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Articles by W Zhang
Total Records ( 30 ) for W Zhang
  A Qian , S Di , X Gao , W Zhang , Z Tian , J Li , L Hu , P Yang , D Yin and P. Shang
 

The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has been widely applied in many fields. In this study, a special designed superconducting magnet, which can produce three apparent gravity levels (0, 1, and 2 g), namely high magneto-gravitational environment (HMGE), was used to simulate space gravity environment. The effects of HMGE on osteoblast gene expression profile were investigated by microarray. Genes sensitive to diamagnetic levitation environment (0 g), gravity changes, and high magnetic field changes were sorted on the basis of typical cell functions. Cytoskeleton, as an intracellular load-bearing structure, plays an important role in gravity perception. Therefore, 13 cytoskeleton-related genes were chosen according to the results of microarray analysis, and the expressions of these genes were found to be altered under HMGE by real-time PCR. Based on the PCR results, the expressions of WASF2 (WAS protein family, member 2), WIPF1 (WAS/WASL interacting protein family, member 1), paxillin, and talin 1 were further identified by western blot assay. Results indicated that WASF2 and WIPF1 were more sensitive to altered gravity levels, and talin 1 and paxillin were sensitive to both magnetic field and gravity changes. Our findings demonstrated that HMGE can affect osteoblast gene expression profile and cytoskeleton-related genes expression. The identification of mechanosensitive genes may enhance our understandings to the mechanism of bone loss induced by microgravity and may provide some potential targets for preventing and treating bone loss or osteoporosis.

  B Xiang , M Yi , L Wang , W Liu , W Zhang , J Ouyang , Y Peng , W Li , M Zhou , H Liu , M Wu , R Wang , X Li and G. Li
 

Oxidored-nitro domain containing protein 1 (NOR1) gene is a novel nitroreductase gene first isolated from nasopharyngeal carcinoma (NPC). It plays an important role in the formation of chemical carcinogen and the carcinogenesis of NPC for its nitrosation function. Overexpression of the wild-type NOR1 gene in nasopharyngeal carcinoma cells is effective to inhibit cell growth and proliferation. In this study, for the first time, we generated a highly specific NOR1 antibody and analyzed NOR1 distribution in the human tissues and NPC biopsies. The results showed that NOR1 protein is predominantly expressed in human nasopharynx and tracheal tissues. Human heart, liver, spleen, stomach, colon, kidney, skeletal muscle, thymus, and pancreas are all deficient of NOR1 protein. More importantly, we performed immunohistochemistry assay of NOR1 protein expression in the NPC tissues, and the result showed that NOR1 protein is frequently down-expressed in NPC. These data shed light on the selectivity of potential physiological functions of NOR1 and provides an indispensable reference to the carcinogenesis process of NPC and to identify or validate tissue-specific drug targets.

  Y Wu , W Zhang , Y Yang , B Yu and A. Huang
 

In this study, we scanned the whole hepatitis B virus (HBV) genome for the identification of potential regulatory elements located on the S-(+)-strand. With pCDNA3.1-HBV1.3 as template which contains 1.3-fold HBV whole genome, HBV fragments were amplified by PCR methods, and then inserted into the upstream of a heterologous luciferase reporter vector (pGL3control) in antisense orientation, allowing the HBV expression from the S-(+)-strand. We found that the reporter plasmid containing nt 509-1(3182)-2639 of HBV inhibited luciferase gene transcription and expression in HepG2 cells. Our results strongly suggested that nt 453–250 of HBV may act as a novel negative regulatory element, which has not been reported before. Serial deletion analyses further indicated that nt 453–250 sequence of HBV genome would be the minimal sequence essential for the inhibitory effect of the novel negative regulatory element.

  X Wu , W Zhang , X Shi , P An , W Sun and Z. Wang
 

In this study, we investigated the therapeutic effect of artemisinin (Art) on lupus nephritis mice and its mechanisms by comparing the differences between lupus nephritis (LN) mice given Art and control mice in molecular biology, immunohistochemistry, and histopathology. The results showed that Art could remarkably relieve the symptoms, decrease the level of urine protein/24 h, and alleviate pathological renal lesions. The differences among the four groups in the expression of the NF-Bp65 protein, nuclear factor-B (NF-B) activity, and the expression of transforming growth factor-β1 (TGF-β1) mRNA in renal tissue suggested that Art can lower the serum levels of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) and inhibit the expression of the NF-Bp65 protein and NF-B and TGF-β1 mRNA in the renal tissues of LN mice. These results proved that it is reliable and effective to use Art to treat LN mice, and its therapeutic mechanisms should closely be related to the fact that Art can obviously decrease the serum levels of TNF- and IL-6 and down-regulate the expression of the NF-Bp65 protein and NF-B and TGF-β1 mRNA in renal tissues.

