics demonstrated mixed inhibition, suggesting the presence of multiple inhibitors or more than one inhibitory mechanism. "In cell" aromatase activity and cell proliferation were measured using MCF-7aro, an aromatase-transfected breast cancer cell line. Phytochemicals in the mushroom aqueous extract inhibited aromatase activity and proliferation of MCF-7aro cells. These results suggest that diets high in mushrooms may modulate the aromatase activity and function in chemoprevention in postmenopausal women by reducing the in situ production of estrogen.

Agaricus↗
PubMed2001

Amyloid beta peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation.

Amyloid beta peptide (A beta), a 39 to 43 amino acid fragment of the beta-amyloid precursor protein (betaAPP), forms insoluble fibrillar accumulation in neurofibrillary tangles and vascular plaques. A beta has been implicated in neuronal and vascular degeneration in brain regions susceptible to plaque formation because of its cytotoxic effect on neurons and endothelial cells (ECs). The authors used a murine cerebral endothelial cell (CEC) line and primary cultures of bovine CECs to explore the cytotoxic mechanism of A beta. A beta 1-40 and A beta 25-35 peptides caused cell death in a dose-dependent and time-dependent manner. Exposure to either A beta 25-35 or A beta 1-40 at 10 micromol/L for 48 hours caused at least 40% cell death. Cerebral endothelial cell death was characterized by nuclear condensation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage. A beta 25-35 activated both caspase-8 and caspase-3 in murine CECs. zVAD-fmk, a broad-spectrum caspase inhibitor, prevented A beta 25-35-induced increase in caspase-3 activity and CEC death. N-acetyl-cysteine, an antioxidant, also prevented A beta-induced cell death. Together, these findings indicate that A beta-mediated CEC death is an apoptotic process that is characterized by increased oxidative stress, caspase activation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage.

Acetylcysteine↗
PubMed2001

Long-distance phloem transport of glucosinolates in Arabidopsis.

Glucosinolates are a large group of plant secondary metabolites found mainly in the order Capparales, which includes a large number of economically important Brassica crops and the model plant Arabidopsis. In the present study, several lines of evidence are provided for phloem transport of glucosinolates in Arabidopsis. When radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) and sucrose were co-applied to the tip of detached leaves, both tracers were collected in the phloem exudates at the petioles. Long-distance transport of [(14)C]p-OHBG was investigated in wild-type and transgenic 35S::CYP79A1 plants, synthesizing high amounts of p-OHBG, which is not a natural constituent of wild-type Arabidopsis. In both wild-type and 35S::CYP79A1 plants, radiolabeled p-OHBG was rapidly transported from the application site into the whole plant and intact p-OHBG was recovered from different tissues. The pattern of distribution of the radioactivity corresponded to that expected for transport of photoassimilates such as sucrose, and was consistent with translocation in phloem following the source-sink relationship. Radiolabeled p-OHBG was shown to accumulate in the seeds of wild-type and 35S::CYP79A1 plants, where p-OHBG had been either exogenously applied or endogenously synthesized from Tyr in the leaves. p-OHBG was found in phloem exudates collected from cut petioles of leaves from both wild-type and 35S::CYP79A1 plants. Phloem exudates were shown to contain intact glucosinolates, and not desulphoglucosinolates, as the transport form. It is concluded that intact glucosinolates are readily loaded into and transported by the phloem.

Arabidopsis↗
PubMed2001

Solubilization and disaggregation of polyglutamine peptides.

A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q20 have been reported to be insoluble in water, but dissolution in--and evaporation from--a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q44 to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies-including in vitro aggregation experiments--on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.

Peptides↗
PubMed2001

Regulation of RpoS by a novel small RNA: the characterization of RprA.

