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Biomedical subjects

C Chen

Publications and source records attributed to C Chen.

At least 145 records · Page 8Linked to original sources
PubMed2001

Heterosynaptic LTP in early development.

The formation of precise synaptic connections in the developing central nervous system involves activity-dependent control of synaptic strength. Correlated activity between the afferent input and postsynaptic neuron strengthens connections through long-term potentiation (LTP), while uncorrelated synaptic inputs are selectively diminished. In this issue of Neuron, Tao et al. report that LTP in young animals has less synaptic specificity than in older animals, indicating that the properties of LTP change during development.

Animals↗
PubMed2001

Multiple phosphorylation sites in RGS16 differentially modulate its GAP activity.

Regulators of G-protein signaling (RGS) are GTPase-activating proteins (GAP) for activated Galpha subunits. We found that mouse RGS16, when expressed in HEK293T cells, is phosphorylated constitutively at serine 194 based on in vivo orthophosphate labeling experiments, while serine 53 is phosphorylated in a ligand-dependent manner upon stimulation by epinephrine in cells expressing the alpha2A adrenergic receptor. Phosphorylation on both sites impairs its GAP activity and subsequent attenuation on heterotrimeric G-protein-stimulated extracellular signal-regulated protein kinase activity. This is the first report of RGS functional downregulation by phosphorylation via a G-protein-coupled receptor.

Cell Line↗
PubMed2001

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Antibodies↗
PubMed2001

Measurement of the top quark p(T) distribution.

We have measured the p(T) distribution of top quarks that are pair produced in pp collisions at sqrt[s] = 1.8 TeV using a sample of tt decays in which we observe a single high- p(T) charged lepton, a neutrino, and four or more jets. We use a likelihood technique that corrects for the experimental bias introduced due to event reconstruction and detector resolution effects. The observed distribution is consistent with the standard model prediction. We use these data to place limits on the production of high- p(T) top quarks suggested in some models of anomalous top quark pair production.

Journal Article↗
PubMed2001

[Model of development of diabetes mellitus in adult Chinese].

OBJECTIVE: To explore the model for development of non-insulin dependent diabetes and the roles of insulin resistance and insulin secretion impairment in the initiation of diabetes in adult Chinese. METHODS: 432 non-diabetics aged 24-74, 126 with normal glucose tolerance (NGT) and 307 with impaired glucose tolerance (IGT), were selected by standard oral glucose tolerance test (OGTT) and plasma insulin concentration measurement in Daqing, Heilongjiang Province, in 1986, and then followed up for six years. OGTT was conducted every two years. The subjects with fasting plasma glucose (FPG) > or = 7.8 mmol/L and/or two hour plasma glucose (PG2h) > or = 11.1 mmol/L were diagnosed as with diabetes. The effect of insulin resistance and insulin secretion at baselie on worsening of glucose tolerance in both NGT and IGT groups were analyzed using SAS software. RESULTS: 70% of DM cases developed from the insulin-resistant IGT subgroup at the end of the study. About 30% of DM cases were not insulin-resistant in their IGT stage. Proportional hazard analysis showed that the baseline PG2h and BMI significantly positively correlated to the development of diabetes during the six years follow-up period (RR = 1.35 and 1.32 respectively, P < 0.01). Dietary and exercise intervention significantly decreased such risk. With these confounders being controlled, baseline insulin sensitivity and insulin secretion index significantly negatively correlated to the development of diabetes in the IGT group (RR = 0.41 and 0.39 respectively, P < 0.01). Incidence of IGT (or DM) in the insulin resistant NGT subgroup was much higher than that in the non-insulin resistant NGT subgroup (34.5% vs 13.4%, P < 0.05), stepwise logistic regression analysis showed that only the baseline insulin sensitivity entered the model and negatively associated to the risk of worsening of glucose tolerance (P < 0.05). CONCLUSION: A two-step model of development of diabetes mellitus is shown among most adult Chinese diabetics. In the first step NGT develops to IGT when insulin resistance play a major role. In the second step, IGT develops to diabetes when both insulin resistance and beta-cell secretion impairment play a major role.

Adult↗
PubMed2001

A small molecule ligand of the glucagon-like peptide 1 receptor targets its amino-terminal hormone binding domain.

