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Articles by X Wang
Total Records ( 68 ) for X Wang
  Y Peng , H Li , M Wu , X Wang , S Fan , F Liu , B Xiang , Q Guo , X Tang and S. Shen
 

Colorectal cancer (CRC) is a common malignant tumor that is associated with an increased incidence of morbidity and mortality. Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate suppressor gene of tumor metastasis, which is down-regulated in CRC. In the present study, we constructed a colorectal tissue microarray to examine the expression profiles of NGX6, phospho-c-Jun N-terminal kinase (p-JNK), and phospho-extracellular signal-regulated kinase (p-ERK ) in CRC tissues. We found that the NGX6 expression was lower in CRC tissues and metastatic lymph nodes, whereas the expressions of p-JNK and p-ERK were higher in CRC tissues, than in normal intestinal mucosa. The expressions of NGX6, p-JNK, and p-ERK were associated with the clinical pathological features of colorectal tissues. NGX6 overexpression inhibited the activation and nuclear translocation of JNK1, which led to an accumulation of p-JNK in the cytoplasm, but did not inhibit the activation and nuclear translocation of ERK1/2. NGX6 also inhibited the expression of the transcription factors AP-1 (c-jun and c-fos) and Ets-1. In addition, NGX6 overexpression decreased the expression of cyclin D1 and dramatically suppressed the transcriptional efficiency of the cyclin D1 promoter. We propose that NGX6 expression is lost in the multi-step process of human colorectal carcinogenesis. Its overexpression can inhibit the expression of transcription factors AP-1 and Ets-1, and down-regulate the transcriptional activity of the cyclin D1 promoter in human CRC.

  J Xiao , S Yin , Y Li , S Xie , D Nie , L Ma , X Wang , Y Wu and J. Feng
 

S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  J Gu , D Sun , Q Zheng , X Wang , H Yang , J Miao , J Jiang and W. Wei
 

Elongator complex has been associated with hyperphosphorylated RNA polymerase II and is known to play critical roles in transcriptional elongation, as well as in tRNA modification and exocytosis. However, the specific mechanism of how human Elongator complex regulates cell growth and cell cycle remains unclear. To investigate the composition of human Elongator complex and its effects on cell growth, 293T cells were established that stably overexpressed Flag-Elp3 and Flag-Elp4. By using anti-Flag M2 antibody-bound resin, a core Elongator complex was purified from cells that stably overexpressed Flag-Elp3. No Elongator complex was purified from cells stably transfected with pFlagCMV4-Elp4. Interestingly, the cell growth was inhibited in 293T cells transfected with pFlagCMV4-Elp3. Flow cytometry analysis showed that most of the cells stably overexpressing Flag-Elp3 were found in G1 stage, indicating a role of the core Elongator in the G1 checkpoint for the regulation of cell cycle. We observed increased basal transcription and remarkably enhanced transcription stimulated by VP16 in 293T cells overexpressing Flag-Elp3. The transcription could also be synergistically activated by overexpressing both Elp3 and Elp4. Taken together, our results suggested that the core Elongator complex formed by Elp1, Elp2, and Elp3 was rather stable, whereas Elp4, Elp5, and Elp6 might loosely contact and work together with the core Elongator to regulate cell functions.

  X Li , C Dong , S Shi , G Wang , Y Li , X Wang , Q Shi , C Tian , R Zhou , C Gao and X. Dong
 

Prion protein (PrP) is considered to associate with microtubule and its major component, tubulin. In the present study, octarepeat region of PrP (PrP51–91) was expressed in prokaryotic-expressing system. Using GST pull-down assay and co-immunoprecipitation, the molecular interaction between PrP51–91 and tubulin was observed. Our data also demonstrated that PrP51–91 could efficiently stimulate microtubule assembly in vitro, indicating a potential effect of PrP on microtubule dynamics. Moreover, PrP51–91 was confirmed to be able to antagonize Cu2+-induced microtubule-disrupting activity in vivo, partially protecting against Cu2+ intoxication to culture cells and stabilize cellular microtubule structure. The association of the octarepeat region of PrP with tubulin may further provide insight into the biological function of PrP in the neurons.

  D Han , Y Ding , S. L Liu , G Wang , I. C Si , X Wang , L Cui and D. Huang
 

Fas ligand (FasL) may play an important role in maintaining the immune privilege of intervertebral disc (IVD). Besides, it is closely related to the apoptosis of degenerative disc cells. Nowadays, lots of reports have described about the paradoxical effects of FasL, although the effect of FasL on IVD cells is still under debate. In this study, we tried to investigate the effects of FasL on Fas expression and on the apoptosis of nucleus pulposus (NP) cells in Sprague–Dawley rats. The results showed that the expression of Fas in NP cells was significantly increased by the recombinant FasL. Meanwhile, the apoptosis of NP cells increased markedly in a FasL dose-dependent manner. Interestingly, RNA interference results indicated that the increase of Fas expression and the NP cell apoptosis described previously were inhibited by Fas siRNA, suggesting that RNA interference might be one of novel strategies to prevent IVD cells from apoptosis.

  D Ouyang , X He , L Xu , X Wang , Q Gao and H. Guo
 

The major histocompatibility complex class I allele Mamu-B*17 of rhesus macaques is an elite controller of simian immunodeficiency virus (SIV) infection whereas Mamu-B*01 has no inhibitory effect on SIV replication. The mechanism is still elusive. In this study, the so-called ‘missing G’ in the leading peptide sequence of Mamu-B*1703 allele was artificially inserted back through PCR amplification, and the new sequence was renamed as Mamu-B*1703(+G). The plasmids harboring Mamu-B*1703, Mamu-B*1703(+G) and Mamu-B*0101 cDNA sequence fused to EGFP gene were transfected into K562 and Cos-7 cells, respectively. Our data showed that these plasmids had similar transfection efficiencies and expression potentials in K562 cells, but the surface density of Mamu-B*1703 complexes, which was slightly influenced by the artificial change of the leading peptide length, was much higher than that of Mamu-B*0101 molecules. These results might partially account for the differential effects of Mamu-B*17 and Mamu-B*01 alleles on SIV replication in rhesus macaques.

  L Tie , X. J Li , X Wang , K. M Channon and A. F. Chen
 

Refractory wound is a severe complication that leads to limb amputation in diabetes. Endothelial nitric oxide synthase (eNOS) plays a key role in normal wound repair but is uncoupled in streptozotocin (STZ)-induced type 1 diabetes because of reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that overexpression of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for de novo BH4 synthesis, retards NOS uncoupling and accelerates wound healing in STZ mice. Blood glucose levels were significantly increased in both male endothelium-specific GTPCH I transgenic mice (Tg-GCH; via a tie-2 promoter) and wild-type (WT) littermates 5 days after STZ regimen. A full-thickness excisional wound was created on mouse dorsal skin by a 4-mm punch biopsy. Wound closure was delayed in STZ mice, which was rescued in STZ Tg-GCH mice. Cutaneous BH4 level was significantly reduced in STZ mice vs. WT mice, which was maintained in STZ Tg-GCH mice. In STZ mice, constitutive NOS (cNOS) activity and nitrite levels were decreased compared with WT mice, paralleled by increased superoxide anion (O2) level and inducible NOS (iNOS) activity. In STZ Tg-GCH mice, nitrite level and cNOS activity were potentiated and O2 level and iNOS activity were suppressed compared with STZ mice. Thus endothelium-specific BH4 overexpression accelerates wound healing in type 1 diabetic mice by enhancing cNOS activity and suppressing oxidative stress.

