Biomedical subjects

Y Xiang

Publications and source records attributed to Y Xiang.

At least 37 records · Page 2Linked to original sources

PubMed2001

Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis.

The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1beta and has an important role in defense against microbial invaders. As its name implies, the hIL-18BP binds to IL-18 with high affinity and prevents the interaction of IL-18 with its receptor. We genetically modified the C terminus of hIL-18BP by appending a 15-amino acid biotinylation recognition site and a six-histidine tag and then performed site-directed mutagenesis to determine the functional epitopes that mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on streptavidin-coated chips. Using surface plasmon resonance, we identified seven amino acids of hIL-18BP which, when changed individually to alanine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted beta-strand c and d of hIL-18BP immunoglobulin-like domain, and most had hydrophobic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, contributed approximately 50% of the binding free energy. Information obtained from these studies could contribute to the design of molecular antagonists of IL-18 for treatment of inflammatory diseases.

Alanine↗

PubMed2001

Orthopoxvirus IL-18 binding proteins: affinities and antagonist activities.

The affinities of purified recombinant human IL-18 binding protein (BP) and ectromelia and cowpox virus homologs for human and murine IL-18 were compared by plasmon resonance. The dissociation constants of human IL-18BP were similar for murine and human IL-18. By contrast, the dissociation constants of the viral proteins for murine IL-18 were 12- to 50-fold lower than that for human IL-18. The ectromelia and cowpox virus proteins were biologically active, as judged by their ability to inhibit induction of interferon-gamma by murine and human IL-18. The relative affinities of the orthopoxvirus IL-18BPs are consistent with the rodent host range of the viruses.

Animals↗

PubMed2001

The anti-proliferative effect of inhibitor of telomerase on cultured retinal pigment epithelial cells.

In order to provide a new method for treating proliferative vitreoretinopathy (PVR), the effects of anti-proliferation and apoptosis induction of inhibitors of telomerase and heat shock protein 90 (Hsp90) on the cultured retinal pigment epithelial (RPE) cells were investigated. The rate of apoptosis cells was measured by using TUNEL on the cultured RPE cells, the co-cultured RPE cells with inhibitor of telomerase (camptothecin) or the co-cultured RPE cells with inhibitor of Hsp90 (geldanamycin). The cell proliferation status was measured in the above three groups by using MTT method. The rate of apoptosis in the RPE cells co-cultured with camptothecin or geldanamycin was increased remarkably (P < 0.05). MTT showed the rate of growth inhibition was 8.4%, 32.3% and 72.3% at the concentrations of camptothecin 1 mumol/L, 5 mumol/L, 10 mumol/L, respectively, and 6.5%, 30.9%, 71.9% at the concentrations of geldanamycin 1 mumol/L, 5 mumol/L, 10 mumol/L, respectively. It was concluded that telomerase and Hsp90 can promote the proliferation of the cultured RPE cells, while the inhibitor of them can induce apoptosis and inhibit the growth of the RPE cells.

Apoptosis↗

PubMed2001

Identification and structural determination of a potent P-selectin inhibitor.

Small quantities of a potent P-selectin inhibitor, 2 (IC50 = 0.2 microM), were isolated and an initial structure proposed based on 1D proton NMR. A reterosynthetic analysis of the proposed structure led us to a total synthesis of 2. NMR studies using the 2-D homo-TOCSY and NOESY and 2-D hetero-HMQC helped to confirm the structure of 2.

Animals↗

PubMed2001

Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity.

The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430-6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.

Amino Acid Sequence↗

PubMed2001

Correspondence of the functional epitopes of poxvirus and human interleukin-18-binding proteins.

Molluscum contagiosum virus, a human poxvirus that causes persistent small benign skin tumors, encodes a variety of putative immune defense proteins. Three such proteins, MC51L, MC53L, and MC54L, have 20 to 35% amino acid sequence identities with human interleukin-18 (hIL-18)-binding protein (hIL-18BP), a naturally occurring antagonist of the proinflammatory cytokine IL-18. We previously demonstrated that seven amino acids within the immunoglobulin-like domain of hIL-18BP were important for high-affinity binding to hIL-18. Model building indicated that MC54L, which has been shown to bind hIL-18, contains five of the seven amino acids at corresponding positions in its immunoglobulin-like domain, the exceptions being the conservative substitution of isoleucine for a leucine and the nonconservative substitution of valine for a phenylalanine. We found that individual alanine substitutions for these six identical or highly conserved amino acids of MC54L caused changes in affinity and binding free energy for hIL-18 that were quantitatively similar to those produced by mutagenesis of hIL-18BP. Furthermore, when the nonconserved valine of MC54L was mutated to phenylalanine, making it more like hIL-18BP, its affinity for hIL-18 increased more than 10-fold. In addition, the carboxyl-terminal half of MC54L, which has no similarity with hIL-18BP, was dispensable for hIL-18 binding. Thus, despite their relatively low overall sequence identity, MC54L and hIL-18BP have similar hIL-18 binding sites and functional epitopes. On the other hand, MC51L and MC53L have nonconservative substitutions of three to six of the seven critical amino acids of hIL-18BP and neither protein bound hIL-18, suggesting that they may interact with unidentified ligands.