The glucagon-like peptide 1 receptor (GLP-1R) belongs to a distinct subgroup of G protein-coupled peptide hormone receptors (class B) that has been difficult to target by small molecule drugs. Here, we report that a non-peptide compound, T-0632, binds with micromolar affinity to the human GLP-1R and blocks GLP-1-induced cAMP production. Furthermore, the observation that T-0632 has almost 100-fold selectivity for the human versus the highly homologous rat GLP-1R provided an opportunity to map determinants of non-peptide binding. Radioligand competition experiments utilizing a series of chimeric human/rat GLP-1R constructs revealed that partial substitution of the amino terminus of the rat GLP-1R with the corresponding sequence from the human homolog was sufficient to confer high T-0632 affinity. Follow-up analysis of receptors where individual candidate amino acids had been exchanged between the human and rat GLP-1Rs identified a single residue that explained species selectivity of non-peptide binding. Replacement of tryptophan 33 in the human GLP-1R by serine (the homologous amino acid in the rat GLP-1R) resulted in a 100-fold loss of T-0632 affinity, whereas the converse mutation in the rat GLP-1R led to a reciprocal gain-of-function phenotype. These observations suggest that in a class B receptor, important determinants of non-peptide affinity reside within the extracellular amino-terminal domain. Compound T-0632 may mimic, and thereby interfere with, the putative "pseudo-tethering" mechanism by which the amino terminus of class B receptors initiates the binding of cognate hormones.

Allosteric Regulation↗
PubMed2001

Intra-supraoptic nucleus sulpiride improves anorexia in tumor-bearing rats.

Previous studies suggest that the dopaminergic system in the supraoptic nucleus (SON) is involved not only in the water balance control but also in the food intake regulation. Since we reported that an injection of the D2 receptor antagonist, sulpiride, into specific hypothalamic nuclei (e.g. the LHA, or the VMN) increases food intake in anorectic tumor-bearing rats, as well as in normal rats, we hypothesized that an injection of sulpiride into the SON would also improve cancer anorexia. Sulpiride injection (4 microg/0.5 microl) into bilateral SON of anorectic tumor-bearing male rats significantly improved food intake via increases in both meal size and meal number. These data suggest that pharmacological manipulation of the hypothalamic dopaminergic system is feasible in amelioration of cancer anorexia.

Animals↗
PubMed2001

Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc.

Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

Animals↗
PubMed2001

Vanadium, molybdenum, and sodium triethanolamine complexes derived from an assembly system containing tetrathiometalate and triethanolamine.

The reaction system composed of triethanolamine (TEA) and tetrathiovanadate in the presence of MeONa (MeOLi) or NaNH2 was studied to afford vanadium and alkali metal TEA complexes. Complexes [M subsetV6O6[N(CH2CH2O)2(CH2CH2OH)]6]2S6 (M = Na (1), Li (2)) contain two cyclic V((IV)) [12]metallacrown-6 cations linked by a polysulfide S6(2-) anion. Also separated from the reaction system is a novel sodium TEA complex [[Na(TEA)]2S6]n (3), in which parallel coordination chains [Na(TEA)+]n are connected by O-H...S hydrogen bonds forming 3-D network structure. Variable-temperature conductance of 3 was determined to display semiconductor feature. The desulfurization of VS4(3-) was observed to form S6(2-) anion and/or S8 molecule in the synthetic reactions of 1-3. A striking contrast was noted that MoS4(2-) did not desulfurize in the similar reaction to that of VS4(3-) with TEA, and a Na/TEA complex containing MoS4(2-), (Et4N)2[Na2(mu-TEA)2(CH3OH)2](MoS4)2 (4), was obtained instead. The S...H-O and O...H-O hydrogen bonds play an important role in forming the one- or three-dimensional structures for all these complexes by the linkages between clusters and chains. The IR spectrum indicates the structural similarity of complexes 1 and 2. For complex 4, the Mo=S stretch vibration at 468 cm(-1) showing widening and slight red-shift and the slight movement of the 1H NMR signals of TEA ligand to downfield are considered to be the influence of the S...H-O hydrogen bonds. Variable temperature magnetic susceptibility data were collected for complex 1. The effect magnetic moment per V6 unit varies gradually from 4.04 muB at 300 K to 6.24 muB at 5 K, exhibiting ferromagnetic interaction. Heisenberg-type vector-coupling model and molecular field approximation were used to treat the interaction between the paramagnetic sites and gave a good fitting result: J = 3.97 cm(-1), J' = 1.99 cm(-1), g = 1.99, F = 3.64 x 10(-3). Density functional calculation was also performed to complex 1 and its Li, K analogues. The lowest total bonding energy of -874.001 eV was obtained for [Na subsetV6(mu3-O)6] complex containing six unpaired electrons, indicating the stability of a V6 system with six independent spins S = 1/2. When the alkali metal ion is inserted into the [V6(mu3-O)6] cavity, the positive values of the bonding interaction (E(b)) indicate that the insertion depresses the total energy and that complexes 1 and 2 together with K+ complex may exist. However, the lowest E(b) value of the K+ complex implies its instability.

