Biomedical subjects

S Chen

Publications and source records attributed to S Chen.

At least 523 records · Page 29Linked to original sources

PubMed1999

Characterization of fractalkine in rat brain cells: migratory and activation signals for CX3CR-1-expressing microglia.

Molecular analyses of the chemokine fractalkine and its receptor CX3C-R1 in the rat brain have revealed a striking polarization: fractalkine is expressed constitutively in neurons and is up-regulated by TNF-alpha and IL-1beta in astrocytes. Expression of its specific receptor, CX3C-R1, is restricted to astrocytes and microglia. We have analyzed the functional correlates of this expression and demonstrate that fractalkine induces microglial cell migration and activation. However, the activity of this chemokine on astrocytes may also be highly relevant in inducing astrocyte-microglia cell interactions through cytokine/mediator release leading to microglial activation.

Actins↗

PubMed1999

Direct cloning of cell differential expression genes with full-length by a new strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction.

The paper described a new cDNA subtractive cloning strategy. This strategy was based on the 'cap-finder' method, 'long distance' polymerase chain reaction (PCR), streptavidin magnetic beads mediated subtraction, and spin column chromatography. When PCR products were resolved on agarose gel after three rounds of subtraction, the 'single gene difference' group displayed a predominantly enriched band, but the 'multiple gene difference' group did not display any apparent difference. Of 200 clones inserted with 0.7-2 kb fragments from the 'multiple gene difference' group, 50% were identified to be new sequences and 35% were known. Of 100 new sequences, 35% contained coding regions and 75% were confirmed by dot-blotting to be differentially expressed by genes of the target cell. The results suggested that this strategy might be very efficient for full-length cloning of differentially expressed genes of the cell.

Cloning, Molecular↗

PubMed1999

An Approach of a Nonlinear Dynamic Model for Micellar Effects on Bimolecular Reactions.

The effects of micellar solutions on bimolecular reactions were studied in detail using a newly established nonlinear dynamic model with a far-from-equilibrium reaction scheme, by which the incorporation of organic substrates with the micellar phase was imitated. Through the linear stability analysis of the steady state solutions given by the rate equation of the change of substrate concentration within a micellar phase with time, it leads to some explicit relationships between the incorporated substrate concentration and the relevant parameters. Based on this model the micellar effects on the kinetics of the alkaline hydrolysis of esters have been interpreted. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Phe310 in transmembrane VI of the alpha1B-adrenergic receptor is a key switch residue involved in activation and catecholamine ring aromatic bonding.

Pharmacophore mapping of adrenergic receptors indicates that the phenyl ring of catecholamine agonists is involved in receptor binding and activation. Here we evaluated Phe310, Phe311, and Phe303 in transmembrane VI (TMVI), as well as Tyr348 in TMVII of the alpha1B-adrenergic receptor (alpha1B-AR), which have been implicated in a catechol-ring interaction. Neither catecholamine docking studies nor mutagenesis studies of Phe311, Phe303, or Tyr348 supported a role for these residues in catechol-ring binding. By contrast, docking studies indicated that the Phe310 side chain is well positioned to interact with the catechol-ring, and substituted cysteine accessibility method studies revealed that the side chain of the 310, but not 311 residue, is both solvent accessible and directed into the agonist-binding pocket. Also, saturation mutagenesis of both Phe310 and Phe311 revealed for the former, but not for the latter, a direct relationship between side chain volume and agonist affinity, and that aromaticity is essential for wild-type agonist binding, and for both wild-type agonist potency and efficacy. Moreover, studies of Phe310 mutants combined with a previously described constitutively active alpha1B-AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R', interaction of Phe310 with catecholamine agonists is essential for isomerization from R' to the fully activated state, R.

2-Hydroxyphenethylamine↗

PubMed1999

[Effects of unarmed marching in dry-heat desert environment on the heat shock protein 27 in human blood].

To explore the variation of blood heat shock protein 27(HSP27) for unarmed marching in dry-heat environment, plasma HSP27 levels of 57 marchers and 14 controls were detected. The results revealed a significant difference between exposed and control groups (P < 0.01), and no significant difference among different time exposures at the same speed. HSP27 levels increased slightly with the increase of speed. During the second hour of marching, HSP27 levels were slightly lower, and kept steady in the third hour.

Adolescent↗

PubMed1999

Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae.

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.

Bacterial Proteins↗

PubMed1999

Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB.

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-kappaB) and bound RICK, a CARD-containing kinase that also induces NF-kappaB activation. Nod1 mutants inhibited NF-kappaB activity induced by RICK, but not that resulting from tumor necrosis factor-alpha stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-kappaB activation pathways.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice.

Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic beta cells is upregulated by cytotoxic cytokines like IL-1beta. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent beta-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1beta, TNF-alpha, and IFN-gamma for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-gamma + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin-induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide.

Animals↗

PubMed1999

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.