  Y Wang , W. B Lau , E Gao , L Tao , Y Yuan , R Li , X Wang , W. J Koch and X. L. Ma
 

Adiponectin (APN) has traditionally been viewed as an adipocyte-specific endocrine molecule with cardioprotective effects. Recent studies suggest that APN is also expressed in cardiomyocytes. However, biological significances of this locally produced APN remain completely unknown. The aim of this study was to investigate the pathological and pharmacological significance of cardiac-derived APN in cardiomyocyte pathology. Adult cardiomyocytes from wild-type littermates (WT) or gene-deficient mice were pretreated with vehicle (V) or rosiglitazone (RSG) for 6 h followed by simulated ischemia-reperfusion (SI/R, 3 h/12 h). Compared with WT cardiomyocytes, myocytes from APN knockout (APN-KO) mice sustained greater SI/R injury, evidenced by greater oxidative/nitrative stress, caspase-3 activity, and lactate dehydrogenase (LDH) release (P < 0.05). Myocytes from adiponectin receptor 1 knockdown (AdipoR1-KD) or AdipoR1-KD/AdipoR2-KO mice had slightly increased SI/R injury, but the difference was not statistically significant. RSG significantly (P < 0.01) increased APN mRNA and protein expression, upregulated AdipoR1/AdipoR2 expression, reduced SI/R-induced apoptosis, and decreased LDH release in WT cardiomyocytes. However, the anti-oxidative/anti-nitrative and cell protective effects of RSG were completely lost in APN-KO cardiomyocytes (P > 0.05 vs. vehicle group), although a comparable degree of AdipoR1/AdipoR2 upregulation was observed. The upregulatory effect of RSG on APN mRNA and protein expression was significantly potentiated in AdipoR1-KD/AdipoR2-KO cardiomyocytes. However, the cellular protective effects of RSG were significantly blunted, although not completely lost, in these cells. These results demonstrated that cardiomyocyte APN is biologically active in protecting cells against SI/R injury. Moreover, this locally produced APN achieves its protective effect primarily through paracrine/autocrine activation of APN receptors.

  L He , H Zeng , F Li , J Feng , S Liu , J Liu , J Yu , J Mao , T Hong , A. F Chen , X Wang and G. Wang
 

Hyperhomocysteinemia (HHcy) has been associated with impaired vascular endothelial function. Our previous study demonstrated significantly higher secretion of the chemokine monocyte chemoattractant protein-1 from monocytes in response to lipopolysaccharide in patients with HHcy. In the present study, we investigated whether coronary endothelial function was damaged in patients with chronic HHcy (plasma level of homocysteine >15 µmol/l) and, if so, whether this impaired endothelial function is induced by the uncoupling of endothelial nitric oxide synthase (eNOS). When tetrahydrobiopterin levels are inadequate, eNOS is no longer coupled to l-arginine oxidation, which results in reactive oxygen species rather than nitric oxide production, thereby inducing vascular endothelial dysfunction. The 71 participants were divided into two groups, control (n = 50) and HHcy (n = 21). Quantification of coronary flow velocity reserve (CFVR) was after rest and after adenosine administration done by noninvasive Doppler echocardiography. Plasma levels of nitric oxide and tetrahydrobiopterin were significantly lower in patients with HHcy than in controls (99.54 ± 32.23 vs. 119.50 ± 37.68 µmol/l and 1.43 ± 0.46 vs. 1.73 ± 0.56 pmol/ml, all P < 0.05). Furthermore, CFVR was significantly lower in the HHcy than the control group (2.76 ± 0.49 vs. 3.09 ± 0.52, P < 0.05). In addition, plasma level of homocysteine was negatively correlated with CFVR. Chronic HHcy may contribute to coronary artery disease by inducing dysfunction of the coronary artery endothelium. The uncoupling of eNOS induced by HHcy in patients with chronic HHcy may explain this adverse effect in part.

  S Togo , X Liu , X Wang , H Sugiura , K Kamio , S Kawasaki , T Kobayashi , R. F Ertl , Y Ahn , O Holz , H Magnussen , K Fredriksson , C. M Skold and S. I. Rennard
 

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-β1, a mediator of fibrosis, can potentially modulate cAMP by altering PGE2 metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-β1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was ~10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-β1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through cAMP-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of PGE2 and TGF-β1-induced PGE2 production. PDE4 inhibitors together with TGF-β1 resulted in augmented PGE2 production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-β1-induced fibroblast stimulation.

  C Xia , Q Tong , Q Wang , Z Tang , L Qi , S Chi , M Zhang , X Wang , H Li and G. Xu
  Background

The in vitro directive of the European Union requires traceability to the international recommended reference procedures. The application of the reference procedures is necessary in order to evaluate the accuracy of -glutamyltransferase (GGT) assays of routine measurement systems in China.

Methods

Five frozen patient-pooled serum samples were assigned values by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure in order to evaluate the traceability of the results of GGT catalytic activity from six homogeneous systems. One of the serum samples was used to calibrate seven non-homogeneous systems.

Results

All of the homogeneous systems, except the Dade system (Dade Bering Inc, IL, USA), achieved traceability within the measurement range. The Roche and Hitachi systems were better than the other systems. After calibration, the variance of the non-homogeneous systems decreased dramatically from between 14.50% and 25.23% to between 1.25% and 3.09% and the bias decreased from between –11.4% and –4.1% to between 0.5% and 3.5%.

Conclusion

Manufacturers in China should ensure that their calibration systems correspond to the IFCC reference procedures. Fresh frozen pooled patient serum assigned by reference laboratories can be used to calibrate non-homogeneous systems in order to achieve traceability.

  J. M Weinberg , L. J Appel , G Bakris , J. J Gassman , T Greene , C. A Kendrick , X Wang , J Lash , J. A Lewis , V Pogue , D Thornley Brown , R. A Phillips and for the African American Study of Hypertension and Kidney Disease Collaborative Research Group
 

Background  The incidence and factors associated with hyperkalemia in patients with chronic kidney disease (CKD) treated with angiotensin converting enzyme inhibitors (ACEIs) and other antihypertensive drugs was investigated using the African American Study of Kidney Disease and Hypertension (AASK) database.

Methods  A total of 1094 nondiabetic adults with hypertensive CKD (glomerular filtration rate [GFR], 20-65 mL/min/1.73 m2) were followed for 3.0 to 6.4 years in the AASK trial. Participants were randomly assigned to ACEI, β-blocker (BB), or dihydropyridine calcium channel blocker (CCB). The outcome variables for this analysis were a serum potassium level higher than 5.5 mEq/L (to convert to millimoles per liter, multiply by 1.0), or a clinical center initiated hyperkalemia stop point.