Amino Acid Sequence↗

PubMed2001

[Integrin-ligands binding reaction upregulates the antioxidant activity of rabbit bronchial epithelial cells].

Antioxidant activity of bronchial epithelial cells (BECs) plays an essential role in preventing the airway epithelium integrity from damage in structure and function. Integrin expressed by BECs is the receptor of extracellular matrix such as fibronectin (Fn), and it is involved in modulation of proliferation, differentiation and metabolism of the cells. In order to test the hypothesis that integrin-ligand binding reaction supports the ability of cells to withstand oxidant attack, the present study evaluated the antioxidant activity of primary cultured rabbit BECs treated with fibronectin or its sequence Arg-Gly-Asp (RGD peptide), by determining changes in the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) and in the level of glutathione (GSH). The results are as follows: (1) Fn (10 micrograms/ml) increased significantly the activity unit of GSH-Px (P < 0.05, n = 5), which was inhibited by calmodulin-inhibitor W7 (10(-5) mol/L) (P < 0.05). Both Fn (5-20 micrograms/ml) and RGD (15-60 micrograms/ml) showed a dose-dependent upregulatory effect (respectively r = 0.93 and r = 0.73). (2) Treatment with Fn increased SOD activity (P < 0.01, n = 7), which was abolished by W7 (P < 0.01). (3) Catalase activity was also stimulated by Fn (P < 0.05, n = 6) and reversed by W7 (P < 0.01). (4) A dose-dependent increase of GSH level was observed in both Fn (r = 0.82) and RGD treatment (r = 0.84). The data suggest that the binding of integrin with extracellular matrix can upregulate activity of antioxidant enzymes, and increase the content of GSH and improve the ability of BECs to resist oxidant injury.

Animals↗

PubMed2001

Beta-adrenergic receptor subtype-specific signaling in cardiac myocytes from beta(1) and beta(2) adrenoceptor knockout mice.

The sympathetic nervous system modulates cardiac contractility and rate by activating beta-adrenergic receptors (beta AR) expressed on cardiac myocytes and specialized cells in the sinoatrial node and the conduction system. Recent clinical studies have suggested that beta-adrenergic receptors also play a role in cardiac remodeling that occurs in the pathogenesis of cardiomyopathy. Both beta(1) and beta(2) adrenergic receptors are expressed in human and murine hearts. We have examined the effect of beta AR activation on the spontaneous contraction rate of neonatal myocyte cultures from wild-type and beta receptor knockout (KO) mice (beta(1)AR-KO, beta(2)AR-KO and beta(1)beta(2)AR-KO mice). Stimulation of the beta(1)AR in beta(2)AR-KO myocytes produces the greatest increase in contraction rate through a signaling pathway that requires protein kinase A (PKA) activation. In contrast, stimulation of the beta(2)AR in beta(1)AR-KO myocytes results in a biphasic effect on contraction rate with an initial increase in rate that does not require PKA, followed by a decrease in rate that involves coupling to a pertussis toxin sensitive G protein. A small isoproterenol-induced decrease in contraction rate observed in beta(1)beta(2)AR-KO myocytes can be attributed to the beta(3)AR. These studies show that all three beta AR subtypes are expressed in neonatal cardiac myocytes, and the beta(1)AR and beta(2)AR couple to distinct signaling pathways.

Adrenergic beta-Agonists↗

PubMed2001

[Clinical analysis of intracranial metastases in gestational trophoblastic tumour].