  W Zhang , A Majumder , X Wu and M. W. Mulholland
 

Ghrelin is a 28-amino-acid hormone derived from the endoproteolytic processing of its prehormone proghrelin. Although ghrelin has been reported to regulate food intake and body weight, it is still unknown whether proghrelin exercises any biological function. Here we show that recombinant proghrelin alters food intake and energy metabolism in mice. After intraperitoneal administration of recombinant proghrelin (100 nmol/kg body wt), cumulative food intake was significantly increased at days 1, 2, and 3 (6 ± 0.3, 13 ± 0.5, and 20 ± 0.8 g vs. 5 ± 0.2, 10 ± 0.2, and 16 ± 0.3 g of the control mice receiving normal saline, respectively, n = 6, P < 0.05). Twelve-hour cumulative food intake in the light photo period in mice treated with proghrelin increased significantly relative to the control (2.1 ± 0.04 vs. 1.3 ± 0.2 g, n = 6, P < 0.05). No change in 12-h cumulative food intake in the dark photo period was observed between mice treated with proghrelin and vehicle (4.2 ± 0.6 vs. 4.3 ± 0.6 g, n = 6, P > 0.05). This is associated with a decrease in body weight (0.42 ± 0.04 g) for mice treated with proghrelin, whereas control animals gained body weight (0.31 ± 0.04 g). Mice treated with proghrelin demonstrate a significant decrease in respiratory quotient, indicating an increase in fat consumption. Recombinant proghrelin is functionally active with effects on food intake and energy metabolism.

  H Wang , W Zhang , C Zhu , C Bucher , B. R Blazar , C Zhang , J. F Chen , J Linden , C Wu and Y. Huo
 

Background— Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall. The A2A receptor (A2AR) plays a central role in many antiinflammatory effects of adenosine. However, the role of A2AR in atherosclerosis is not clear.

Methods and Results— The knockout of A2AR in apolipoprotein E–deficient (Apoe–/–/A2AR–/–) mice led to an increase in body weight and levels of blood cholesterol and proinflammatory cytokines, as well as the inflammation status of atherosclerotic lesions. Unexpectedly, Apoe–/–/A2AR–/– mice developed smaller lesions, as did chimeric Apoe–/– mice lacking A2AR in bone marrow–derived cells (BMDCs). The lesions of those mice exhibited a low density of foam cells and the homing ability of A2AR-deficient monocytes did not change. Increased foam cell apoptosis was detected in atherosclerotic lesions of Apoe–/–/A2AR–/– mice. In the absence of A2AR, macrophages incubated with oxidized LDL or in vivo–formed foam cells also exhibited increased apoptosis. A2AR deficiency in foam cells resulted in an increase in p38 mitogen–activated protein kinase (MAPK) activity. Inhibition of p38 phosphorylation abrogated the increased apoptosis of A2AR-deficient foam cells.

Conclusion— Inactivation of A2AR, especially in BMDCs, inhibits the formation of atherosclerotic leisons, suggesting that A2AR inactivation may be useful for the treatment of atherosclerosis.

  H Wang , W Zhang , R Tang , R. P Hebbel , M. A Kowalska , C Zhang , J. D Marth , M Fukuda , C Zhu and Y. Huo
 

Objective— Core2 1 to 6-N-glucosaminyltransferase-I (C2GlcNAcT-I) plays an important role in optimizing the binding functions of several selectin ligands, including P-selectin glycoprotein ligand. We used apolipoprotein E (ApoE)-deficient atherosclerotic mice to investigate the role of C2GlcNAcT-I in platelet and leukocyte interactions with injured arterial walls, in endothelial regeneration at injured sites, and in the formation of arterial neointima.

Methods and Results— Arterial neointima induced by wire injury was smaller in C2GlcNAcT-I-deficient apoE–/– mice than in control apoE–/– mice (a 79% reduction in size). Compared to controls, apoE–/– mice deficient in C2GlcNAcT-I also demonstrated less leukocyte adhesion on activated platelets in microflow chambers (a 75% reduction), and accumulation of leukocytes at injured areas of mouse carotid arteries was eliminated. Additionally, endothelial regeneration in injured lumenal areas was substantially faster in C2GlcNAcT-I-deficient apoE–/– mice than in control apoE–/– mice. Endothelial regeneration was associated with reduced accumulation of platelet factor 4 (PF4) at injured sites. PF4 deficiency accelerated endothelial regeneration and protected mice from neointima formation after arterial injury.