Results  A total of 6497 potassium measurements were obtained, and 80 events in 51 subjects were identified (76 events driven by a central laboratory result and 4 driven by a clinical center–initiated hyperkalemia stop point). Compared with a GFR higher than 50 mL/min/1.73 m2, after multivariable adjustment, the hazard ratio (HR) for hyperkalemia in patients with a GFR between 31 and 40 mL/min/1.73 m2 and a GFR lower than 30 mL/min/1.73 m2 was 3.61 (95% confidence interval [CI], 1.42-9.18 [P = .007]) and 6.81 (95% CI, 2.67-17.35 [P < .001]), respectively; there was no increased risk of hyperkalemia if GFR was 41 to 50 mL/min/1.73 m2. Use of ACEIs was associated with more episodes of hyperkalemia compared with CCB use (HR, 7.00; 95% CI, 2.29-21.39 [P < .001]) and BB group (HR, 2.85; 95% CI, 1.50-5.42 [P = .001]). Diuretic use was associated with a 59% decreased risk of hyperkalemia.

Conclusions  In nondiabetic patients with hypertensive CKD treated with ACEIs, the risk of hyperkalemia is small, particularly if baseline and follow-up GFR is higher than 40 mL/min/1.73 m2. Including a diuretic in the regimen may markedly reduce risk of hyperkalemia.

  X Wang and J. Shen
 

We study a class of monotone univariate regression estimators. We use B-splines to approximate an underlying regression function and estimate spline coefficients based on grouped data. We investigate asymptotic properties of two monotone estimators: a grouped Brunk estimator and a penalized monotone estimator. These estimators are consistent at the boundary and their mean square errors achieve optimal convergence rates under suitable assumptions of the true regression function. Asymptotic distributions are developed and are shown to be independent of spline degrees and the number of knots. Simulation results and car data illustrate performance of the proposed estimators.

  Y Liao , J Tang , M Ma , Z Wu , M Yang , X Wang , T Liu , X Chen , P. C Fletcher and W. Hao
 

Ketamine abuse has been shown to have a deleterious impact on brain function. However, the precise mechanisms of ketamine dependence-induced pathological change remain poorly understood. Although there is evidence for white matter changes in drug abuse, the presence of white matter abnormalities in chronic ketamine users has not been studied. White matter volumes were measured using in vivo diffusion tensor magnetic resonance imaging data in 41 ketamine-dependent subjects and 44 drug-free healthy volunteers. White matter changes associated with chronic ketamine use were found in bilateral frontal and left temporoparietal cortices. There was also evidence that frontal white matter fractional anisotropy correlated with the severity of drug use (as measured by estimated total ketamine consumption). We provide direct evidence for dose-dependent abnormalities of white matter in bilateral frontal and left temporoparietal regions following chronic ketamine use. The findings suggest a microstructural basis for the changes in cognition and experience observed with prolonged ketamine use. Moreover, the similarities of these changes to those observed in chronic schizophrenia have implications for the glutamate model of this illness.

  L. E Kelemen , X Wang , Z. S Fredericksen , V. S Pankratz , P. D.P Pharoah , S Ahmed , A. M Dunning , D. F Easton , R. A Vierkant , J. R Cerhan , E. L Goode , J. E Olson and F. J. Couch
 

Background: Gene amplification leading to overexpression is a common event in breast tumors that is linked to tumor development and progression. The 17q23 region is amplified in >40% of breast tumors and contains several candidate oncogenes. Because common genetic variation in several oncogenes has been associated with cancer risk, we assessed the relevance of common variants in the 17q23 candidate oncogenes to breast cancer.

Methods: We investigated 60 polymorphisms in the TUBD1, SEPT4, PRKCA, TBX2, TBX4, TEX14, TLK2, YPEL2, and PPM1E genes from this amplicon for association with breast cancer risk among 798 Caucasian breast cancer cases and 843 unaffected Caucasian controls from the Mayo Clinic.

Results: Eight polymorphisms in PRKCA, TBX4, TLK2, and YPEL2 displayed significant dose-response associations with breast cancer risk (Ptrend < 0.05). Of these, PRKCA rs7342847 and TLK2 rs2245092 and rs733025 were also associated with hormone receptor–positive breast cancer: PRKCA rs7342847 (odds ratio, 0.7; 95% confidence interval, 0.6-0.9; Ptrend = 0.002) and TLK2 rs733025 and rs2245092 (both: odds ratio, 0.8; 95% confidence interval, 0.7-1.0; Ptrend = 0.03). Interactions between SEPT4 rs758377 and TEX14 rs302864 (Pinteraction = 0.0003) and between TLK2 rs733025 and YPEL2 rs16943468 (Pinteraction = 0.05) for risk of breast cancer were also observed.

Conclusion: These findings suggest that single polymorphisms and combinations of polymorphisms within candidate oncogenes from the 17q23 amplicon may influence risk of breast cancer overall and possibly specific molecular subtypes of breast tumors. The findings are discussed within the context of the results from two independent data sets. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1864–8)

  P Jin , X. j Lu , J. q Sheng , L Fu , X. m Meng , X Wang , T. p Shi , S. r Li and J. Rao
 

Estrogen is reported to have a protective effect on colon cancer; however, the underlying mechanism is unclear. Impaired mismatch repair plays an important role in colonic carcinogenesis. The purpose of this study was to investigate the association of estrogen on regulating mismatch repair expression in colonic epithelial cells. In cultured COLO205 cells, the effect of estradiol (E2) and antagonist ICI182.780 on the expression of hMLH1 and hMSH2 was studied using reverse transcription-PCR and Western blotting. The correlation between serum level E2 and the expression of hMLH1 and hMSH2 in colonic mucosal tissue of 42 healthy individuals was also examined using reverse transcription-PCR and immunohistochemical staining. E2 increased the expression of hMLH1 in COLO205 cells, which was suppressed by ICI182.780. However, the effect of E2 on hMSH2 expression was not significant in COLO205 cells. In healthy individuals, a strong positive correlation of E2 level with hMLH1 expression in normal colonic epithelial cell was observed when serum E2 level was >45 pg/mL, but no correlation was seen between E2 and hMSH2 expression. E2 affects the expression of hMLH1 but not hMSH2 in vitro, and high serum E2 level correlates with hMLH1 expression in vivo. These findings suggest that the anticolonic cancer effect of estrogen may be related to hMLH1 regulation. Cancer Prev Res; 3(8); 910–6. ©2010 AACR.

  T Adamovic , D McAllister , V Guryev , X Wang , J. W Andrae , E Cuppen , H. J Jacob and S. L. Sugg
 

The presence of copy number variants in normal genomes poses a challenge to identify small genuine somatic copy number changes in high-resolution cancer genome profiling studies due to the use of unpaired reference DNA. Another problem is the well-known rearrangements of immunoglobulin and T-cell receptor genes in lymphocytes (a commonly used reference), which may misdirect the researcher to a locus with no relevance in tumorigenesis. We here show real gains of the IgG heavy chain V gene region in carcinogen-induced rat mammary tumor samples after normalization to paired mammary gland, a tissue without lymphocyte infiltration. We further show that the segmental duplication region encompassing the IgG heavy chain V genes is a copy number variant between the susceptible (SS) and the resistant (BN) to mammary tumor development inbred rat strains. Our data suggest that the already inherently unstable genomic region is a convenient target for additional structural rearrangements (gains) at the somatic level when exposed to a carcinogen (7,12-dimethylbenz[a]anthracene), which subsequently seem to benefit tumor development in the mammary gland of the susceptible strain. Thus, the selection of an appropriate reference DNA enabled us to identify immunoglobulin genes as novel cancer targets playing a role in mammary tumor development. We conclude that control DNA in array-based comparative genomic hybridization experiments should be selected with care, and DNA from pooled spleen (contains immature lymphocytes and is used as reference in animal studies) or blood may not be the ideal control in the study of primary tumors. [Cancer Res 2009;69(12):5159–67]