Adenosine Diphosphate↗

PubMed1999

cDNA expression array reveals heterogeneous gene expression profiles in three glioblastoma cell lines.

Tumor cell lines are an indispensable tool for cancer research. However, among cell lines of the same pathological group, heterogeneity has been detected in gene expression, gene mutation, and cellular response to various treatments. In this study, we systematically investigated the extent of heterogeneity of gene expression in three glioblastoma cell lines using cDNA array technology in which the expression of 588 cellular genes is studied simultaneously. Comparison of the expression profiles revealed substantial qualitative and quantitative heterogeneity. Among the 588 genes, 197 genes were expressed in all three lines and 56 genes were not expressed in any of the three lines; total of 222 genes were expressed in only two of the three cell lines, and 113 genes were expressed in only one of the three cell lines. These results provide molecular evidence that cell lines of the same pathological origin can be highly heterogeneous.

DNA, Complementary↗

PubMed1999

Identification of the individual residues that determine human CD59 species selective activity.

Formation of the cytolytic membrane attack complex of complement on host cells is inhibited by the membrane-bound glycoprotein, CD59. The inhibitory activity of CD59 is species restricted, and human CD59 is not effective against rat complement. Previous functional analysis of chimeric human/rat CD59 proteins indicated that the residues responsible for the species selective function of human CD59 map to a region contained between positions 40 and 66 in the primary structure. By comparative analysis of rat and human CD59 models and by mutational analysis of candidate residues, we now identify the individual residues within the 40-66 region that confer species selective function on human CD59. All nonconserved residues within the 40-66 sequence were substituted from human to rat residues in a series of chimeric human/rat CD59 mutant proteins. Functional analysis revealed that the individual human to rat residue substitutions F47A, T51L, R55E, and K65Q each produced a mutant human CD59 protein with enhanced rat complement inhibitory activity with the single F47A substitution having the most significant effect. Interestingly, the side chains of the residues at positions 47, 51, and 55 are all located on the short single helix (residues 47-55) of CD59 and form an exposed continuous strip parallel to the helix axis. A single human CD59 mutant protein containing rat residue substitutions at all three helix residues produced a protein with species selective activity comparable to that of rat CD59. We further found that synthetic peptides spanning the human CD59 helix sequence were able to inhibit the binding of human CD59 to human C8, but had little effect on the binding of rat CD59 to rat C8.

Amino Acid Sequence↗

PubMed1999

Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly.

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8-9-fold by cotransfected hRXRalpha. Induction by K386A, in the presence or absence of RXRalpha, was only slightly lower than that seen with wild type VDR. On the other hand, I384R/Q385R alone displayed no stimulatory activity and less than 2-fold induction in the presence of hRXRalpha. Qualitatively similar findings were observed with the negative regulation of the human atrial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.

Amino Acid Sequence↗

PubMed1999

CIPER, a novel NF kappaB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10.

We have identified and characterized CIPER, a novel protein containing a caspase recruitment domain (CARD) in its N terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or Apaf-1. Expression of CIPER induced NF-kappaB activation, which was inhibited by dominant-negative NIK and a nonphosphorylable IkappaB-alpha mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was sufficient and necessary for NF-kappaB-inducing activity. Point mutations in highly conserved residues in the CARD of CIPER disrupted the ability of CIPER to activate NF-kappaB and to form homodimers, indicating that the CARD is essential for NF-kappaB activation and dimerization. We propose that CIPER acts in a NIK-dependent pathway of NF-kappaB activation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

B7-2 is required for the progression but not the initiation of the type 2 immune response to a gastrointestinal nematode parasite.

T cells require CD28/CTLA-4 costimulatory molecule interactions in addition to Ag-specific signals through the TCR for in vivo effector Th cell function. Some studies have suggested that the ligands for these costimulatory molecules may differentially influence effector T cell function with B7-2 favoring a type 2 response and B7-1 favoring a type 1 response, while other studies have suggested that these molecules may be redundant. The recent development of B7-2-deficient mice permits the direct analysis of the requirement of B7-2 during a type 2 immune response to an infectious pathogen. We have examined, in B7-2-deficient mice, effector Th cell function and the associated type 2 immune response following infection with Heligmosomoides polygyrus, a natural murine parasitic nematode. Elevations in cytokine gene expression and protein secretion were pronounced and comparable in inoculated B7-2-/- and B7-2+/+ mice at day 8 after H. polygyrus inoculation. However, by day 14 after infection, increases in T cell cytokine expression were markedly inhibited in H. polygyrus-inoculated B7-2-/- mice. Furthermore, elevations in serum IgE and germinal center formation were inhibited at later stages of the immune response, while elevations in serum IgG1 persisted. These findings suggest that certain T-dependent components vary in their B7-2-dependency during the type 2 immune response. They further demonstrate that B7-2 interactions are not necessary for the initiation of the type 2 immune response, but are instead required for its progression after the development of effector T cells.