Conclusions— C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation, and this effect is attributable to decreased leukocyte recruitment to injured vascular walls and increased endothelial regeneration. Both C2GlcNAcT-I and PF4 are promising targets for the treatment of arterial restenosis.

  W Torres Garcia , W Zhang , G. C Runger , R. H Johnson and D. R. Meldrum
 

Motivation: Gene expression profiling technologies can generally produce mRNA abundance data for all genes in a genome. A dearth of proteomic data persists because identification range and sensitivity of proteomic measurements lag behind those of transcriptomic measurements. Using partial proteomic data, it is likely that integrative transcriptomic and proteomic analysis may introduce significant bias. Developing methodologies to accurately estimate missing proteomic data will allow better integration of transcriptomic and proteomic datasets and provide deeper insight into metabolic mechanisms underlying complex biological systems.

Results: In this study, we present a non-linear data-driven model to predict abundance for undetected proteins using two independent datasets of cognate transcriptomic and proteomic data collected from Desulfovibrio vulgaris. We use stochastic gradient boosted trees (GBT) to uncover possible non-linear relationships between transcriptomic and proteomic data, and to predict protein abundance for the proteins not experimentally detected based on relevant predictors such as mRNA abundance, cellular role, molecular weight, sequence length, protein length, guanine-cytosine (GC) content and triple codon counts. Initially, we constructed a GBT model using all possible variables to assess their relative importance and characterize the behavior of the predictive model. A strong plateau effect in the regions of high mRNA values and sparse data occurred in this model. Hence, we removed genes in those areas based on thresholds estimated from the partial dependency plots where this behavior was captured. At this stage, only the strongest predictors of protein abundance were retained to reduce the complexity of the GBT model. After removing genes in the plateau region, mRNA abundance, main cellular functional categories and few triple codon counts emerged as the top-ranked predictors of protein abundance. We then created a new tuned GBT model using the five most significant predictors. The construction of our non-linear model consists of a set of serial regression trees models with implicit strength in variable selection. The model provides variable relative importance measures using as a criterion mean square error. The results showed that coefficients of determination for our nonlinear models ranged from 0.393 to 0.582 in both datasets, providing better results than linear regression used in the past. We evaluated the validity of this non-linear model using biological information of operons, regulons and pathways, and the results demonstrated that the coefficients of variation of estimated protein abundance values within operons, regulons or pathways are indeed smaller than those for random groups of proteins.

  T Ishii , J Wang , W Zhang , J Mascarenhas , R Hoffman , Y Dai , N Wisch and M. Xu
 

Pruritus is a common symptom in patients with Philadelphia chromosome–negative myeloproliferative disorders (MPDs). The pathophysiology of MPD-associated pruritus is unclear. We have demonstrated that MPD mast cells (MCs) are involved by the malignant process. In the present study, we explored the hypothesis that MCs play an important role in the development of pruritogenesis in MPDs. We found that MPD MCs released significantly greater amounts of pruritogenic factors, including histamine, leukotrienes, and interleukin-31 (IL-31) than normal MCs. Elevated levels of IL-31 were also observed in MPD CD3+ cell-conditioned media. MPD MCs exhibited increased migratory behavior in response to stem cell factor or interleukin-8, which was associated with increased filamentous-actin content. Furthermore, the presence of pruritus in MPDs was statistically correlated with a greater number of MCs being generated by CD34+ cells, a greater number of MC colonies being formed by CD34+ cells, decreased apoptosis and prostaglandin D2 release by cultured MCs, and higher plasma levels of IL-31. These data demonstrate that functional abnormalities of MPD MCs probably lead to pruritogenesis in patients with MPDs. These studies provide cellular and molecular targets for the development of antipruritus drugs for patients with MPDs.

  S. R Morey Kinney , W Zhang , M Pascual , J. M Greally , B. M Gillard , E Karasik , B. A Foster and A. R. Karpf
 

Green tea polyphenols (GTP) have been reported to inhibit DNA methylation in cultured cells. Here, we tested whether oral consumption of GTPs affects normal or cancer-specific DNA methylation in vivo, using mice. Wild-type (WT) and transgenic adenocarcinoma of mouse prostate (TRAMP) mice were given 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate.