  X Wang and A. Schneider
 

Despite their individual key roles in promoting head and neck squamous cell carcinoma (HNSCC) progression and treatment resistance, little is known about the impact of intratumoral hypoxia on the activity of the epidermal growth factor receptor (EGFR) signaling pathway in this cancer type. Here, we show that in highly EGFR-expressing HNSCC cells, hypoxic stress triggers the activation of the EGFR and downstream targets, including Akt and phospholipase C (PLC) 1. In support of these findings, we also demonstrate that EGFR activation takes place within hypoxic foci in a subset of human HNSCC tissues. Whereas hypoxia had no major effect on HNSCC cell proliferation, it markedly altered tumor cell shape by inducing morphological changes consistent with a more spindle-shaped, fibroblast-like morphology together with an enhanced migratory capacity. We found that hypoxia-induced EGFR activation and cell migration could be prevented by targeting EGFR signaling with the tyrosine kinase inhibitor tyrphostin, the phospholipase C inhibitor U73122, or by inhibiting the expression of the subunit of hypoxia-inducible factor 2 via RNA interference or the topoisomerase II inhibitor etoposide. Our results position hypoxia-inducible factor-2 as a novel regulator of EGFR activation under low oxygen conditions, and suggest that hypoxia-induced EGFR signaling may promote a more aggressive phenotype in a fraction of HNSCC tumors. Because EGFR continues in the forefront as a highly attractive target in clinical oncology, further studies are warranted to define the mechanistic and therapeutic implications of the hypoxic response relative to the EGFR signaling pathway in head and neck cancer.

  S Pinkert , D Westermann , X Wang , K Klingel , A Dorner , K Savvatis , T Grossl , S Krohn , C Tschope , H Zeichhardt , K Kotsch , K Weitmann , W Hoffmann , H. P Schultheiss , O. B Spiller , W Poller and H. Fechner
 

Background— Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach.

Methods and Results— We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59±3.8 versus 45.4±2.7 mm Hg, median 59 versus 45.8 mm Hg, P<0.01; dP/dtmax 3645.1±443.6 versus 2057.9±490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P<0.01; and dP/dtmin –2125.5±330.5 versus –1310.2±330.3 mm Hg/s, median –2083.7 versus –1295.9 mm Hg/s, P<0.01) and improved contractility if induced concomitantly with infection (left ventricular pressure 76.4±19.2 versus 56.8±10.3 mm Hg, median 74.8 versus 54.4 mm Hg, P<0.05; dP/dtmax 5214.2±1786.2 versus 3011.6±918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P<0.05), respectively. Importantly, hemodynamics of animals treated with AdG12 (plus doxycycline) were similar to uninfected controls. Preinfection induction of sCAR-Fc completely blocked and concomitant induction strongly reduced cardiac CVB3 infection, myocardial injury, and inflammation.

Conclusion— AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions.

  J Qian , X Ren , X Wang , P Zhang , W. K Jones , J. D Molkentin , G. C Fan and E. G. Kranias
 

Rationale: The levels of a small heat shock protein (Hsp)20 and its phosphorylation are increased on ischemic insults, and overexpression of Hsp20 protects the heart against ischemia/reperfusion injury. However, the mechanism underlying cardioprotection of Hsp20 and especially the role of its phosphorylation in regulating ischemia/reperfusion–induced autophagy, apoptosis, and necrosis remain to be clarified.

Objective: Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20S16A). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation.

Methods and Results: Our results demonstrated that Hsp20S16A hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20S16A hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20S16A hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20S16A hearts.

Conclusions: Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.

  V Kandalam , R Basu , T Abraham , X Wang , P. D Soloway , D. M Jaworski , G. Y Oudit and Z. Kassiri
 

Rationale: Myocardial infarction (MI) results in remodeling of the myocardium and the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) are critical regulators of ECM integrity via inhibiting matrix metalloproteinases (MMPs). TIMP2 is highly expressed in the heart and is the only TIMP that, in addition to inhibiting MMPs, is required for cell surface activation of pro-MMP2. Hence, it is difficult to predict the function of TIMP2 as protective (MMP-inhibiting) or harmful (MMP-activating) in heart disease.

Objective: We examined the role of TIMP2 in the cardiac response to MI.

Methods and Results: MI was induced in 11- to 12-week-old male TIMP2–/– and age-matched wild-type mice. Cardiac function was monitored by echocardiography at 1 and 4 weeks post-MI. ECM fibrillar structure was visualized using second harmonic generation and multiphoton imaging of unfixed/unstained hearts. Molecular analyses were performed at 3 days and 1 week post-MI on flash-frozen infarct, periinfarct, and noninfarct tissue. Membrane type 1 (MT1)-MMP levels and activity were measured in membrane protein fractions. TIMP2–/–-MI mice exhibited a 25% greater infarct expansion, markedly exacerbated left ventricular dilation (by 12%) and dysfunction (by 30%), and more severe inflammation compared to wild-type MI mice. Adverse ECM remodeling was detected by reduced density and enhanced disarray of fibrillar collagen in TIMP2–/–-MI compared to wild-type MI hearts. TIMP2 deficiency completely abrogated MMP2 activation but markedly increased collagenase activity, particularly MT1-MMP activity post-MI.

Conclusions: The MMP-inhibitory function of TIMP2 is a key determinant of post-MI myocardial remodeling primarily because of its inhibitory action on MT1-MMP. TIMP2 replenishment in diseased myocardium could provide a potential therapy in reducing or preventing disease progression.

  H Ding , B Wu , H Wang , Z Lu , J Yan , X Wang , J. R Shaffer , R Hui and D. W. Wang
 

Rationale: Asymmetrical dimethylarginine (ADMA), an endogenous arginine analogue, inhibits nitric oxide synthases and plays an important role in endothelial dysfunction.

Objective: In the present study, we tested whether a novel genetic variant in dimethylarginine dimethylaminohydrolase 1 (DDAH1), an important ADMA hydrolyzing gene, was associated with stroke and coronary heart disease (CHD) susceptibility in the Chinese Han population.

Methods and Results: By resequencing, we identified a novel 4-nucleotide deletion/insertion variant in the DDAH1 promoter. The insertion allele disrupted binding of metal-regulatory transcription factor 1, which resulted in significant reduction of in vitro DDAH1 transcriptional activity and in vivo DDAH1 mRNA level, and in turn, increased plasma ADMA level and the ratio of ADMA to l-arginine. We initially genotyped the polymorphism in 1388 stroke patients and 1027 controls as well as 576 CHD patients and 557 controls and then replicated our study in additional independent case-control cohorts comprising 961 stroke patients and 822 controls and 482 CHD patients and 1072 controls. We identified that the –396 4N ins allele was significantly associated with increased risk of thrombosis stroke and CHD after adjusting for environmental factors in both samples for both diseases (thrombosis stroke discovery set: odds ratio [OR]=1.35, P=0.032; replication set: OR=1.51, P=0.006; CHD discovery set: OR=1.45, P=0.035; replication set: OR=1.47, P=0.003).

Conclusions: Our results suggest that the DDAH1 loss-of-function polymorphism is associated with both increased risk of thrombosis stroke and CHD.