OBJECTIVE: To evaluate characteristics of patients with intracranial metastases of gestational trophoblastic tumour (GTT) and determine the prognostic factors and therapeutic modality. METHODS: We retrospectively reviewed the records of 814 GTT patients treated at Peking Union Medical College Hospital from 1984 to 1998. Of them, 382 were choriocarcinoma and 61 developed brain metastases (16.0%); 432 were invasive mole and 8 of them presented brain metastases (1.9%). Patients with brain metastases were divided into three categories: Group A, individuals with no prior chemotherapy (30 cases); Group B, patients who had received chemotherapy before transferred to our hospital (31 cases); Group C, individuals who developed brain metastases during therapy in our hospital (8 cases). Apart from 12 patients died before or during the first cycle of chemotherapy, the remaining 57 patients were treated with 5-FU combined chemotherapy or etopside, methotrexate, kengshengmycin, /vincristine, cyclophosphamide (EMA/CO) regimen, the number of courses varied from 3 to 17 cycles. The median number of chemotherapy for each patient was 8.2. Intrathecal methotrexate chemotherapy was utilized for all patients. Emergency surgical decompression was performed in 4 cases who had symptoms of highly increased intracranial pressure. RESULTS: Apart from 12 patients died before they received regular therapy in our hospital, remission rate of other 57 patients was 71.9%. The cumulative survival rate for these 57 patients at 5 years was 45.8%. Women with no prior chemotherapy (group A) had outcomes significantly better than those who had been treated before transfer to our hospital (group B) and there were no survivors among the patients who developed brain metastases during active chemotherapy (group C) [P < 0.05 (A Vs B); P < 0.01 (A or B Vs C)]. CONCLUSIONS: Multiagent systemic chemotherapy combined with intrathecal methotrexate chemotherapy still play the key role in the management of brain metastatic GTT patients; Surgical decompression should be performed if significant neurologic symptoms are present.

Adult↗

PubMed2001

[Establishment of the drug resistant cell line of choriocarcinoma and the reversal of drug resistance by transfection of human interleukin 2 gene].

OBJECTIVE: To establish the drug-resistant cell line of choriocarcinoma and to study the transfection of the human interleukin 2 (hIL-2) gene into the established drug resistant cell line and investigate the reversal of the multidrug resistance. METHODS: The resistant cell line was established by pulse exposed choriocarcinoma cell line JEG-3 to etopside (VP-16) for ten months. The recombinant plasmid containing pcDNA3.1(+)-hIL-2 gene was constructed. The drug resistant cell line was transfected with the constructed plasmid by lipofectin, and the tumor cell colonies containing the IL-2 sequence were selected by genetin. The expression of hIL-2 and drug resistant-related genes was detected by reverse transcript polymerase chain reaction. The chemosensitivity of the gene-transfected tumor cells and the non transfected cell lines to methetraxate, VP-16, kengshengmycine, paclitaxol and 5-fluorouracil was determined by the methyl thiazolyl tetrazolium cytotoxicity assay. RESULTS: The transfected cells expressed human hIL-2 gene, and showed the reversal of multidrug resistance by methyl thiazolyl tetrazolium assay. The transfected cells expressed no multidrug resistance gene-1 (MDR1) on mRNA level. Drug resistance index to VP-16 decreased from 38.7 to 6.0 and 6.1, the index to methetraxate decreased from 14.5 to 2.6 and 2.5, to methetraxate from 13.0 to 2.0. CONCLUSION: The transfection of hIL-2 gene into the drug resistance cell line of choriocarcinoma can modulate the MDR1 expression on the mRNA level, and reverse the drug resistance.

Choriocarcinoma↗

PubMed2001

[Diagnosis and treatment of ectopic molar pregnancy].

OBJECTIVE: To summerize the experience in the diagnosis and treatment of ectopic molar pregnancy. METHODS: Clinical data of 3 cases of women with ectopic hydatidiform mole in Peking Union Medical College Hospital were analyzed retrospectively. RESULTS: The clinical characteritics of ectopic molar pregnancy depend upon the site of the disease. It can be metastasized distantly in early stage. Digital subtraction angiography, colour ultrosound and laparotomy may play very important roles in the diagnosis of ectopic molar pregnancy. Regular chemotherapy and resection of drug-resistant focus are still the main methods of treatment. CONCLUSIONS: Prophylactic chemotherapy for ectopic molar pregnancy should be emphasized. It is very important to make an early diagnosis of malignant metastasis and to give a regular treatment for the patients.

Angiography, Digital Subtraction↗

PubMed2000

Characterization of cucurbita maxima phloem serpin-1 (CmPS-1). A developmentally regulated elastase inhibitor.

We report on the molecular, biochemical, and functional characterization of Cucurbita maxima phloem serpin-1 (CmPS-1), a novel 42-kDa serine proteinase inhibitor that is developmentally regulated and has anti-elastase properties. CmPS-1 was purified to near homogeneity from C. maxima (pumpkin) phloem exudate and, based on microsequence analysis, the cDNA encoding CmPS-1 was cloned. The association rate constant (k(a)) of phloem-purified and recombinant His(6)-tagged CmPS-1 for elastase was 3.5 +/- 1.6 x 10(5) and 2.7 +/- 0.4 x 10(5) m(-)(1) s(-)(1), respectively. The fraction of complex-forming CmPS-1, X(inh), was estimated at 79%. CmPS-1 displayed no detectable inhibitory properties against chymotrypsin, trypsin, or thrombin. The elastase cleavage sites within the reactive center loop of CmPS-1 were determined to be Val(347)-Gly(348) and Val(350)-Ser(351) with a 3:2 molar ratio. In vivo feeding assays conducted with the piercing-sucking aphid, Myzus persicae, established a close correlation between the developmentally regulated increase in CmPS-1 within the phloem sap and the reduced ability of these insects to survive and reproduce on C. maxima. However, in vitro feeding experiments, using purified phloem CmPS-1, failed to demonstrate a direct effect on aphid survival. Likely roles of this novel phloem serpin in defense against insects/pathogens are discussed.