  Y Wu , X Feng , Y Jin , Z Wu , W Hankey , C Paisie , L Li , F Liu , S. H Barsky , W Zhang , R Ganju and X. Zou
 

The natural compound indole-3-carbinol (I3C; found in vegetables of the genus Brassica) is a promising cancer prevention or therapy agent. The cell division cycle 25A (Cdc25A) phosphatase is overexpressed in a variety of human cancers and other diseases. In the present study, I3C induced degradation of Cdc25A, arrest of the G1 cell cycle, and inhibition of the growth of breast cancer cells. We also showed that the Ser124 site of Cdc25A, which is related to cyclin-dependent kinase 2, is required for I3C-induced degradation of Cdc25A in breast cancer cells, and that interruption of the ATM-Chk2 pathway suppressed I3C-induced destruction of Cdc25A. Our in vivo studies of different mutated forms of Cdc25A found that the mutation Cdc25AS124A (Ser124 to Ala124), which confers resistance to I3C-induced degradation of Cdc25A, attenuated I3C inhibition of breast tumorigenesis in a mouse xenograft model. The present in vitro and in vivo studies together show that I3C-induced activation of the ATM-Chk2 pathway and degradation of Cdc25A represent a novel molecular mechanism of I3C in arresting the G1 cell cycle and inhibiting the growth of breast cancer cells. The finding that I3C induces Cdc25A degradation underscores the potential use of this agent for preventing and treating cancers and other human diseases with Cdc25A overexpression. Cancer Prev Res; 3(7); 818–28. ©2010 AACR.

  M Wang , W Zhang , L Yuan , G Fu , Q Wei and Z. Zhang
 

A recent genome-wide association study identified two common variants that confer susceptibility to bladder cancer. We hypothesized that these variants are associated with risk of bladder cancer in Chinese populations. We genotyped rs9642880 G>T on 8q24 and rs710521 A>G on 3q28 in a two-stage case–control study of bladder cancer to evaluate the association and further examined the expression of MYC. We found that the rs9642880 G>T, but not the rs710521 A>G polymorphism, was associated with an increased risk of bladder cancer. Compared with the rs9642880 GG genotype, the GT/TT genotypes were associated with an odds ratio of 1.65 (95% confidence interval = 1.25–2.17), and this risk was more pronounced in young men and for low-risk tumors. Additional experiments revealed that the rs9642880 GT/TT genotypes were associated with enhanced levels of both MYC mRNA and protein in bladder tissues. Our findings suggested that the rs9642880 G>T polymorphism on 8q24 was independently associated with the risk of bladder cancer in Chinese populations.

  J Wang , Q Gu , M Li , W Zhang , M Yang , B Zou , S Chan , L Qiao , B Jiang , S Tu , J Ma , I. F Hung , H. Y Lan and B. C.Y. Wong
 

Background and aims: X-linked inhibitor of apoptosis-associated factor 1 (XAF1) was first recognized as an antagonist of X-linked inhibitor of apoptosis in suppressing caspase 3 activity. It has lower expression in cancer cells than normal tissue. Overexpression of XAF1 can inhibit cancer cell growth and sensitize tumor necrosis factor-related apoptosis-inducing ligand- or etoposide-induced apoptosis. The aim of this study is to elucidate the mechanism of XAF1 in regulating cell growth. Methods: Stable transfectants of gastrointestinal (GI) cancer cell lines AGS and SW1116 expressing XAF1 and vector control were generated. Cell growth, apoptosis, mitotic status and cell cycle distribution were assessed. The interaction between XAF1 and G2/M checkpoint proteins was evaluated by immunoblotting, kinase assay and co-immunoprecipitation assay. Mitotic catastrophe was identified by occurrence of aberrant nuclei and centrosomal amplification. Results: Our results showed that overexpression of XAF1 suppressed serum-dependent cancer cell growth, induced mitotic catastrophe and G2/M cell cycle arrest. Interestingly, XAF1 was predominantly expressed in G2/M phase after cell cycle synchronization. XAF1 interacted with and activated checkpoint kinase 1 (Chk1), inactivated Cdc25C and lead to inactivation of Cdc2–cyclin B complex. Suppression of Chk1 abrogated XAF1-induced G2/M arrest. Conclusions: Our findings implicate XAF1 as a novel cell cycle modulator that is recruited in G2/M phase and thus unravel a novel function pathway of XAF1, suggesting the potential role of XAF1 as the target for the management of GI cancers.

  L Zhang , T Deng , X Li , H Liu , H Zhou , J Ma , M Wu , M Zhou , S Shen , Z Niu , W Zhang , L Shi , B Xiang , J Lu , L Wang , D Li , H Tang and G. Li
 