  X Wang , W Xie , Y Zhang , P Lin , L Han , P Han , Y Wang , Z Chen , G Ji , M Zheng , N Weisleder , R. P Xiao , H Takeshima , J Ma and H. Cheng
 

Rationale: Unrepaired cardiomyocyte membrane injury causes irreplaceable cell loss, leading to myocardial fibrosis and eventually heart failure. However, the cellular and molecular mechanisms of cardiac membrane repair are largely unknown. MG53, a newly identified striated muscle-specific protein, is involved in skeletal muscle membrane repair. But the role of MG53 in the heart has not been determined.

Objective: We sought to investigate whether MG53 mediates membrane repair in cardiomyocytes and, if so, the cellular and molecular mechanism underlying MG53-mediated membrane repair in cardiomyocytes. Moreover, we determined possible cardioprotective effect of MG53-mediated membrane repair.

Methods and Results: We demonstrated that MG53 is crucial to the emergency membrane repair response in cardiomyocytes and protects the heart from stress-induced loss of cardiomyocytes. Disruption of the sarcolemmal membrane by mechanical, electric, chemical, or metabolic insults caused rapid and robust translocation of MG53 toward the injury sites. Ablation of MG53 prevented sarcolemmal resealing after infrared laser–induced membrane damage in intact heart, and exacerbated mitochondrial dysfunction and loss of cardiomyocytes during ischemia/reperfusion injury. Unexpectedly, the MG53-mediated cardiac membrane repair was mediated by a cholesterol-dependent mechanism: depletion of membrane cholesterol abolished, and its recovery restored injury-induced membrane translocation of MG53. The redox status of MG53 did not affect initiation of MG53 translocation, whereas MG53 oxidation conferred stability to the membrane repair patch.

Conclusions: Thus, cholesterol-dependent MG53-mediated membrane repair is a vital, heretofore unappreciated cardioprotective mechanism against a multitude of insults and may bear important therapeutic implications.

  Y Yao , B. J Bennett , X Wang , M. E Rosenfeld , C Giachelli , A. J Lusis and K. I. Bostrom
  Rationale:

The bone morphogenetic proteins (BMPs), a family of morphogens, have been implicated as mediators of calcification and inflammation in the vascular wall.

Objective:

To investigate the effect of altered expression of matrix Gla protein (MGP), an inhibitor of BMP, on vascular disease.

Methods and Results:

We used MGP transgenic or MGP-deficient mice bred to apolipoprotein E mice, a model of atherosclerosis. MGP overexpression reduced vascular BMP activity, atherosclerotic lesion size, intimal and medial calcification, and inflammation. It also reduced expression of the activin-like kinase receptor 1 and the vascular endothelial growth factor, part of a BMP-activated pathway that regulates angiogenesis and may enhance lesion formation and calcification. Conversely, MGP deficiency increased BMP activity, which may explain the diffuse calcification of vascular medial cells in MGP deficient aortas and the increase in expression of activin-like kinase receptor 1 and vascular endothelial growth factor. Unexpectedly, atherosclerotic lesion formation was decreased in MGP-deficient mice, which may be explained by a dramatic reduction in expression of endothelial adhesion molecules limiting monocyte infiltration of the artery wall.

Conclusions:

Our results indicate that BMP signaling is a key regulator of vascular disease, requiring careful control to maintain normal vascular homeostasis.

  H Ding , Y Xu , X Wang , Q Wang , L Zhang , Y Tu , J Yan , W Wang , R Hui , C. Y Wang and D. W. Wang
 

Background— Recent studies on genome-wide association have identified common variants on chromosome 9p21 associated with coronary artery disease (CAD). Given that ischemic stroke and CAD share several aspects of etiology and pathogenesis, we investigated the association of variants on chromosome 9p21 with ischemic stroke and CAD in the Chinese Han population by capturing the majority of diversity in this locus using haplotype-tagging single-nucleotide polymorphisms.

Methods and Results— We performed a shared control-cases study using 15 tagging single-nucleotide polymorphisms and 2 previously reported susceptibility single-nucleotide polymorphisms spanning 58 kb of the chromosome of 9p21 in a set of 558 patients with ischemic stroke, 510 patients with CAD, and 557 unaffected participants (controls) in the Chinese Han population. The association analyses were performed at both SNP and haplotype levels. We further verified our findings in an independent cohort of 442 ischemic stroke cases and 502 control subjects. In the first study, rs2383206, rs1004638, and rs10757278 in block 3 were significantly associated with CAD but not with ischemic stroke independent of traditional cardiovascular risk factors in additive model (P=0.002 to 0.0001, q=0.026 to 0.004). Analysis from all blocks revealed that haplotype profiles of block 3 on 9p21 were significantly different between shared control and cases of CAD (P=1.3x10–10, q=1.2x10–9) and ischemic stroke (P=1.7x10–6, q=7.7x10–6). In the expanded second case-control study, block 3 on 9p21 remained associated with ischemic stroke (P=2.6x10–4, q=6.3x10–4).

Conclusions— Our results suggest for the first time that 9p21 is a shared susceptibility locus, strongly for CAD and weakly for ischemic stroke, in a Chinese Han population.

  S. S Wang , L. J Martin , E. E Schadt , H Meng , X Wang , W Zhao , L Ingram Drake , M Nebohacova , M Mehrabian , T. A Drake and A. J. Lusis
 

Background— Disruption of the elastic lamina, as an early indicator of aneurysm formation, and vascular calcification frequently occur together in atherosclerotic lesions of humans.

Methods and Results— We now report evidence of shared genetic basis for disruption of the elastic lamina (medial disruption) and medial calcification in an F2 mouse intercross between C57BL/6J and C3H/HeJ on a hyperlipidemic apolipoprotein E (ApoE–/–) null background. We identified 3 quantitative trait loci (QTLs) on chromosomes 6, 13, and 18, which are common to both traits, and 2 additional QTLs for medial calcification on chromosomes 3 and 7. Medial disruption, including severe disruptions leading to aneurysm formation, and medial calcification were highly correlated and occurred concomitantly in the cross. The chromosome 18 locus showed a striking male sex-specificity for both traits. To identify candidate genes, we integrated data from microarray analysis, genetic segregation, and clinical traits. The chromosome 7 locus contains the Abcc6 gene, known to mediate myocardial calcification. Using transgenic complementation, we show that Abcc6 also contributes to aortic medial calcification.

Conclusions— Our data indicate that calcification, though possibly contributory, does not always lead to medial disruption and that in addition to aneurysm formation, medial disruption may be the precursor to calcification.

  P. S Gargalovic , A Erbilgin , O Kohannim , J Pagnon , X Wang , L Castellani , R LeBoeuf , M. L Peterson , B. T Spear and A. J. Lusis
 

Background— We previously mapped a quantitative trait locus on chromosome 15 in mice contributing to high-density lipoprotein cholesterol and triglyceride levels and now report the identification of the underlying gene.

Methods and Results— We first fine-mapped the locus by studying a series of congenic strains derived from the parental strains BALB/cJ and MRL/MpJ. Analysis of gene expression and sequencing followed by transgenic complementation led to the identification of zinc fingers and homeoboxes 2 (Zhx2), a transcription factor previously implicated in the developmental regulation of -fetoprotein. Reduced expression of the protein in BALB/cJ mice resulted in altered hepatic transcript levels for several genes involved in lipoprotein metabolism. Most notably, the Zhx2 mutation resulted in a failure to suppress expression of lipoprotein lipase, a gene normally silenced in the adult liver, and this was normalized in BALB/cJ mice carrying the Zhx2 transgene.