Amino Acid Sequence↗

PubMed2000

Trends in colorectal cancer rates in urban shanghai, 1972-1996, in relation to dietary changes.

PURPOSE: In urban Shanghai, the largest industrial and commercial city in China, the age-adjusted (world standard) incidence rates for colorectal cancer increased from 14.5 to 23.3 per 10(5) men and from 12.1 to 20.3 per 10(5) women between 1972 and 1996. This change was even more pronounced for colon cancer, whose incidence rates doubled from 5.95 to 13.7 per 10(5) men and from 5.77 to 12.5 per 10(5) women. The reasons for the rapid increases in cancer rates are not fully understood, but may involve dietary exposures that have changed substantially over the past two decades.METHODS: We calculated Pearson correlation coefficients (r) between colorectal cancer rates and the dietary factors of grain, vegetable oil, pork, poultry and vegetable consumption over the period of 1972 through 1996 in urban Shanghai.RESULTS: Statistically significant positive associations were observed between colon cancer rates and per capita consumption of vegetable oil (r = 0.91 for men, r = 0.94 for women), poultry (r = 0.90 for men, r = 0.90 for women), and pork (r = 0.78 for men, r = 0.81 for women). The correlation coefficients were not statistically significant between colon cancer and per capita consumption of grain (r = 0.38 for men, r = 0.37 for women) or vegetables (r = 0.16 for men, r = 0.14 for women). Similar weaker associations were observed between rectal cancer rates and vegetable oil, pork and poultry consumption.CONCLUSIONS: The findings in our study suggest that increases in dietary fat, poultry and pork intake may play a role in the rising colorectal cancer rates in Shanghai.

Journal Article↗

PubMed2000

Transcription elongation activity of the vaccinia virus J3 protein in vivo is independent of poly(A) polymerase stimulation.

Prior genetic analysis suggests that the vaccinia virus J3 gene product, previously characterized as a bifunctional (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor, is a postreplicative positive transcription elongation factor. To test this hypothesis, viruses bearing mutations in the J3 gene were characterized with respect to viral protein and RNA synthesis in infected cells. The analysis reveals that compared to wt virus infections, J3 mutants synthesize reduced amounts of large late viral proteins and shorter-than-normal intermediate and late mRNAs. Structural analysis of one late mRNA shows that it is specifically truncated from the 3' end, thus accounting for its shorter than normal chain length. Thus J3 mutant viruses are defective in elongation of transcription of postreplicative viral genes, strongly suggesting that the J3 gene product normally acts as a positive transcription elongation factor. Biochemical analysis of one J3 missense mutant demonstrates that it retains poly(A) stimulatory activity but is defective in (nucleoside-2'-O-)-methyltransferase activity. Thus the elongation factor activity of the J3 gene product is independent of the poly(A) stimulatory activity. It remains to be determined whether the (nucleoside-2'-O-)-methyltransferase and elongation factor activities of the J3 protein are linked or can be uncoupled by mutation.

Animals↗

PubMed2000

The vaccinia virus bifunctional gene J3 (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor is implicated as a positive transcription elongation factor by two genetic approaches.

Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3' ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBT(d)) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBT(d) mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2'-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection.

Animals↗

PubMed2000

Immune-defense molecules of molluscum contagiosum virus, a human poxvirus.

Molluscum contagiosum virus encodes more than 150 proteins including some involved in host interactions that might contribute to prolonged viral replication in the skin. These include homologs of a selenocysteine-containing glutathione peroxidase, a death effector domain protein, a chemokine, a major histocompatibility complex class I molecule and an interleukin-18-binding protein.

Humans↗

PubMed2000

The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments.

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.

Amino Acid Motifs↗

PubMed2000

Efficiency of generalized simulated annealing

We have explored the evolution of the efficiency of generalized simulated annealing (GSA) through a comparative study with classical simulated annealing (CSA) and fast simulated annealing (FSA). Our calculations on the Thomson model and nickel clusters show that the relative efficiency of GSA compared to CSA and FSA increases with the number of variables of the objective function. Thus, relative to CSA and FSA, the more complex the system, the more efficient is the GSA method.

Journal Article↗