Journal Article↗
PubMed2001

Comparison of the amino acid residues in the sixth transmembrane domains accessible in the binding-site crevices of mu, delta, and kappa opioid receptors.

We have mapped the residues in the sixth transmembrane domains (TMs 6) of the mu, delta, and kappa opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the mu and kappa receptors and the wild-type delta receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat mu receptor and 9 of 22 mutants of the human delta receptor and 10 of 22 mutants of the human kappa receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C mu mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the mu receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the delta receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the kappa receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the mu, delta, and kappa opioid receptors are consistent with the notion that the TMs 6 are in alpha-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure.

Amino Acid Substitution↗
PubMed2001

Identification of a nonpeptide ligand that releases bioactive insulin-like growth factor-I from its binding protein complex.

Insulin-like growth factor-I (IGF-I) has both metabolic and mitogenic activities mediated through interaction with the type 1 IGF receptor. The circulation of IGF-I in blood and interstitial fluid is not free but bound mostly to a family of six high affinity IGF-binding proteins, which form stable complexes with IGF and neutralize its bioactivity. Therefore, displacement of this large pool of endogenous IGF from the binding proteins could elevate "free" IGF levels to elicit beneficial effects in diabetes and other IGF-responsive diseases comparable with those produced by administration of exogenous IGF-I. We report here the identification of a nonpeptide ligand NBI-31772, which displaces IGF-I from all six IGF-binding proteins at low nanomolar concentrations from screening of the in-house chemical libraries. Furthermore, the released free IGF-I was shown to be biologically active in an in vitro bioassay. Thus, NBI-31772 could serve as a valuable lead molecule for the design of novel therapeutics to treat diabetes and other IGF-responsive diseases.

Animals↗
PubMed2001

Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae.

Gross chromosome rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions and inversions, are often observed in cancer cells. Spontaneous GCRs are rare in Saccharomyces cerevisiae; however, the existence of mutator mutants with increased genome instability suggests that GCRs are actively suppressed. Here we show by genetic analysis that these genome rearrangements probably result from DNA replication errors and are suppressed by at least three interacting pathways or groups of proteins: S-phase checkpoint functions, recombination proteins and proteins that prevent de novo addition of telomeres at double-strand breaks (DSBs). Mutations that inactivate these pathways cause high rates of GCRs and show synergistic interactions, indicating that the pathways that suppress GCRs all compete for the same DNA substrates.

DNA Damage↗
PubMed2001

HIV envelope proteins differentially utilize CXCR4 and CCR5 coreceptors for induction of apoptosis.