Translational regulation of the stationary phase sigma factor RpoS is mediated by the formation of a double-stranded RNA stem-loop structure in the upstream region of the rpoS messenger RNA, occluding the translation initiation site. The interaction of the rpoS mRNA with a small RNA, DsrA, disrupts the double-strand pairing and allows high levels of translation initiation. We screened a multicopy library of Escherichia coli DNA fragments for novel activators of RpoS translation when DsrA is absent. Clones carrying rprA (RpoS regulator RNA) increased the translation of RpoS. The rprA gene encodes a 106 nucleotide regulatory RNA. As with DsrA, RprA is predicted to form three stem-loops and is highly conserved in Salmonella and Klebsiella species. Thus, at least two small RNAs, DsrA and RprA, participate in the positive regulation of RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. RprA is non-essential. Mutations in the gene interfere with the induction of RpoS after osmotic shock when DsrA is absent, demonstrating a physiological role for RprA. The existence of two very different small RNA regulators of RpoS translation suggests that such additional regulatory RNAs are likely to exist, both for regulation of RpoS and for regulation of other important cellular components.

Bacterial Proteins↗
PubMed2001

Protection of xenogeneic cells from human complement-mediated lysis by the expression of human DAF, CD59 and MCP.

CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.

3T3 Cells↗
PubMed2001

Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR.

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding approximately 500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.

Animals↗
PubMed2001

Randomised controlled trial of oral vitamin A supplementation in preterm infants to prevent chronic lung disease.

BACKGROUND: Intramuscular supplementation with vitamin A in large doses may reduce the incidence of chronic lung disease. AIM: To investigate whether oral supplementation with vitamin A would reduce the incidence of chronic lung disease in a group of extremely low birthweight infants. METHODS: Infants with birth weight < 1000 g were randomised at birth to receive oral vitamin A supplementation (5000 IU/day) or placebo for 28 days. The primary outcome was oxygen dependency at 28 days of age or death. RESULTS: A total of 154 infants were randomised; 77 received vitamin A (median birth weight (interquartile range) 806 (710-890) g), and 77 received placebo (median birth weight (interquartile range) 782 (662-880) g). Plasma vitamin A concentrations in the supplemented group were significantly higher at 24 hours of age but did not differ significantly at birth, 12 hours of age, 7 days, or 28 days of life. There were no significant differences in the proportion of infants who survived, required oxygen at 28 days, required oxygen at 36 weeks postmenstrual age, survived without chronic lung disease at 36 weeks, survived without significant retinopathy, or who survived without significant intraventricular haemorrhage. CONCLUSIONS: Oral supplementation with 5000 IU vitamin A in extremely low birthweight infants does not significantly alter the incidence of chronic lung disease. However, this dose may have been inadequate to achieve optimal serum retinol concentrations.

Chronic Disease↗
PubMed2001

High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism.

Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ET(B) antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ET(A) inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.

Actins↗
PubMed2001

Accelerated inactivation in a mutant Na(+) channel associated with idiopathic ventricular fibrillation.

Idiopathic ventricular fibrillation (IVF) can cause sudden death in both adults and children. One form of IVF (Brugada syndrome), characterized by S-T segment elevation (STE) in the electrocardiogram, has been linked to mutations of SCN5A, the gene encoding the voltage-gated cardiac Na(+) channel. A missense mutation of SCN5A that substitutes glutamine for leucine at codon 567 (L567Q, in the cytoplasmic linker between domains I and II) is identified with sudden infant death and Brugada syndrome in one family. However, neither the functional effect of the L567Q mutation nor the molecular mechanism underlying the pathogenicity of the mutation is known. Patch-clamp analysis of L567Q channels expressed in human embryonic kidney cells revealed a marked acceleration and a negative shift in the voltage dependence of inactivation. Unlike other Brugada mutations, this phenotype was expressed independently of temperature or auxiliary beta(1)-subunits. These results support a proposed linkage between Brugada syndrome and some instances of sudden infant death and the hypothesis that reduced Na(+) conductance is the primary cause of IVF with STE.

Cell Line↗
PubMed2001

Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells.

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.

Actins↗
PubMed2001

Prevalence of sexually transmitted infections (Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis and human papillomavirus) in female attendees of a sexually transmitted diseases clinic in Ulaanbaatar, Mongolia.

