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S Chen

Publications and source records attributed to S Chen.

At least 685 records · Page 38Linked to original sources
PubMed1998

pICln can regulate swelling-induced Cl- currents in either layer of rabbit ciliary epithelium.

Swelling-induced Cl- currents were investigated in freshly prepared non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells of the rabbit ciliary body using the whole-cell patch clamp technique. Exposure of both NPE and PE cells to hypotonic stress induced Cl- currents that exhibited outward rectification and were insensitive to Ca+2. We found that swelling-induced Cl- currents in PE cell are observed shortly after isolation. The swelling-induced Cl- current showed little or no inactivation at positive membrane voltages and was sensitive to 100 microM NPPB and 100 microM DIDS. Injection of cRNA encoded rabbit pICln into Xenopus oocytes produced an outwardly rectifying Cl- current displaying features consistent with the swelling-induced Cl- current in epithelium. pICln is ubiquitous in the ciliary epithelium. It participates in the equilibration of short term tonicity alterations, a phenomenon underlying mechanisms with larger and slower amplitudes for aqueous secretion by these cells.

Animals↗
PubMed1998

CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor.

DFF45 is a subunit of the DNA fragmentation factor (DFF) that is cleaved by caspase-3 during apoptosis. However, the mechanism by which DFF45 regulates apoptotic cell death remains poorly understood. Here we report the identification and characterization of two mammalian genes, CIDE-A and CIDE-B, encoding highly related proteins with homology to the N-terminal region of DFF45. CIDE-A and CIDE-B were found to activate apoptosis in mammalian cells, which was inhibited by DFF45 but not by caspase inhibitors. Expression of CIDE-A induced DNA fragmentation in 293T cells, which was inhibited by DFF45, further suggesting that DFF45 inhibits the apoptotic activities of CIDEs. In addition to mammalian CIDE-A and CIDE-B, we identified DREP-1, a Drosophila melanogaster homolog of DFF45 that could inhibit CIDE-A-mediated apoptosis. Mutant analysis revealed that the C-terminal region of CIDE-A was necessary and sufficient for killing whereas the region with homology to DFF45 located in the N-terminus was required for DFF45 to inhibit CIDE-A-induced apoptosis. CD95/Fas-mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. These studies suggest that DFF45 is evolutionarily conserved and implicate CIDEs as DFF45-inhibitable effectors that promote cell death and DNA fragmentation.

Amino Acid Sequence↗
PubMed1998

Modifying the mechanical property and shear threshold of L-selectin adhesion independently of equilibrium properties.

Interactions between adhesion molecules on two different cells differ from interactions between receptors and soluble ligands in that the adhesion molecule interaction (bond) is often subjected to force. It is widely assumed by cell biologists that the 'strength' of a bond is a simple function of the affinity of one adhesion molecule for the other, whereas biophysicists suggest that bonds have 'mechanical properties' that affect their strength. Mechanical properties are a function of the shape of the energy landscape related to bond formation and dissociation, whereas affinity is related only to the net energy change. Mechanical properties determine the amount by which the kinetics and affinity of bonds are altered by applied force. To date there has been no experimental manipulation of an adhesion molecule that has been shown to affect mechanical properties. L-selectin is an adhesion molecule that mediates lymphocyte binding to, and rolling on, high endothelial venules; these are prerequisites for the emigration of lymphocytes from the bloodstream into lymph nodes. Here we report a selective and reversible chemical modification of a mucin-like ligand that alters the mechanical properties of its bond with L-selectin. The effect of force on the rate of bond dissociation, that is, on a mechanical property, is altered, whereas there is little or no effect of the modification on the rate of bond dissociation in the absence of force. Moreover, the puzzling requirement for hydrodynamic shear flow above a threshold level for L-selectin interactions is dramatically altered.

Antigens, CD34↗
PubMed1998

Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography.

Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of ribonucleotides to deoxyribonucleotides. It is composed of two homodimeric subunits, R1 and R2. R2 contains the diferric-tyrosyl radical cofactor essential for the nucleotide reduction process. The in vitro mechanism of assembly of this cluster starting with apo R2 or with a diferrous form of R2 has been examined by time-resolved physical biochemical methods. An intermediate, Fe3+/Fe4+ cluster (intermediate X), has been identified that is thought to be directly involved in the oxidation of Y122 to the tyrosyl radical (*Y122). An R2 mutant in which phenylalanine has replaced Y122 has been used to accumulate intermediate X at sufficient levels that it can be studied using a variety of spectroscopic methods. The details of the reconstitution of the apo and diferrous forms of Y122F R2 have been examined by stopped-flow UV/vis spectroscopy and by rapid freeze quench electron paramagnetic resonance, and Mössbauer spectroscopies. In addition the structure of this mutant, crystallized at pH 7.6 in the absence of mercury, at 2.46 A resolution has been determined. These studies suggest that Y122F R2 is an appropriate model for the examination of intermediate X in the assembly process. Studies with two mutants, Y356F and double mutant Y356F and Y122F R2, are interpreted in terms of the possible role of Y356 in the putative electron transfer reaction between the R1 and R2 subunits of this RNR.

