Biomedical subjects

X Chen

Publications and source records attributed to X Chen.

At least 415 records · Page 23Linked to original sources

PubMed2000

The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the beta 2-adrenergic receptor.

Previous work in the beta(2)-adrenergic receptor demonstrated critical interactions between Ser-204 and Ser-207 in the fifth membrane-spanning segment and the meta-OH and para-OH, respectively, of catecholamine agonists (Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. (1989) J. Biol. Chem. 264, 13572-13578). Using the substituted cysteine accessibility method in the beta(2)-adrenergic receptor, we have found that in addition to Ser-204 and Ser-207, Ser-203 is also accessible on the surface of the binding-site crevice and is occluded by bound agonist. Mutation of Ser-203 to Ala, Val, or Cys reduced the binding affinity and adenylyl cyclase-activating potency of agonists containing a meta-OH, whereas their affinities and potencies were largely preserved by mutation of Ser-203 to Thr, which maintained an OH at this position. Thus both Ser-203 and Ser-204 appear to interact with the meta-OH of catecholamines, perhaps through a bifurcated H bond. Furthermore, the removal of the OH at position 203 led to a significant loss of affinity of antagonists with nitrogen in their heterocyclic ring structure. The greatest effect was seen with pindolol, a partial agonist, suggesting that a H bond between the heterocyclic ring and Ser-203 may play a role in partial agonism. In contrast, the affinities of antagonists such as propranolol or alprenolol, which have cyclic structures without H-bonding capability, were unaltered after mutation of Ser-203.

Adrenergic beta-2 Receptor Antagonists↗

PubMed2000

Key role for the epsilon isoform of protein kinase C in painful alcoholic neuropathy in the rat.

Chronic alcohol consumption produces a painful peripheral neuropathy for which there is no reliably successful therapy, attributable to, in great part, a lack of understanding of the underlying mechanisms. We tested the hypothesis that neuropathic pain associated with chronic alcohol consumption is a result of abnormal peripheral nociceptor function. In rats maintained on a diet to simulate chronic alcohol consumption in humans, mechanical hyperalgesia was present by the fourth week and maximal at 10 weeks. Thermal hyperalgesia and mechanical allodynia were also present. Mechanical threshold of C-fibers in ethanol fed rats was lowered, and the number of action potentials during sustained stimulation increased. The hyperalgesia was acutely attenuated by intradermal injection of nonselective protein kinase C (PKC) or selective PKCepsilon inhibitors injected at the site of nociceptive testing. Western immunoblot analysis indicated a higher level of PKCepsilon in dorsal root ganglia from alcohol-fed rats, supporting a role for enhanced PKCepsilon second-messenger signaling in nociceptors contributing to alcohol-induced hyperalgesia.

Action Potentials↗

PubMed2000

Purification, characterization, and cDNA sequence of halysetin, a disintegrin-like/cysteine-rich protein from the venom of Agkistrodon halys Pallas.

By means of DEAE-Sepharose CL-6B column chromatography, gel filtration on Sephadex G-75 and Superose 12 FPLC, halysetin, an antiplatelet protein, was purified from the venom of Agkistrodon halys Pallas with molecular mass of 29 kDa on SDS-PAGE and 23,168 Da by mass spectrometry. The p1 was about 5.0. Halysetin was devoid of phospholipase A2, fibrino-(geno)lytic, esterase, hemorrhagenic activities. Halysetin dose-dependently inhibited the aggregation of human platelet, which was stimulated by collagen with IC50 of 420 nM, but not that stimulated by ADP. The N-and C-terminal sequences of halysetin were characterized. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. It encoded a protein of 212-amino-acid residues with disintegrin-like/cysteine-rich domains and was highly homologous with SYMPs (snake venom metalloprotease).

Adenosine Diphosphate↗

PubMed2000

Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers.

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.

Amino Acid Substitution↗

PubMed2000

Induction of inducible nitric oxide synthase by ginsenosides in cultured porcine endothelial cells.

Mechanism of Nitric oxide (NO) production by ginsenosides was investigated in cultured porcine endothelial cells. Beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) staining showed that the NO production was significantly enhanced by the presence of 40 microg/ml ginsenosides with 10 microM L-arginine after 12 h incubation. NO production was suppressed by addition of 0.5 microM Nomega-Nitro-L-arginine (L-NNA), an inhibitor of NO synthases (NOSs), to the incubation medium. In addition, the immunoreactive signals of inducible NOS (iNOS) were appeared in endothelial cells after 12-h incubation of ginsenosides, whereas the signals were not observed in non-treated cells. Our findings suggest that ginsenosides can enhance NO production by induction of iNOS in addition to its direct effect on endothelial cells by increasing intracellular Ca2+ concentration.