The involvement of CXCR4 and CCR5 coreceptors in apoptosis induced by the HIV envelope (Env) proteins has not been well defined. We found that simian human immunodeficiency virus (SHIV) virus-like particles (VLPs) containing HIV Env proteins preferentially induce apoptosis of cells corresponding to their coreceptor usage in a CD4+ T cell line. We also demonstrated that induction of apoptosis by SHIV VLPs is correlated with coreceptor usage in a non-T cell line. We examined the effects of SHIV VLPs containing Env proteins derived from either a T-cell-tropic HIV (BH10) strain or a dual-tropic HIV (89.6) strain on induction of apoptosis in recombinant CD4+ human osteosarcoma (HOS) cells expressing either CXCR4 (HOS-CD4.CXCR4) or CCR5 coreceptors (HOS-CD4.CCR5). HOS-CD4.CXCR4 or HOS-CD4.CCR5 cells were activated with concanavalin A and cocultured with VLPs. By TUNEL (TdT-mediated dUTP-X nick end labeling) fluorescence staining and flow cytometry assays, SHIV BH10 VLPs were found to preferentially induce apoptosis in HOS-CD4.CXCR4 cells but not in HOS-CD4 or HOS-CD4.CCR5 cells. On the other hand, SHIV 89.6 VLPs induced an elevated level of apoptosis in both HOS-CD4.CXCR4 and HOS-CD4.CCR5 cells in a dose-dependent fashion. These data demonstrate that T-cell-tropic BH10 Env preferentially utilizes CXCR4, but not CCR5, for induction of apoptosis, whereas dual-tropic 89.6 Env induces apoptosis in both CXCR4- and CCR5-containing cell lines.

Apoptosis↗
PubMed2001

Tofu consumption and blood lead levels in young Chinese adults.

Tofu is a commonly consumed food in China. Tofu may interfere with lead absorption and retention because of its high calcium content. In this observational study, the authors examined whether dietary tofu intake was associated with blood lead levels among young adults in Shenyang, China. The analyses included 605 men and 550 women who completed baseline questionnaires and had blood lead measurements taken in 1996-1998 as part of a prospective cohort study on reproductive health. Mean blood lead levels were 13.2 microg/dl in men and 10.1 microg/dl in women. Blood lead levels were negatively associated with tofu intake in both genders. A linear trend test showed a 3.7% (0.5-microg/dl) decrease in blood lead level with each higher category of tofu intake (p = 0.003). The highest tofu intake group (> or =750 g/week) had blood lead levels 11.3% lower (95% confidence interval: 4.1, 18.0) than those of the lowest tofu intake group (<250 g/week). In all regression models, data were adjusted for gender, age, height, body mass index, district, cigarette smoking, alcohol drinking, education, occupation, use of vitamin supplements, season, and dietary intake of meat, fish, vegetables, eggs, and milk. In conclusion, the authors found a significant inverse dose-response relation between tofu consumption and blood lead levels in this Chinese population.

Adult↗
PubMed2001

Three-dimensional interaction of Phi29 pRNA dimer probed by chemical modification interference, cryo-AFM, and cross-linking.

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer --> tetramer --> hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81 of the left stem were found to be oriented toward the head loop and proximate to bases 26-31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45-71 and 82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.

Bacillus Phages↗
PubMed2001

Location and mechanism of alpha 2,6-sialyltransferase dimer formation. Role of cysteine residues in enzyme dimerization, localization, activity, and processing.

A significant proportion of the alpha2,6-sialyltransferase of protein Asn-linked glycosylation (ST6Gal I) forms disulfide-bonded dimers that exhibit decreased activity, but retain the ability to bind asialoglycoprotein substrates. Here, we have investigated the subcellular location and mechanism of ST6Gal I dimer formation, as well as the role of Cys residues in the enzyme's trafficking, localization, and catalytic activity. Pulse-chase analysis demonstrated that the ST6Gal I disulfide-bonded dimer forms in the endoplasmic reticulum. Mutagenesis experiments showed that Cys-24 in the transmembrane region is required for dimerization, while catalytic domain Cys residues are required for trafficking and catalytic activity. Replacement of Cys-181 and Cys-332 generated proteins that are largely retained in the endoplasmic reticulum and minimally active or inactive, respectively. Replacement of Cys-350 or Cys-361 inactivated the enzyme without compromising its localization or processing, suggesting that these amino acids are part of the enzyme's active site. Replacement of Cys-139 or Cys-403 generated proteins that are catalytically active and appear to be more stably localized in the Golgi, since they exhibited decreased cleavage and secretion. The Cys-139 mutant also exhibited increased dimer formation suggesting that ST6Gal I dimers may be critical in the oligomerization process involved in stable ST6Gal I Golgi localization.

Amino Acid Substitution↗
PubMed2001

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense.

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Cloning, Molecular↗