Conclusions— We identified the gene underlying the chromosome 15 quantitative trait locus, and our results show that Zhx2 functions as a novel developmental regulator of key genes influencing lipoprotein metabolism.

  Z Kassiri , J Zhong , D Guo , R Basu , X Wang , P. P Liu , J. W Scholey , J. M Penninger and G. Y. Oudit
 

Background— Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that metabolizes Ang II into Ang 1-7, thereby functioning as a negative regulator of the renin-angiotensin system. We hypothesized that ACE2 deficiency may compromise the cardiac response to myocardial infarction (MI).

Methods and Results— In response to MI (induced by left anterior descending artery ligation), there was a persistent increase in ACE2 protein in the infarct zone in wild-type mice, whereas loss of ACE2 enhanced the susceptibility to MI, with increased mortality, infarct expansion, and adverse ventricular remodeling characterized by ventricular dilation and systolic dysfunction. In ACE2-deficient hearts, elevated myocardial levels of Ang II and decreased levels of Ang 1-7 in the infarct-related zone was associated with increased production of reactive oxygen species. ACE2 deficiency leads to increased matrix metalloproteinase (MMP) 2 and MMP9 levels with MMP2 activation in the infarct and peri-infarct regions, as well as increased gelatinase activity leading to a disrupted extracellular matrix structure after MI. Loss of ACE2 also leads to increased neutrophilic infiltration in the infarct and peri-infarct regions, resulting in upregulation of inflammatory cytokines, interferon-, interleukin-6, and the chemokine, monocyte chemoattractant protein-1, as well as increased phosphorylation of ERK1/2 and JNK1/2 signaling pathways. Treatment of Ace2/y-MI mice with irbesartan, an AT1 receptor blocker, reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, infarct size, MMP activation, and myocardial inflammation, ultimately resulting in improved post-MI ventricular function.

Conclusions— We conclude that loss of ACE2 facilitates adverse post-MI ventricular remodeling by potentiation of Ang II effects by means of the AT1 receptors, and supplementing ACE2 can be a potential therapy for ischemic heart disease.

  D. S Lee , N Ghosh , J. S Floras , G. E Newton , P. C Austin , X Wang , P. P Liu , T. A Stukel and J. V. Tu
 

Background— Higher blood pressure in acute heart failure has been associated with improved survival; however, the relationship between blood pressure and survival in stabilized patients at hospital discharge has not been established.

Methods and Results— In 7448 patients with heart failure (75.2±11.5 years; 49.9% men) discharged from the hospital in Ontario, Canada, we examined the association of systolic blood pressure (SBP) and diastolic blood pressure with long-term survival. Parametric survival analysis was performed, and survival time ratios were determined according to discharge blood pressure group. A total of 25 427 person-years of follow-up were examined. In those with left ventricular ejection fraction ≤40%, median survival was decreased by 17% (survival time ratio, 0.83; 95% CI, 0.71 to 0.98; P=0.029) when discharge SBP was 100 to 119 mm Hg and decreased by 23% (survival time ratio, 0.77; 95% CI, 0.62 to 0.97; P=0.024) when discharge SBP was <100 mm Hg, compared with those in the reference range of 120 to 139 mm Hg. Survival time ratios were 0.75 (95% CI, 0.60 to 0.92; P=0.007) and 0.75 (95% CI, 0.53 to 1.07; P=0.12) when discharge SBPs were 140 to 159 and ≥160 mm Hg, respectively. In those with left ventricular ejection fraction >40%, survival time ratios were 0.69 (95% CI, 0.51 to 0.93), 0.83 (95% CI, 0.71 to 0.99), 0.95 (95% CI, 0.80 to 1.14), and 0.76 (95% CI, 0.61 to 0.95) for discharge SBPs <100, 100 to 119, 140 to 159, and ≥160 mm Hg, respectively.

Conclusions— In this long-term population-based study of patients with heart failure, the association of discharge SBP with mortality followed a U-shaped distribution. Survival was shortened in those with reduced or increased values of discharge SBP.

  P. A Kavsak , X Wang , D. T Ko , A. R MacRae and A. S. Jaffe
 

Background: The next-generation, high-sensitivity cardiac troponin assays can measure quantifiable concentrations of cTn in a majority of individuals, but there are few studies assessing these assays for risk stratification. The present study was undertaken to determine if a research hs-cTnI assay can be useful for predicting death/myocardial infarction (MI), both short- and long-term, in an emergency department acute coronary syndrome (ACS) population.

Methods: In a cohort of 383 subjects, originally recruited in 1996, presenting to the emergency department with symptoms suggestive of ACS, the heparin plasma obtained at initial presentation was thawed and measured in 2007 with a research hs-cTnI assay. AccuTnI (Beckman Coulter) measurements were made on these same samples in 2003. The population was divided into 4 groups by hs-cTnI: <5.00, 5.00–9.99, 10.00–40.00, and >40.00 ng/L. Kaplan–Meier, Cox proportional hazards, ROC curves, and logistic regression analyses were used to identify which hs-cTnI concentrations were predictive of death/MI within 10 years after presentation.

Results: There were significant differences between the hs-cTnI groups for the probability of death/MI up to 10 years after presentation (P < 0.05). At 6 months, patients with hs-cTnI ≥10.00 ng/L were at higher risk for death/MI (hazard ratio >3.7; P < 0.05) compared with those having hs-cTnI <5.00 ng/L. ROC curve analysis for death/MI at 30 days with the hs-cTnI assay had an area under the curve of 0.74 (95% CI 0.65–0.82), with logistic models yielding an optimal assay threshold of 12.68 ng/L.

Conclusions: This research hs-cTnI assay appears useful for risk stratification for death/MI in an ACS population.

  J Jiang , N. L. S Tang , C Ohlsson , A. L Eriksson , L Vandenput , C Liao , X Wang , F. W. K Chan , A Kwok , E Orwoll , T. C. Y Kwok , J Woo and P. C. Leung
  BACKGROUND:

Results of recent studies have demonstrated that genetic variants of the enzyme steroid 5 reductase type II (SRD5A2) are associated with serum concentrations of major androgen metabolites such as conjugates of androstane-3,17β-diol-glucuronide (3-diol-G). However, this association was not consistently found among different ethnic groups. Thus, we aimed to determine whether the association with SRD5A2 genetic variations exists in a cohort of healthy Chinese elderly men, by examining 2 metabolite conjugates: androstane-3,l7β-diol-3-glucuronide (3-diol-3G) and androstane-3,17β-diol-17-glucuronide (3-diol-17G).

METHODS:

We used GC-MS and LC-MS to measure serum sex steroid concentrations, including testosterone and dihydrotestosterone, and 3-diol-3G and 3-diol-17G in 1182 Chinese elderly men age 65 and older. Genotyping of the 3 SRD5A2 tagSNPs [rs3731586, rs12470143, and rs523349 (V89L)] was performed by using melting-temperature–shift allele-specific PCR.

RESULTS:

The well-described SRD5A2 missense variant rs523349 (V89L) was modestly associated with the 3-diol-17G concentration (P = 0.040). On the other hand, SNP rs12470143 was found to be significantly correlated with 3-diol-3G concentration (P = 0.021). Results of haplotype analysis suggested that the presence of an A-C-G haplotype leads to an increased 3-diol-3G concentration, a finding consistent with results of single SNP analysis.