Amino Acid Substitution↗
PubMed1998

ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases.

We have identified and characterized ARC, apoptosis repressor with caspase recruitment domain (CARD). Sequence analysis revealed that ARC contains an N-terminal CARD fused to a C-terminal region rich in proline/glutamic acid residues. The CARD domain of ARC exhibited significant homology to the prodomains of apical caspases and the CARDs present in the cell death regulators Apaf-1 and RAIDD. Immunoprecipitation analysis revealed that ARC interacts with caspase-2, -8, and Caenorhabditis elegans CED-3, but not with caspase-1, -3, or -9. ARC inhibited apoptosis induced by caspase-8 and CED-3 but not that mediated by caspase-9. Further analysis showed that the enzymatic activity of caspase-8 was inhibited by ARC in 293T cells. Consistent with the inhibition of caspase-8, ARC attenuated apoptosis induced by FADD and TRADD and that triggered by stimulation of death receptors coupled to caspase-8, including CD95/Fas, tumor necrosis factor-R1, and TRAMP/DR3. Remarkably, the expression of human ARC was primarily restricted to skeletal muscle and cardiac tissue. Thus, ARC represents an inhibitor of apoptosis expressed in muscle that appears to selectively target caspases. Delivery of ARC by gene transfer or enhancement of its endogenous activity may provide a strategy for the treatment of diseases that are characterized by inappropriately increased cell death in muscle tissue.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL.

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.

Amino Acid Sequence↗
PubMed1998

Proteolytic regulation of the zinc finger transcription factor YY1, a repressor of muscle-restricted gene expression.

Regulated proteolysis has been postulated to be critical for proper control of cell functions. Muscle development, in particular, involves a great deal of structural adaptation and remodeling mediated by proteases. The transcription factor YY1 represses muscle-restricted expression of the sarcomeric alpha-actin genes. Consistent with this repressor function of YY1, the nuclear regulator is down-regulated at the protein level during skeletal as well as cardiac muscle cell differentiation. However, the YY1 message remains relatively unaltered throughout the myoblast-myotube transition, implicating a post-translational regulatory mechanism. We show that YY1 can be a substrate for cleavage by the calcium-activated neutral protease calpain II (m-calpain) and the 26 S proteasome. The calcium ionophore A23187 destabilized YY1 in cultured myoblasts, and the decrease in YY1 protein levels could be prevented by calpain inhibitor II and calpeptin. Treatment with the proteasome inhibitors MG132 and lactacystin resulted in the stabilization of YY1 protein, which is consistent with the finding that YY1 is readily polyubiquitinated in reticulocyte lysates. We further show that proteolytic targeting by calpain II and the proteasome involves different structural elements of YY1. This study thus illustrates two proteolytic pathways through which the transcriptional regulator can be differentially targeted under different cell growth conditions.

Animals↗
PubMed1998

Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids.

Potent and selective dipeptidyl boronic acid proteasome inhibitors are described. As compared to peptidyl aldehyde compounds, boronic acids in this series display dramatically enhanced potency. Compounds such as 15 are promising new therapeutics for treatment of cancer and inflammatory diseases.

Boronic Acids↗
PubMed1998

A pineal regulatory element (PIRE) mediates transactivation by the pineal/retina-specific transcription factor CRX.

The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein we recently identified, is produced as a truncated form of the Wilson disease gene (Atp7b) product. To identify the regulatory elements required for pineal-specific gene expression, we isolated sequences upstream of the rat PINA gene and discovered a cis-acting element that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5' regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE in vitro and transactivates PIRE-reporter constructs. These data suggest that Crx may play a crucial role in regulating pineal gene expression through interactions with PIRE.

Acetylserotonin O-Methyltransferase↗
PubMed1998

Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2.

Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormö, M., Nordlund, P., & Sjöberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

Ascorbic Acid↗
PubMed1998

Cost-effectiveness and cost-benefit analysis of using methotrexate vs Goeckerman therapy for psoriasis. A pilot study.