Animals↗

PubMed2000

Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3.

Overexpression of ErbB2 has been found in approximately 25-30% of human breast cancers and has been shown to render the cancer cells more resistant to chemotherapy. However, it is not clear whether ErbB2 overexpression renders the cells more resistant to specific anti-cancer drugs or renders the cells more resistant to a broad range of anti-cancer drugs. It is not clear how the function of ErbB2 in drug resistance is related to expression and activation of the other ErbB receptors. In this communication, we showed that several breast cancer cell lines including BT20, BT474, MCF-7, MDA-MB-453, and SKBR-3 cells had a similar pattern of resistance to a broad range of anti-cancer drugs including 5-Fluorouracil, Cytoxan, Doxorubincin, Taxol, and Vinorelbin, suggesting a mechanism of multidrug resistance. High expression of P-glycoprotein and the ErbB receptors contribute to drug resistance of these breast cancer cells; however, overexpression of ErbB2 alone is not a major factor in determining drug resistance. To further determine the role of the ErbB receptors in drug resistance, we selected various NIH 3T3 cell lines that specifically expressed EGF receptor (EGFR), ErbB2, ErbB3, EGFR/ErbB2, EGFR/ErbB3, or ErbB2/ErbB3. A cytotoxicity assay showed that expression of ErbB2 alone did not significantly enhance drug resistance, whereas coexpression of either EGFR or ErbB3 with ErbB2 significantly enhanced drug resistance. Moreover, ErbB2 was highly phosphorylated in NIH 3T3 cells that coexpress ErbB2 with either EGFR or ErbB3, but not in NIH 3T3 cells that express ErbB2 alone. Together, our results suggest that coexpression of EGFR or ErbB3 with ErbB2 induces high phosphorylation of ErbB2 and renders the cells more resistant to various anti-cancer drugs.

3T3 Cells↗

PubMed2000

Determination of proteins at nanogram levels based on their enhancement effects of Rayleigh light scattering on dibromomethylchlorophosphonazo.

A new Rayleigh light scattering (RLS) assay of protein was conducted in this paper. At the optimum pH conditions, and in the presence of Tween-20, the weak RLS of dibromomethylchlorophosphonazo (DBM-CPA) can be enhanced greatly by the addition of proteins. Based on this, the reactions of DBM-CPA and proteins were studied. A new quantitative determination method for proteins has been developed. The method is simple, practical and relatively free from interference from coexisting substances, as well as much more sensitive (the dynamic ranges of 0.065-40.05 microg ml(-1) and detection limit of 30 ng ml(-1) for bovine serum albumin (BSA)) than most of the existing assays. The determination results of human body serum samples are identical to those by the CBB method, with relative S.D. of six determination of 0.5-2.2%.

Blood Proteins↗

PubMed2000

Signal peptidase and oligosaccharyltransferase interact in a sequential and dependent manner within the endoplasmic reticulum.

We demonstrate that the signal peptides of prepro-alpha-factor and preinvertase must be cleaved before Asn-X-Ser/Thr acceptor tripeptides located near the signal peptides of these precursors can be efficiently glycosylated within the endoplasmic reticulum of the yeast Saccharomyces cerevisiae. The data support a model whereby the interaction of a signal peptide with the membrane prevents an acceptor tripeptide juxtaposed to the signal peptide from accessing the oligosaccharyltransferase active site until the signal peptide is cleaved.

Amino Acid Sequence↗

PubMed2000

[The effect of FHIT gene on renal cell carcinomas].

OBJECTIVE: To study the potential role of FHIT gene in renal cell carcinomas. METHODS: Cancerous tissues derived from 23 patients with renal cell carcinomas and an established cell line were examined for the alteration of FHIT gene transcripts and structure of genomic DNA by using RT-PCR and Southern blot hybridization assays. RESULTS: Eight point three percent(2/24) of cancerous tissues exhibited aberrant transcripts. The transcript in the renal-cell cancer cell line appeared a small-size one when cDNA was amplified with outside nest primer; absent expression of transcript was shown when amplified with inside nest primer. About twenty percent(5/24) cancerous tissues showed genomic rearrangement in FHIT gene locus. CONCLUSION: FHIT gene exhibited aberrant transcripts and genomics in some of renal cell cancers. Therefore change of FHIT gene may play a role in development of renal cell carcinomas.

Acid Anhydride Hydrolases↗

PubMed2000

CYP17 promoter polymorphism and breast cancer in Australian women under age forty years.