CONCLUSIONS:

The genetic variation of SRD5A2 is associated with circulating 3-diol-3G and 3-diol-17G concentrations in Chinese elderly men. In addition, we showed that SRD5A2 haplotypic association, rather than a single SNP alone, might be a better predictor of the 3-diol-G concentration. Thus, the effect of either the haplotype itself or of other ungenotyped SNPs in linkage disequilibrium with the haplotype is responsible for the interindividual variation of 3-diol-G.

  J. M Young , N Terrin , X Wang , T Greene , G. J Beck , J. W Kusek , A. J Collins , M. J Sarnak and V. Menon
 

Background and objectives: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, reduces bioavailability of nitric oxide and induces endothelial dysfunction. This dimethylated amino acid accumulates in chronic kidney disease and may be involved in the pathophysiology of cardiovascular disease (CVD) in this population.

Design, settings, participants, & methods: The Modification of Diet in Renal Disease Study was a randomized, controlled trial conducted between 1989 and 1993. We measured ADMA in frozen samples collected at baseline (n = 820) and obtained survival status, up to December 31, 2000, from the National Death Index. We examined the relationship of ADMA with prevalent CVD and performed multivariable Cox models to examine the relationship of ADMA with all-cause and CVD mortality.

Results: Mean (SD) age was 52 (12) yr, GFR was 32 ± 12 ml/min per 1.73 m2, and ADMA was 0.70 ± 0.25 µmol/L. A 1-SD increase in ADMA was associated with a 31% increased odds of prevalent CVD in an adjusted logistic regression model. During the 10-yr follow-up period, 202 (25%) participants died of any cause, 122 (15%) from CVD, and 545 (66%) reached kidney failure. In multivariable Cox models, a 1-SD increase in ADMA was associated with a 9% increased risk for all-cause and 19% increased risk for CVD mortality.

Conclusions: In this cohort of patients with predominantly nondiabetic, stages 3 to 4 chronic kidney disease, there was a strong association of ADMA with prevalent CVD and a modest association with all-cause and CVD mortality.

  Z Xing , C Lu , D Hu , Y. y Yu , X Wang , C Colnot , M Nakamura , Y Wu , T Miclau and R. S. Marcucio
  Zhiqing Xing, Chuanyong Lu, Diane Hu, Yan-yiu Yu, Xiaodong Wang, Celine Colnot, Mary Nakamura, Yalei Wu, Theodore Miclau, and Ralph S. Marcucio

Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory cells to sites of injury occurs in response to specific signaling pathways. The CC chemokine receptor type 2 (CCR2) is crucial for recruiting macrophages, as well as regulating osteoclast function. In this study, we examined fracture healing in Ccr2–/– mice. We first demonstrated that the expression of Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2–/– mice compared with wild-type controls at 3 days after injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after injury in mutant mice. At day 14, Ccr2–/– mice had less bone in their calluses. At day 21, Ccr2–/– mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2–/– osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2–/– mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.

  C Xu , X Wang and J. L. Staudinger
 

The liver- and intestine-enriched carboxylesterase 2 (CES2) enzyme catalyzes the hydrolysis of several clinically important anticancer agents administered as prodrugs. For example, irinotecan, a carbamate prodrug used in the treatment of colorectal cancer, is biotransformed in vivo by CES2 in intestine and liver, thereby producing a potent topoisomerase I inhibitor. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of ligand-activated transcription factors, mediate gene activation in response to xenobiotic stress. Together, these receptors comprise a protective response in mammals that coordinately regulate hepatic transport, metabolism, and elimination of numerous xenobiotic compounds. In the present study, microarray analysis was used to identify PXR target genes in duodenum in mice. Here, we show that a gene encoding a member of the CES2 subtype of liver- and intestine-enriched CES enzymes, called Ces6, is induced after treatment with pregnenolone 16-carbonitrile in a PXR-dependent manner in duodenum and liver in mice. Treatment of mice with the CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene also induced expression of Ces6 in duodenum and liver in a CAR-dependent manner, whereas treatment with phenobarbital produced induction of Ces6 exclusively in liver. These data identify a key role for PXR and CAR in regulating the drug-inducible expression and activity of an important CES enzyme in vivo. Future studies should focus on determining whether these signaling pathways governing drug-inducible CES expression in intestine and liver are conserved in humans.

  C Xu , X Wang and J. L. Staudinger
 

The liver- and intestine-enriched carboxylesterase 2 (CES2) enzyme catalyzes the hydrolysis of several clinically important anticancer agents administered as prodrugs. For example, irinotecan, a carbamate prodrug used in the treatment of colorectal cancer, is biotransformed in vivo by CES2 in intestine and liver, thereby producing a potent topoisomerase I inhibitor. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of ligand-activated transcription factors, mediate gene activation in response to xenobiotic stress. Together, these receptors comprise a protective response in mammals that coordinately regulate hepatic transport, metabolism, and elimination of numerous xenobiotic compounds. In the present study, microarray analysis was used to identify PXR target genes in duodenum in mice. Here, we show that a gene encoding a member of the CES2 subtype of liver- and intestine-enriched CES enzymes, called Ces6, is induced after treatment with pregnenolone 16-carbonitrile in a PXR-dependent manner in duodenum and liver in mice. Treatment of mice with the CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene also induced expression of Ces6 in duodenum and liver in a CAR-dependent manner, whereas treatment with phenobarbital produced induction of Ces6 exclusively in liver. These data identify a key role for PXR and CAR in regulating the drug-inducible expression and activity of an important CES enzyme in vivo. Future studies should focus on determining whether these signaling pathways governing drug-inducible CES expression in intestine and liver are conserved in humans.

  X Wang , Y. P Hsueh , W Li , A Floyd , R Skalsky and J. Heitman
 

Cosuppression is a silencing phenomenon triggered by the introduction of homologous DNA sequences into the genomes of organisms as diverse as plants, fungi, flies, and nematodes. Here we report sex-induced silencing (SIS), which is triggered by tandem integration of a transgene array in the human fungal pathogen Cryptococcus neoformans. A SXI2a-URA5 transgene array was found to be post-transcriptionally silenced during sexual reproduction. More than half of the progeny that inherited the SXI2a-URA5 transgene became uracil-auxotrophic due to silencing of the URA5 gene. In vegetative mitotic growth, silencing of this transgene array occurred at an ~250-fold lower frequency, indicating that silencing is induced during the sexual cycle. Central components of the RNAi pathway—including genes encoding Argonaute, Dicer, and an RNA-dependent RNA polymerase—are all required for both meiotic and mitotic transgene silencing. URA5-derived ~22-nucleotide (nt) small RNAs accumulated in the silenced isolates, suggesting that SIS is mediated by RNAi via sequence-specific small RNAs. Through deep sequencing of the small RNA population in C. neoformans, we also identified abundant small RNAs mapping to repetitive transposable elements, and these small RNAs were absent in rdp1 mutant strains. Furthermore, a group of retrotransposons was highly expressed during mating of rdp1 mutant strains, and an increased transposition/mutation rate was detected in their progeny, indicating that the RNAi pathway squelches transposon activity during the sexual cycle. Interestingly, Ago1, Dcr1, Dcr2, and Rdp1 are translationally induced in mating cells, and Ago1, Dcr1, and Dcr2 localize to processing bodies (P bodies), whereas Rdp1 appears to be nuclear, providing mechanistic insights into the elevated silencing efficiency during sexual reproduction. We hypothesize that the SIS RNAi pathway operates to defend the genome during sexual development.