BACKGROUND: Epidemiological data suggest that the prevalence of syphilis, gonorrhea and trichomoniasis has increased in both urban and rural areas of Mongolia. These data are primarily substantiated by notifications of cases of clinically apparent disease in both rural and urban areas, plus laboratory diagnoses from the AIDS/STD Reference Center, Ulaanbaatar. In the past 5 years, however, there has been a marked decline in the total number of patients being screened for sexually transmitted infections (STIs). An assessment of true prevalence of STIs in a female population attending an urban sexually transmitted diseases (STD) clinic was therefore commenced. METHODS: Consecutive women attending an STD clinic in Ulaanbaatar had genital samples collected by the insertion and immediate removal of a tampon, which was then tested for the presence of Neisseria gonorrhoeae, Chlamydia trachomatis, human papillomavirus (HPV) and Trichomonas vaginalis, using polymerase chain reaction (PCR) amplification. RESULTS: A total of 110 women were studied (mean age 26.7 years). Overall, 58 (53%) patients had one or more pathogens identified; 43 (39%) had a single pathogen, while 15 (14%) had mixed pathogens. C. trachomatis was found in 15 (14%), N. gonorrhoeae in 12 (11%), T. vaginalis in nine (8%) and HPV in 39 (36%). Among the 39 HPV-positive patients, oncogenicgenotypes (16, 18, 31, 33, 35, 39, 45, 51, 52) were found in 17(44%) patients. CONCLUSIONS: Sexually transmitted infections as defined by PCR were common, and found in 53% of female attendees of an urban STD clinic in Mongolia. As infections with conventional STIs increase the risk of human immunodeficiency virus (HIV) transmission, it is imperative that strategies be introduced to reduce the prevalence of STIs. Furthermore, detection of oncogenic HPV was common, indicating that it is vital that a strategy to reduce cervical cancer such as a pre-cancer cervical cytology screening program also be introduced.

Adult↗
PubMed2001

Utilization of MO6-G3 immortalized odontoblast cells in studies regarding dentinogenesis.

Tooth formation is the result of reciprocal instructive interactions between oral epithelium and cranial neural-crest-derived ectomesenchymal tissues. These interactions lead to the cytodifferentiation of highly specialized matrix-forming cell types, the ameloblast, odontoblast, and cementoblast, that produce the mineralized tissues enamel, dentin, and cementum, respectively. Our laboratory has been developing immortalized dental cell lines representative of these various cell types to facilitate studies on gene regulation, cell differentiation, matrix formation, and mineralization. Odontoblasts are solely responsible for the synthesis and secretion of the dentin extracellular matrix bilayer that consists of non-mineralized predentin and mineralized dentin. The mouse immortalized MO6-G3 cell line expresses the major matrix proteins associated with the odontoblast phenotype, producing a matrix that is capable of mineralization. This cell line serves as a useful tool in studies designed to explore the various processes of dentinogenesis. In this paper, we present studies using the mouse odontoblast cell line MO6-G3 as examples of the various research applications. Studies highlighted are: in vitro promoter studies investigating the tooth-specific gene regulation of the major non-collagenous dentin matrix protein, dentin sialophosphoprotein; regulation of tertiary dentin formation by cytokines, such as transforming growth factor-Beta 1; and the utilization of dentally relevant cells in dental material biocompatibility testing.

Ameloblasts↗
PubMed2001

Primary synovial sarcoma of the kidney: a case report with literature review.

We describe a case of primary renal synovial sarcoma (SS) in a 48-year-old man. The patient presented with hematuria and was found to have a large tumor in his left kidney on computed tomography scan. Histology revealed a highly cellular spindle cell neoplasm with minimal pleomorphism. The major differential diagnoses included leiomyosarcoma, hemangiopericytoma, and SS. The presence of focal areas with a biphasic pattern, uniformly positive immunostain for bcl-2, focally positive immunostains for epithelial membrane antigen and cytokeratin, and negative immunostains for CD-34, smooth muscle actin and S-100 established the diagnosis. This was subsequently confirmed by molecular testing for t(X;18) translocation. Since the existence of primary SS in the kidney was first suggested in 1999, to the best of our knowledge a total of 19 cases including the present case have been reported to date. Although primary renal SS is rare, these findings indicate that it should be included in the differential diagnosis of spindle cell tumors of the kidney.

Biomarkers, Tumor↗