BACKGROUND: The cytochrome P450c17alpha enzyme functions in the steroid biosynthesis pathway, and altered endogenous steroid hormone levels have been reported to be associated with a T to C polymorphism in the 5' promoter region of the CYP17 gene. Because steroid hormone exposure is known to influence breast cancer risk, we conducted a population-based, case-control-family study to assess the relationship between the CYP17 promoter polymorphism and early-onset breast cancer. METHODS: Case subjects under 40 years of age at diagnosis of a first primary breast cancer, population-sampled control subjects, and the relatives of both case and control subjects were interviewed to record family history of breast cancer and other risk factors. CYP17 genotype was determined in 369 case subjects, 284 control subjects, and 91 relatives of case subjects. Genotype distributions were compared by logistic regression, and cumulative risk was estimated by a modified segregation analysis. All statistical tests were two-tailed. RESULTS: Compared with the TT genotype (i.e., individuals homozygous for the T allele), the TC genotype was not associated with increased breast cancer risk (P: =.7). Compared with the TT and TC genotypes combined, the CC genotype was associated with a relative risk of 1. 81 (95% confidence interval [CI] = 1.15-2.86; P: =.01) before adjustment for measured risk factors and 1.63 (95% CI = 1.00-2.64; P: =.05) after adjustment. There was an excess of CC genotypes in case subjects who had at least one affected first- or second-degree relative, compared with control subjects unstratified by family history of breast cancer (23% versus 11%; P: =.006), and these case subjects had a threefold to fourfold higher risk than women of other groups defined by genotype and family history of breast cancer. Analysis of breast cancer in first- and second-degree relatives of case subjects with the CC genotype, excluding two known carriers of a deleterious mutation in BRCA1 or BRCA2, gave a relative hazard in women with the CC genotype of 3.48 (95% CI = 1.13-10.74; P: =.04), which is equivalent to a cumulative risk of 16% to age 70 years. CONCLUSIONS: The CC genotype may modify the effect of other familial risk factors for early-onset breast cancer.

Adult↗

PubMed2000

Integrin-linked kinase (ILK): a "hot" therapeutic target.

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.

Animals↗

PubMed2000

Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase. More efficient biocatalysis for synthesis of alpha-Gal epitopes.

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.

Animals↗

PubMed2000

S-nitrosothiols as novel, reversible inhibitors of human rhinovirus 3C protease.

Human rhinovirus (HRV) 3C protease was inactivated by a series of S-nitrosothiols. These compounds exhibited different inhibitory activities in a time- and concentration-dependent manner with second-order rate constants (kinact/K(I)) ranging from 131 to 5360 M(-1) min(-1). The inactive enzyme could be re-activated by DTT, GSH and ascorbate, which indicated the inactivation mechanism was through an S-transnitrosylation process.

3C Viral Proteases↗

PubMed2000

Expression of the Na(+)/Ca(2+) exchanger ameliorates ionomycin-induced cell death.

PC12 cells were stably transfected with cDNA encoding the Na(+)/Ca(2+) exchanger (NCX1.4). A robust Na(+)-dependent Ca(2+) uptake confirmed the functional expression of the protein. When NCX1. 4 expressing cells (NO) and vector transfected control cells (VC) were exposed to 0.5-20 microM ionomycin for 6 h, a dose-dependent increase in LDH release was observed. LDH release was significantly reduced in NO when compared with VC. When either VC and NO were treated with 3 microM ionomycin and 1.1 mM EGTA, the increase in LDH release was nearly abolished. However, when VC and NO were treated with ionomycin and then EGTA was added 2 min later, LDH release remained elevated. These data suggest ionomycin-induced cell death was Ca(2+) dependent and expressing NCX1.4 may have ameliorated cell death by reducing elevated [Ca(2+)](I).

Animals↗

PubMed2000

Activation of the estrogen-signaling pathway by p21(WAF1/CIP1) in estrogen receptor-negative breast cancer cells.

BACKGROUND: Estrogen stimulates the proliferation of cells in normal mammary glands and most estrogen receptor (ER)-positive mammary carcinomas by binding to the ER and promoting the transcription of ER-responsive genes. In cells with functional ERs, estrogen mediates the transition of cells from the G(1) to S phase of the cell cycle. Several cell cycle regulatory proteins have been implicated in the ER-signaling pathway involved in estrogen-mediated growth stimulation and antiestrogen-mediated growth arrest. We sought to determine whether p21, a cyclin-dependent kinase inhibitor, is a component of this pathway and, if so, whether it can mediate estrogen's action in ER-negative breast cancer cells. METHODS: We overexpressed p21 with a tetracycline-inducible system in ER-negative, p21-negative breast cancer cells. Activity of the ER-signaling pathway was monitored in transient transfection assays by using constructs in which the ER promoter or the estrogen-response element (ERE) controls Luciferase expression. The growth-modulating effects of estradiol and antiestrogens on p21-overexpressing clones were assessed. All P: values are from two-sided tests. RESULTS: A strong positive association was found between the expression of p21 and ER in nine breast cancer cell lines and in tumor samples from 60 patients with breast cancer (P:<. 001). Overexpression of p21 in a p21-negative, ER-negative cell line induced both the ER and ERE promoters in an estrogen-responsive manner. Last, stable p21 clones that also lack the expression of wild-type ER were responsive to the growth-inhibitory effects of ICI 182,780, a potent antiestrogen, and the growth-stimulatory effects of 17beta-estradiol. CONCLUSION: The ability of p21 to mediate the activation of the estrogen-signaling pathway in ER-negative tumor cells suggests that p21 plays a novel role in this pathway, a finding that also has important clinical implications.