microRNAs (miRNAs) are small non-coding RNAs and have been implicated in the pathology of various diseases, including cancer. Here we report that the miRNA profiles have been changed after knockdown of one of the most important oncogene c-MYC or re-expression of a candidate tumor suppressor gene SPLUNC1 in nasopharyngeal carcinoma (NPC) cells. Both c-MYC knockdown and SPLUNC1 re-expression can down-regulate microRNA-141 (miR-141). miR-141 is up-regulated in NPC specimens in comparison with normal nasopharyngeal epithelium. Inhibition of miR-141 could affect cell cycle, apoptosis, cell growth, migration and invasion in NPC cells. We found that BRD3, UBAP1 and PTEN are potential targets of miR-141, which had been confirmed following luciferase reporter assays and western blotting. BRD3 and UBAP1 are both involved in NPC carcinogenesis as confirmed through our previous studies and PTEN is a crucial tumor suppressor in many tumor types. BRD3 is involved in the regulation of the Rb/E2F pathway. Inhibition of miR-141 could affect some important molecules in the Rb/E2F, JNK2 and AKT pathways. It is well known that carcinogenesis of NPC is involved in the networks of genetic and epigenetic alteration events. We propose that miR-141- and tumor-related genes c-MYC, SPLUNC1, BRD3, UBAP1 and PTEN may constitute a gene–miRNA network to contribute to NPC development.

  W Zhang , B Jiang , Z Guo , C Sardet , B Zou , C. S. C Lam , J Li , M He , H. Y Lan , R Pang , I. F. N Hung , V. P. Y Tan , J Wang and B. C. Y. Wong
 

Background and Aims: Cancer invasion and metastasis may associate with the phenotype transition called epithelial-mesenchymal transition (EMT). We aim to evaluate the impact of four-and-a-half LIM protein 2 (FHL2) on EMT and invasion of colon cancer. Methods: The functional role of FHL2 in EMT was determined by overexpression or small interfering RNA-mediated depletion of FHL2. Mechanisms of FHL2 on expression or activity of E-cadherin and β-catenin were assessed. Results: FHL2 was highly expressed in primary and metastatic colon cancer but not in normal tissues. FHL2 was critical for cancer cell adhesion to extracellular matrix, migration and invasion. FHL2 expression was stimulated by transforming growth factor (TGF)-β1. Moreover, FHL2 acted as a potent EMT inducer by stimulating vimentin and matrix metalloproteinase-9 expressions and causing a loss of E-cadherin, whereas those alterations of EMT markers were not affected by silencing of Smad molecules (typical TGF-β signal mediators) in FHL2 stable transfectant cells. Therefore, FHL2 induced EMT in a TGF-β-dependent and Smad-independent manner. FHL2 downregulated E-cadherin expression and inhibited the formation of membrane-associated E-cadherin–β-catenin complex. FHL2 also stabilized nuclear β-catenin, resulting in enforcement of β-catenin transactivation activity. Conclusion: FHL2 is a potent EMT inducer and might be an important mediator for invasion and/or metastasis of colon cancer.

  Y Yuan , W Zhang , R Yan , Y Liao , L Zhao , C Ruan , X Du and K. Dai
 

Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3 and filamin A. Both 14-3-3 and filamin A associate with the cytoplasmic domain of GPIb, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX.

Objective: To explore the mechanism underlying the roles of 14-3-3 and filamin A in regulating the VWF binding function of GPIb-IX.

Methods and Results: A truncation mutant of GPIb (565) deleting the C-terminal 14-3-3 binding sites retained 14-3-3 binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb totally abolished 14-3-3 binding, indicating that the residues 551 to 564 of GPIb are important in the interaction between 14-3-3 and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIb suggesting phosphorylation of a population of GPIb molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb eliminated the association of 14-3-3 with 565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIb with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions.

Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3 to central filamin A binding site of GPIb, and the dimeric 14-3-3 binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

  T Yang , S. K Chung , W Zhang , J. G.L Mullins , C. H McCulley , J Crawford , J MacCormick , C. A Eddy , A. N Shelling , J. K French , P Yang , J. R Skinner , D. M Roden and M. I. Rees
 

Background— Inherited long-QT syndrome is characterized by prolonged QT interval on the ECG, syncope, and sudden death caused by ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modeling, and phylogenetic alignments; however, biophysical testing offers a higher degree of validating evidence.

Methods and Results— By using in vitro electrophysiological testing of transfected mutant and wild-type long-QT syndrome constructs into Chinese hamster ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand-based long-QT syndrome screening program. We demonstrate through electrophysiology and molecular modeling that 7 of the missense mutations have profound pathological dominant-negative loss-of-function properties, confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed- or fast-activation kinetics. Further investigation of the A46T family has revealed an inconsistent cosegregation of the variant with the clinical phenotype.

Conclusions— Electrophysiological characterization should be used to validate long-QT syndrome pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counseling and screening of such families, and alternative disease-causing mechanisms should be considered.