Animals↗

PubMed1999

Low-dose whole-body irradiation induces alteration of protein expression in mouse splenocytes.

In previous studies, the stimulatory effect on immunological function and adaptive response in splenocytes were found after exposure of mice to low-dose whole-body irradiation (LD-WBI). Protein synthesis was found to be required for these responses. The present study, therefore, attempts to investigate the protein changes in mouse splenocytes after exposure to LD-WBI. Two-dimensional gel electrophoresis was used to show the alterations of proteins in nuclei, cytoplasm and extracellular fluid of the splenocytes at 4 h after mice exposed to 75 mGy X-rays. As compared to control, changed expressions of 20, 15 and 6 proteins, in sizes ranging from 10 to 69 kDa with pI of 5.0-8.2, were found in the nuclei, cytoplasm and extracellular fluid of splenocytes, respectively. One protein, shown in the cytoplasm of splenocytes of control mice, appeared in extracellular fluid of splenocytes after LD-WBI. Two proteins, shown in cytoplasm, and one protein, shown in extracellular fluid of splenocytes in control mice, appeared in the nuclei after LD-WBI. Time-course of protein synthesis varied in different proteins after LD-WBI. These results suggest that alterations of protein expressions and redistribution of proteins between intracellular and extracellular compartments occurred in the splenocytes after LD-WBI. Protein extract from LD-WBI splenocytes was fractionated by Sephadex G-100. Fractions 61-80 contained proteins able to protect lymphocytes in vitro from radiation-induced chromosome aberrations. These findings are of importance in elucidating mechanisms of immuno-enhancement and adaptive response induced by low-dose radiation. although the features and the functions of these proteins remain to be elucidated in future studies.

Animals↗

PubMed1999

Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1.

The effects of L1-Fc and CHL1-Fc fusion proteins on neuronal survival were investigated. Cerebellar granule neurons of mouse and hippocampal neurons of rat embryo undergo apoptosis when cultured in serum-free medium. Treatment with chimeric proteins containing the extracellular domains of the neural adhesion molecules L1 or CHL1 fused to the Fc region of human immunoglobulin significantly enhanced the survival of neurons. Compared to the control, the percentage of surviving neurons increased about 60% and 45% with L1 and CHL1 fusion proteins, respectively. A fusion protein containing the extracellular domain of NCAM had no effect on survival. The L1 and CHL1 fusion proteins were effective both in soluble form or when offered as a substrate, with the maximal effect at about 1 microg/mL. To explore the intracellular events related to the neuronal survival effects of L1-Fc fusion protein, Bcl-2 and c-Jun expression were analyzed by Western blotting. The level of Bcl-2 in cerebellar granule neurons was increased by treatment with L1-Fc at both 1 and 5 days of culture. The level of c-Jun was not significantly affected at the early time point and was reduced by L1-Fc fusion protein after long-term culture. The results demonstrate that the neural adhesion molecule L1 and its relative CHL1 are potential neuronal survival factors for neurons of the central nervous system. Bcl-2 may serve as one of the intracellular mediators of the neuronal survival effects of L1.

Animals↗

PubMed1999

An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear.

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.

Antibodies, Monoclonal↗

PubMed1999

Persistent effect of in utero meso-2,3-dimercaptosuccinic acid (DMSA) on immune function and lead-induced immunotoxicity.

Meso-2,3-dimercaptosuccinic acid (DMSA) is a drug currently employed for cheltion therapy in lead poisoning; however, little is known about its potential effects on the immune system. To examine the effect of DMSA and its capacity to reverse immunotoxicity resulting from exposure to lead in utero, female Fischer 344 rats were administered lead acetate in drinking water from 2 weeks prior to mating until parturition; DMSA was given by gavage on days 6-21 of gestation. The immune function of the female offspring was tested at 13 weeks of age. The results showed that lead (250 ppm) suppressed Th1-type responses (delayed-type hypersensitivity (DTH), interferon gamma (IFN gamma) production), enhanced a Th2-type response (interleukin-4 (IL-4) production), and increased tumor necrosis factor alpha (TNF alpha) production from macrophages. DMSA treatment (60 mg/kg per day) during pregnancy significantly lowered the blood lead levels of both the embryos and the lactating dams as well as the milk lead level of lactating dams. The chelation treatment also reversed the lead-induced alterations in pup body weight, relative spleen weight, TNF alpha, and IL-4 production. But in utero exposure to DMSA alone resulted in decreased DTH response in adult offspring. This was likely due to a reduced cell recruitment, since plasma monocyte chemoattractant protein-1 (MCP-1) levels were decreased. The DMSA-exposed offspring also demonstrated increased interleukin-2 (IL-2) production. These results suggest that DMSA reverses some of the lead-induced immunotoxicity; however, this treatment itself during embryonic development produces subsequent adult immunomodulation.

Animals↗