Antineoplastic Agents, Hormonal↗

PubMed2000

Differential regulation of responsiveness to fMLP and C5a upon dendritic cell maturation: correlation with receptor expression.

The trafficking of immature and mature dendritic cells (DCs) to different anatomical sites in vivo is critical for fulfilling their roles in the induction of Ag-specific immune responses. Although this process is complex and regulated by many mediators, the capacity of DCs to migrate is predominantly dependent on the expression of particular chemotactic receptors on the surface of DCs that enable them to move along chemotactic gradients formed by the corresponding chemokines and/or classical chemoattractants. Here we show that immature DCs (iDCs) respond to both fMLP and C5a as determined by chemotaxis and Ca2+ mobilization, whereas mature DCs (mDCs) respond to C5a, but not fMLP. Additionally, iDCs express the receptors for both fMLP and C5a at mRNA and protein levels. Upon maturation of DCs, fMLP receptor expression is almost completely absent, whereas C5a receptor mRNA and protein expression is maintained. Concomitantly, mDCs migrate chemotactically and mobilize intracellular Ca2+ in response to C5a, but not fMLP. Thus the interaction between C5a and its receptor is likely involved in the regulation of trafficking of both iDCs and mDCs, whereas fMLP mobilizes only iDCs. The differential responsiveness to fMLP and C5a of iDCs and mDCs suggests that they play different roles in the initiation of immune responses.

Animals↗

PubMed2000

Androgen receptor exon 1 CAG repeat length and risk of ovarian cancer.

Epidemiological studies indicate that ovarian cancer is an endocrine-related tumour. We conducted a case-control comparison to assess the androgen receptor (AR) exon 1 polymorphic CAG repeat length (CAG(n)) as a risk factor for epithelial ovarian cancer. AR CAG(n) was determined for 319 case subjects with ovarian adenocarcinoma and 853 unaffected control subjects (comprising 300 unrelated adult female monozygotic twins, and 553 adult females sampled randomly from the population using the electoral rolls). The CAG(n) distributions of case subjects and control subjects were compared as a continuum, and by dichotomising alleles according to different CAG(n) cut-points. Logistic regression was used to calculate age-adjusted odds ratio (OR) estimates. Analyzed as a continuous variable, there was no difference between case subjects and control subjects for the smaller, larger or average allele sizes of the CAG(n) genotype, before or after adjusting for age. The mean (95% CI) for the average CAG(n) was 22.0 (21.8-22.2) for case subjects and 22.0 (21.9-22.1) for control subjects (p>.9). Analysis of CAG(n) as a dichotomous variable showed no difference between case subjects and control subjects for the median cutpoint (>/= 22), or for another cut-point previously reported to act as a modifier of breast cancer risk (>/= 29). Our data provide no evidence for an association between ovarian cancer risk and the genotype defined by the AR exon 1 CAG(n) polymorphism, although we cannot exclude small effects, or threshold effects in a small subgroup.

Adenocarcinoma↗

PubMed2000

Effects of a traditional Chinese medicine, Qing Nao Yi Zhi Fang, on glutamate excitotoxicity in rat fetal cerebral neuronal cells in primary culture.

Qing Nao Yi Zhi Fang (QNYZ), a traditional Chinese medicine, has been developed as a drug to be used for the prevention and treatment of vascular dementia. However, the mechanisms by which this drug affects vascular dementia remain unknown. We examined the effects of QNYZ serum on glutamate excitotoxicity in rat fetal cerebral neuronal cells in primary culture. Exposure of neuronal cells to glutamate leads to a decrease in the activities of cholinesterase, superoxide dismutase, and streptoavidin peroxidase, and an increase in lactate dehydrogenase release. These enzyme activities were restored to the levels in untreated cells by the addition of QNYZ serum. QNYZ serum suppressed the increased nitric oxide production induced by glutamate and prevented glutamate-mediated apoptosis. QNYZ serum also improved mitochondrial energy metabolism after glutamate exposure. These findings suggest that QNYZ has protective effects against glutamate-mediated excitotoxicity in neuronal cells during ischemic brain injury.

Animals↗