  W Wu , W Zhang , R Qiao , D Chen , H Wang , Y Wang , S Zhang , G Gao , A Gu , J Shen , J Qian , W Fan , L Jin , B Han and D. Lu
 

Purpose: Platinum agents cause DNA cross-linking and adducts. Xeroderma pigmentosum group D (XPD) plays a key role in the nucleotide excision repair pathway of DNA repair. Genetic polymorphisms of XPD may affect the capacity to remove the deleterious DNA lesions in normal tissues and lead to greater treatment-related toxicity. This study aimed to investigate the association of three polymorphisms of XPD at codons 156, 312, and 711, with the occurrence of grade 3 or 4 toxicity in advanced non–small cell lung cancer patients.

Experimental Design: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to genotype the three polymorphisms in 209 stage III and IV non–small cell lung cancer patients treated with platinum-based chemotherapy.

Results: The variant homozygotes of XPD p.Arg156Arg (rs238406) polymorphism were associated with a significantly increased risk of grade 3 or 4 hematologic toxicity (adjusted odds ratios, 3.24; 95% confidence interval, 1.35-7.78; P for trend = 0.009), and, more specifically, severe leukopenia toxicity (P for trend = 0.005). No statistically significant association was found for the three polymorphisms and grade 3 or 4 gastrointestinal toxicity. Consistent with these results of single-locus analysis, both the haplotype and the diplotype analyses revealed a protective effect of the haplotype "CG" (in the order of p.Arg156Arg-p.Asp312Asn) on the risk of grade 3 or 4 hematologic toxicity.

Conclusions: This investigation, for the first time, provides suggestive evidence of an effect of XPD p.Arg156Arg polymorphism on severe toxicity variability among platinum-treated non–small cell lung cancer patients.

  N. R Dean , J. R Newman , E. E Helman , W Zhang , S Safavy , D.M Weeks , M Cunningham , L. A Snyder , Y Tang , L Yan , L. R McNally , D. J Buchsbaum and E. L. Rosenthal
 

Purpose: Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb)–based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation.

Experimental Design: Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells.

Results: Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN–expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb–treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1β (IL-1β), IL-6, and IL-8 cytokine production, in vitro and in vivo.

Conclusions: These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.

  W Zhang , K Sun , Y Yang , H Zhang , F. B Hu and R. Hui
 

Background: Hyperuricemia has been positively associated with hypertension, but whether this association is independent of adiposity and other cardiovascular risk factors remains a matter of debate.

Methods: We conducted a community-based prospective cohort study comprising 7220 participants (mean age 37 years; 73.8% men) in the Qingdao Port Health and Nutrition Examination Survey in China, who were free from hypertension at study entry in 1999–2000. During 4-year follow-up, 1370 men (19.0%) and 208 women (11.0%) had developed hypertension.

Results: After adjustment for age, body mass index, and other covariates, the relative risks (RRs) of developing hypertension comparing the highest and lowest uric acid quartiles were 1.55 (95% CI 1.10–2.19; P for trend <0.001) for men and 1.91 (1.12–3.25; P for trend <0.001) for women. After additional adjustment for abdominal obesity, the RRs comparing the participants in the highest and lowest quartiles of uric acid were 1.39 (1.16–1.68; P for trend 0.003) for men and 1.85 (1.06–3.24; P for trend 0.006) for women. In joint analysis, compared with those in the lowest uric acid quartile and without abdominal obesity, participants who were in the highest quartile and also had abdominal obesity had a 3.0- and 3.4-fold greater risk of incident hypertension (1.56–3.97 for men and 2.10–3.81 for women, respectively).

Conclusions: These data suggest a positive association between plasma uric acid and incidence of hypertension during short-term follow-up in a Chinese population. The association between hyperuricemia and hypertension was partly mediated by abdominal obesity.

  S Filipovic Sadic , S Sah , L Chen , J Krosting , E Sekinger , W Zhang , P. J Hagerman , T. T Stenzel , A. G Hadd , G. J Latham and F. Tassone
 

Background: Fragile X syndrome (FXS) is a trinucleotide-repeat disease caused by the expansion of CGG sequences in the 5' untranslated region of the FMR1 (fragile X mental retardation 1) gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on 2 tests, PCR and Southern blotting; however, performance or throughput limitations of these methods currently constrain routine testing.

Methods: We evaluated a novel FMR1 gene–specific PCR technology with DNA templates from 20 cell lines and 146 blinded clinical samples. The CGG repeat number was determined by fragment sizing of PCR amplicons with capillary electrophoresis, and results were compared with those for FMR1 Southern blotting analyses with the same samples.

Results: The FMR1 PCR accurately detected full-mutation alleles up to at least 1300 CGG repeats and consisting of >99% GC character. All categories of alleles detected by Southern blotting, including 66 samples with full mutations, were also identified by the FMR1 PCR for each of the 146 clinical samples. Because all full mutation alleles in samples from heterozygous females were detected by the PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940-CGG allele in a background of 99% 23-CGG allele—a roughly 5- fold greater sensitivity than obtained with Southern blotting.