OBJECTIVE: To analyze the net benefit and cost-effectiveness of methotrexate use and Goeckerman therapy for psoriasis. DESIGN: Net benefit and cost-effectiveness depend on the costs, efficacy, and utilities of therapy. Utilities are quantitative measures of patient preferences. We obtained costs by using resource-based accounting techniques. Efficacy was estimated from literature reports. We surveyed patients with psoriasis, dermatologists, and healthy subjects using utility assessment methods. All assumptions were examined in a sensitivity analysis. MAIN OUTCOME MEASURES: For net benefit, if benefits out-weighed the costs, it was deemed worth providing. For the cost-effectiveness analysis, the ratio of costs-to-effectiveness of less than $35,000 was considered cost-effective. RESULTS: Using utilities from healthy nonexperts, the costs of both therapies exceeded the benefits in mild and moderate psoriasis. In severe psoriasis, only methotrexate demonstrates a net benefit. Both therapies were cost-effective compared with no therapy. Liquid methotrexate should be chosen over the tablet form since it was cheaper and had the same outcome. Goeckerman was cost-effective against liquid methotrexate in severe, but not mild or moderate psoriasis. There was a trend for therapies to be more cost-effective when using patient utilities and less with dermatologist utilities. The results were highly sensitive to efficacy and utilities. CONCLUSIONS: The results of this study need to be confirmed in other settings, but they demonstrate that the tools of cost-effectiveness and cost-benefit analysis have great potential value in dermatology. Once efficacy is better characterized and utilities better quantified, these types of analyses will be crucial for health care policy.

Cost-Benefit Analysis↗
PubMed1998

Experimental destruction of substantia nigra initiated by Parkinson disease immunoglobulins.

BACKGROUND: Increased levels of free radicals and oxidative stress may contribute to the pathogenesis of substantia nigra (SN) injury in Parkinson disease (PD), but the initiating etiologic factors remain undefined in most cases. OBJECTIVE: To determine the potential importance of immune mechanisms in triggering or amplifying neuronal injury, we assayed serum samples from patients with PD to determine the ability of IgG to initiate relatively specific SN injury in vivo. METHODS: IgG purified from the serum of 5 patients with PD and 10 disease control (DC) patients was injected into the right side of the SN in adult rats. Coronal sections were cut from the whole brain at the level of the stereotaxic injections, stained for tyrosine hydroxylase and with cresyl violet, and cellular profiles were counted in identical brain regions at the injection and contralateral sides. The ratio of cell profile counts of the corresponding injected and uninjected regions was used as an internal standard. RESULTS: Four weeks following injection of IgG, a 50% decrease in tyrosine hydroxylase-positive cellular profiles was noted on the injected sides compared with the contralateral sides of the same animals. Similarly, applied DC IgG caused only an 18% decrease. Cresyl violet staining revealed a 35% decrease in neuronal profiles of PD IgG injected into the SN pars compacta compared with the contralateral uninjected side, whereas DC IgG caused a minimal 10% decrease. Even at 4 weeks after the PD IgG injections, perivascular inflammation and significant microglial infiltration were present near injured SN pars compacta neurons. No cytotoxic effects of PD IgG were noted in choline acetyltransferase-positive neurons after stereotaxic injections into the medial septal region. Absorption of PD IgG with mesencephalic membranes and protein A agarose gel beads removed cytotoxicity, while absorption with liver membranes did not change the cytotoxicity. CONCLUSIONS: Our data suggest that PD IgG can initiate a relatively specific inflammatory destruction of SN pars compacta neurons in vivo and demonstrate the potential relevance of immune mechanisms in PD.

Animals↗
PubMed1998

Connexin50, a gap junction protein of macrogliaP6n the mammalian retina and visual pathway.

Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry were used to study the expression of gap junction proteins (connexins; Cx) in the rat and rabbit retina. RT-PCR of rabbit total retinal RNA using primers selected for the human Cx50 (alpha 8 Cx) DNA template yielded cDNA fragments of the predicted base pair size. Western blots of rat and rabbit retinal membrane preparations probed with a monoclonal antibody which recognizes Cx50 in the lens of several mammalian species revealed a single band (MW 50 kD), identical to that recognized in lens membrane extracts. In frozen retinal sections of both species, the same monoclonal antibody as well as two polyclonal antisera raised against a synthetic peptide from the C-terminal region of the human Cx50 polypeptide labeled Müller cells and astrocytes. In Müller cells, labeling was strongest in the endfeet and in the filamentous processes ensheathing the photoreceptors. Extending from the neural retina, Cx50-like immuno-reactivity was detected in astrocytes of the optic nerve and along retinal projections within the CNS. Our data indicate that Müller cells and astrocytes of mammalian retinas and throughout the visual pathway are coupled through gap junctions composed of connexin50.