Conclusions: The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify; reproducibly detect low abundance full mutation alleles; and correctly infer homozygosity in female samples, thus greatly reducing the need for sample reflexing to Southern blotting.

  W Zhang , E Lopez Garcia , T. Y Li , F. B Hu and R. M. van Dam
  OBJECTIVE

Coffee consumption has been linked to detrimental acute metabolic and hemodynamic effects. We investigated coffee consumption in relation to risk of CVDs and mortality in diabetic men.

RESEARCH DESIGN AND METHODS

We conducted a prospective cohort study including 3,497 diabetic men without CVD at baseline.

RESULTS

After adjustment for age, smoking, and other cardiovascular risk factors, relative risks (RRs) were 0.88 (95% CI 0.50–1.57) for CVDs (P for trend = 0.29) and 0.80 (0.41–1.54) for all-cause mortality (P for trend = 0.45) for the consumption of ≥4 cups/day of caffeinated coffee compared with those for non–coffee drinkers. Stratification by smoking and duration of diabetes yielded similar results. RRs for caffeine intake for the highest compared with the lowest quintile were 1.02 (0.70–1.47; P for trend = 0.96) for CVDs and 0.96 (0.64–1.44; P for trend = 0.69) for mortality.

CONCLUSIONS

These data indicate that regular coffee consumption is not associated with increased risk for CVDs or mortality in diabetic men.

  W Zhang , L Wang , Y Liu , J Xu , G Zhu , H Cang , X Li , M Bartlam , K Hensley , G Li , Z Rao and X. C. Zhang
 

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.

  W Zhang and D. Durocher
 

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability, since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. Here we report that Mec1, the ATR ortholog, couples the detection of DNA ends with the inhibition of telomerase. Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, a phosphorylation event that suppresses Cdc13 accumulation at DSBs. Conversely, telomere addition at accidental breaks is promoted by Pph3, the yeast protein phosphatase 4 (PP4). Pph3 is itself modulated by Rrd1, an activator of PP2A family phosphatases. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DNA breaks. These studies therefore identify a mechanism by which the ATR family of kinases enforces genome integrity, and a process that underscores the contribution of Cdc13 to the fate of DNA ends.

  C Li , Z Chen , Z Liu , J Huang , W Zhang , L Zhou , D. L Keefe and L. Liu
 

Mammalian parthenogenetic embryos (pE) are not viable due to placental deficiency, presumably resulting from lack of paternally expressed imprinted genes. Pluripotent parthenogenetic embryonic stem (pES) cells derived from pE could advance regenerative medicine by avoiding immuno-rejection and ethical roadblocks. We attempted to explore the epigenetic status of imprinted genes in the generation of pES cells from parthenogenetic blastocysts, and its relationship to pluripotency of pES cells. Pluripotency was evaluated for developmental and differentiation potential in vivo, based on contributions of pES cells to chimeras and development to day 9.5 of pES fetuses complemented by tetraploid embryos (TEC). Consistently, pE and fetuses failed to express paternally expressed imprinted genes, but pES cells expressed those genes in a pattern resembling that of fertilized embryos (fE) and fertilized embryonic stem (fES) cells derived from fE. Like fE and fES cells, but unlike pE or fetuses, pES cells and pES cell–fetuses complemented by TEC exhibited balanced methylation of Snrpn, Peg1 and U2af1-rs1. Coincidently, global methylation increased in pE but decreased in pES cells, further suggesting dramatic epigenetic reprogramming occurred during isolation and culture of pES cells. Moreover, we identified decreased methylation of Igf2r, Snrpn, and especially U2af1-rs1, in association with increased contributions of pES cells to chimeras. Our data show that in vitro culture changes epigenetic status of imprinted genes during isolation of pES cells from their progenitor embryos and that increased expression of U2af1-rs1 and Snrpn and decreased expression of Igf2r correlate with pluripotency of pES cells.

  Y Ding , L Jiao , W Zhang , L Zhou , X Zhang and L. Zhang
 

Sucrose is a convenient, common, tissue-equivalent material suitable for electron paramagnetic resonance (EPR) dosimetry of ionising radiation. A number of publications have reported on the dosimetric properties of sucrose and their use in radiation accident dose reconstruction. However, previous studies did not include specially the description of measurement methods of sucrose by EPR. The aim of this work is to introduce particularly the EPR measurement methods of sucrose. In this regard, practical considerations of sample size, microwave power, modulation amplitude, EPR spectrum and signal stability are discussed.

  C Jiang , H Zhang , W Zhang , W Kong , Y Zhu , Q Xu , Y Li and X. Wang
 

Adipokines may represent a mechanism linking insulin resistance to cardiovascular disease. We showed previously that homocysteine (Hcy), an independent risk factor for cardiovascular disease, can induce the expression and secretion of resistin, a novel adipokine, in vivo and in vitro. Since vascular smooth muscle cell (VSMC) migration is a key event in vascular disease, we hypothesized that adipocyte-derived resistin is involved in Hcy-induced VSMC migration. To confirm our hypothesis, Sprague-Dawley rat aortic SMCs were cocultured with Hcy-stimulated primary rat epididymal adipocytes or treated directly with increasing concentrations of resistin for up to 24 h. Migration of VSMCs was investigated. Cytoskeletal structure and cytoskeleton-related proteins were also detected. The results showed that Hcy (300–500 µM) increased migration significantly in VSMCs cocultured with adipocytes but not in VSMC cultured alone. Resistin alone also significantly increased VSMC migration in a time- and concentration-dependent manner. Resistin small interfering RNA (siRNA) significantly attenuated VSMC migration in the coculture system, which indicated that adipocyte-derived resistin mediates Hcy-induced VSMC migration. On cell spreading assay, resistin induced the formation of focal adhesions near the plasma membrane, which suggests cytoskeletal rearrangement via an 5β1-integrin-focal adhesion kinase/paxillin-Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway. Our data demonstrate that Hcy promotes VSMC migration through a paracrine or endocrine effect of adipocyte-derived resistin, which provides further evidence of the adipose-vascular interaction in metabolic disorders. The migratory action exerted by resistin on VSMCs may account in part for the increased incidence of restenosis in diabetic patients.

  W Zhang , L Du and S. J. Gunst
 

The small GTPase RhoA increases the Ca2+ sensitivity of smooth muscle contraction and myosin light chain (MLC) phosphorylation by inhibiting the activity of MLC phosphatase. RhoA is also a known regulator of cytoskeletal dynamics and actin polymerization in many cell types. In airway smooth muscle (ASM), contractile stimulation induces MLC phosphorylation and actin polymerization, which are both required for active tension generation. The objective of this study was to evaluate the primary mechanism by which RhoA regulates active tension generation in intact ASM during stimulation with acetylcholine (ACh). RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, in the intact tissues or by treating them with the cell-permeant RhoA inhibitor, exoenzyme C3 transferase. RhoA inactivation reduced ACh-induced contractile force by ~60% and completely inhibited ACh-induced actin polymerization but inhibited ACh-induced MLC phosphorylation by only ~20%. Inactivation of MLC phosphatase with calyculin A reversed the reduction in MLC phosphorylation caused by RhoA inactivation, but calyculin A did not reverse the depression of active tension and actin polymerization caused by RhoA inactivation. The MLC kinase inhibitor, ML-7, inhibited ACh-induced MLC phosphorylation by ~80% and depressed active force by ~70% but did not affect ACh-induced actin polymerization, demonstrating that ACh-stimulated actin polymerization occurs independently of MLC phosphorylation. We conclude that the RhoA-mediated regulation of ACh-induced contractile tension in ASM results from its role in mediating actin polymerization rather than from effects on MLC phosphatase or MLC phosphorylation.

  E. M Neuhaus , W Zhang , L Gelis , Y Deng , J Noldus and H. Hatt
 

Olfactory receptors (ORs) are expressed not only in the sensory neurons of the olfactory epithelium, where they detect volatile substances, but also in various other tissues where their potential functions are largely unknown. Here, we report the physiological characterization of human OR51E2, also named prostate-specific G-protein-coupled receptor (PSGR) due to its reported up-regulation in prostate cancer. We identified androstenone derivatives as ligands for the recombinant receptor. PSGR can also be activated with the odorant β-ionone. Activation of the endogenous receptor in prostate cancer cells by the identified ligands evoked an intracellular Ca2+ increase. Exposure to β-ionone resulted in the activation of members of the MAPK family and inhibition of cell proliferation. Our data give support to the hypothesis that because PSGR signaling could reduce growth of prostate cancer cells, specific receptor ligands might therefore be potential candidates for prostate cancer treatment.

  M. J Benson , R Elgueta , W Schpero , M Molloy , W Zhang , E Usherwood and R. J. Noelle
 

The hypothesis that bystander inflammatory signals promote memory B cell (BMEM) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220+IgG+ BMEM toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent BMEM clonally expand. Surprisingly, proliferating BMEM do not acquire germinal center (GC) B cell markers before generating daughter BMEM and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of BMEM proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of BMEM occurred. The absence of a BMEM response to nonspecific inflammatory signals clearly shows that BMEM proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

 
 
 
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