Animals↗
PubMed1998

I-2190A is a potent immunosuppressive drug for vascularized heart transplantation in rats.

The effect of a new immunosuppressant-I-2190A was tested in a rodent heart allograft model. Grafts were transplanted to recipients heterotopically. There were 5 groups: group 1 received no immunosuppressive agents; group 2 was given CsA (2.0 mg/kg, i.p.); group 3 was administered I-2190A (0.1 mg/kg, i.p.) in carboxymethyl cellulose (CMC); group 4 received injection of I-2190A (0.5 mg/kg, i.p. in CMC); group 5 received the combination treatment of I-2190A (0.1 mg/kg) and CsA (2.0 mg/kg). Immunosuppressants were discontinued 14 days after operation. No statistically significant difference in grafts median survival time (MST) was found between group 2 (9.5 days) and group 1 (9 days). The MSTs of grafts in group 3 (22 days, P < 0.05), group 4 (> 100 days, P < 0.01) and group 5 (> 100 days, P < 0.01) were significantly prolonged compared with control group 1 (9 days). Our results suggest that I-2190A is a potent immunosuppressant able to significantly prolong heart allograft survival in rats after a short time treatment, Low-dose I-2190A could potentiate the effect of sub-therapeutic dose of CsA as well.

Animals↗
PubMed1998

HSP60, HSP70 in the pathogenesis of Kawasaki disease: implication and action.

HSP60, HSP70 in plasma of 11 cases of Kawasaki diseases (KD) and 23 healthy children were determined. The two groups were controlled for age. Determination of HSP60, HSP70 was conducted in lymphocytes of 14 cases of KD and 26 healthy children. The results were compared with those of 12 patients with febrile diseases and 10 patients with tuberculosis. Our results showed that except a significant difference in plasma HSP70 found between acute phase and convalescent phase of KD (P < 0.01), no significant difference was found in HSP60, HSP70 among all groups (P > 0.05). The differences in HSP60, HSP70 in lymphocytes were relatively obvious among all groups. The levels of HSP60, HSP70 in acute phase of KD were significantly higher than those in convalescent phase or in healthy controls (P < 0.01). The levels of HSP60 in KD were significantly higher than those of patients with febrile diseases. HSP60 of KD children was significantly lower than those of children with tuberculosis (P < 0.01). The findings showed that HSP60, HSP70 might contribute to the pathogenesis of KD. Determination of HSP60, HSP70 in lymphocytes is of help in the diagnosis of KD.

Chaperonin 60↗
PubMed1998

Immunohistochemical localisation of alpha1B-adrenergic receptors in the rat iris.

Expression of the alpha1B-adrenergic receptor was investigated immunohistochemically in the rat iris, cornea and superior cervical ganglion by using antibodies raised in chickens immunised with a peptide corresponding to a portion of the 3rd intracellular loop common to the human, hamster and rat alpha1B-adrenergic receptor. Antibodies stained COS and HEK cell membranes of cells transfected with DNA encoding and expressing the hamster alpha1B-adrenergic receptor but not membranes from cells transfected with DNA encoding and expressing the rat alpha1A-adrenergic receptor or the rat alpha1D-adrenergic receptor. Staining was abolished by preincubation of the antibodies with the peptide used for immunisation. The distribution of alpha1B-adrenergic receptor was examined immunohistochemically with this antibody (1BI3) and a previously characterised antibody (Ab506) raised in rabbits against the carboxyl-terminal decapeptide of the receptor. In the iris, alpha1B-adrenergic receptor was detected in the dilator muscle, ciliary processes and posterior epithelium but no staining was observed in the superior cervical ganglion with either antibody. By contrast, differences in tissue staining between 1BI3 and Ab506 were observed for the sphincter muscle of the iris and for the cornea. 1BI3 stained both tissues intensely, whereas Ab506 only stained the cornea weakly and the sphincter not at all. Reverse transcription/polymerase chain reaction and nucleotide sequencing confirmed the presence of mRNA encoding the epitopes recognised by 1BI3 and Ab506 in cornea and other tissues. We conclude that (1) there is a good correlation between alpha1B-adrenergic receptor mRNA and protein expression in the iris, (2) mRNA, but not protein, is detected in the superior cervical ganglion and (3) additional processes may regulate receptor expression in the cornea.

Animals↗