  X Wang , H Tang , J. E Bowers and A. H. Paterson
 

Whole-genome duplication produces massive duplicated blocks in plant genomes. Sharing appreciable sequence similarity, duplicated blocks may have been affected by illegitimate recombination. However, large-scale evaluation of illegitimate recombination in plant genomes has not been possible previously. Here, based on comparative and phylogenetic analysis of the sequenced genomes of rice and sorghum, we report evidence of extensive and long-lasting recombination between duplicated blocks. We estimated that at least 5.5% and 4.1% of rice and sorghum duplicated genes have been affected by nonreciprocal recombination (gene conversion) over nearly their full length after rice–sorghum divergence, while even more (8.7% and 8.1%, respectively) have been converted over portions of their length. We found that conversion occurs in higher frequency toward the terminal regions of chromosomes, and expression patterns of converted genes are more positively correlated than nonconverted ones. Though converted paralogs are more similar to one another than nonconverted ones, elevated nucleotide differences between rice–sorghum orthologs indicates that they have evolved at a faster rate, implying that recombination acts as an accelerating, rather than a conservative, element. The converted genes show no change in selection pressure. We also found no evidence that conversion contributed to guanine-cytosine (GC) content elevation.

  S Li , T Hu , Y Chen , X Wang , T Liu , G Ma and Z. Su
 

Carboxylmethylated konjac glucomannan (CKGM) is a carboxylmethylated polymer of mannose and glucose that is derived from the plant Amorphophallus konjac cultivated in East Asia. The CKGM solution had a high volume-expanding efficacy and was evaluated as a plasma substitute in the present study. Ameliorative hemorrhagic shock rabbits were used as the model animals. The in vivo hemodynamic and hemorheologic properties, including blood pressure, blood viscosity, hematocrit, erythrocyte deformation index and erythrocyte aggregation index, were measured in animals treated in the CKGM solution. The in vitro colloid osmotic pressure (COP) of the CKGM solution was measured to estimate its plasma-expanding efficacy. These parameters of the CKGM-treated group were compared with groups exposed to four other treatments: human serum albumin (HSA), hydroxyethyl starch (HES), polygeline and normal saline. The CKGM solution showed an exceptionally higher COP than other therapy solutions. For example, the COP of 1% (weight in volume [w/v]) CKGM solution is comparable to those of 6% (w/v) HES solution and 5% (w/v) HSA solution. Accordingly, the CKGM solution can be transfused in a much lower dosage while maintaining its plasma-expanding efficacy. The CKGM-treated group showed an improved intravascular persistence and good hemodynamic and hemorheological properties. Biopsy analysis suggested no organ dysfunction in the group treated in CKGM solution. Moreover, the high plasma-expanding efficacy and inexpensive availability of the CKGM solution may facilitate its clinical application as a potential plasma substitute.

  R. L Milne , J Benitez , H Nevanlinna , T Heikkinen , K Aittomaki , C Blomqvist , J. I Arias , M. P Zamora , B Burwinkel , C. R Bartram , A Meindl , R. K Schmutzler , A Cox , I Brock , G Elliott , M. W. R Reed , M. C Southey , L Smith , A. B Spurdle , J. L Hopper , F. J Couch , J. E Olson , X Wang , Z Fredericksen , P Schurmann , M Bremer , P Hillemanns , T Dork , P Devilee , C. J van Asperen , R. A. E. M Tollenaar , C Seynaeve , P Hall , K Czene , J Liu , Y Li , S Ahmed , A. M Dunning , M Maranian , P. D. P Pharoah , G Chenevix Trench , J Beesley , kConFab Investigators , N. N Antonenkova , I. V Zalutsky , H Anton Culver , A Ziogas , H Brauch , C Justenhoven , Y. D Ko , S Haas , P. A Fasching , R Strick , A. B Ekici , M. W Beckmann , G. G Giles , G Severi , L Baglietto , D. R English , O Fletcher , N Johnson , I dos Santos Silva , J Peto , C Turnbull , S Hines , A Renwick , N Rahman , B. G Nordestgaard , S. E Bojesen , H Flyger , D Kang , K. Y Yoo , D. Y Noh , A Mannermaa , V Kataja , V. M Kosma , M Garcia Closas , S Chanock , J Lissowska , L. A Brinton , J Chang Claude , S Wang Gohrke , C. Y Shen , H. C Wang , J. C Yu , S. T Chen , M Bermisheva , T Nikolaeva , E Khusnutdinova , M. K Humphreys , J Morrison , R Platte , D. F Easton and on behalf of the Breast Cancer Association Consortium
  Background

A recent genome-wide association study identified single-nucleotide polymorphism (SNP) 2q35-rs13387042 as a marker of susceptibility to estrogen receptor (ER)–positive breast cancer. We attempted to confirm this association using the Breast Cancer Association Consortium.

Methods

2q35-rs13387042 SNP was genotyped for 31 510 women with invasive breast cancer, 1101 women with ductal carcinoma in situ, and 35 969 female control subjects from 25 studies. Odds ratios (ORs) were estimated by logistic regression, adjusted for study. Heterogeneity in odds ratios by each of age, ethnicity, and study was assessed by fitting interaction terms. Heterogeneity by each of invasiveness, family history, bilaterality, and hormone receptor status was assessed by subclassifying case patients and applying polytomous logistic regression. All statistical tests were two-sided.

Results

We found strong evidence of association between rs13387042 and breast cancer in white women of European origin (per-allele OR = 1.12, 95% confidence interval [CI] = 1.09 to 1.15; Ptrend = 1.0 x 10–19). The odds ratio was lower than that previously reported (P = .02) and did not vary by age or ethnicity (all P ≥ .2). However, it was higher when the analysis was restricted to case patients who were selected for a strong family history (P = .02). An association was observed for both ER-positive (OR = 1.14, 95% CI = 1.10 to 1.17; P = 10–15) and ER-negative disease (OR = 1.10, 95% CI = 1.04 to 1.15; P = .0003) and both progesterone receptor (PR)–positive (OR = 1.15, 95% CI = 1.11 to 1.19; P = 5 x 10–14) and PR-negative disease (OR = 1.10, 95% CI = 1.06 to 1.15; P = .00002).

Conclusion

The rs13387042 is associated with both ER-positive and ER-negative breast cancer in European women.

  K Wang , D Tang , M Wang , J Lu , H Yu , J Liu , B Qian , Z Gong , X Wang , J Chen , M Gu and Z. Cheng
  Kejian Wang, Ding Tang, Mo Wang, Jufei Lu, Hengxiu Yu, Jiafan Liu, Baoxiang Qian, Zhiyun Gong, Xin Wang, Jianmin Chen, Minghong Gu, and Zhukuan Cheng

MER3, a ZMM protein, is required for the formation of crossovers in Saccharomyces cerevisiae and Arabidopsis. Here, MER3, the first identified ZMM gene in a monocot, is characterized by map-based cloning in rice (Oryza sativa). The null mutation of MER3 results in complete sterility without any vegetative defects. Cytological analyses show that chiasma frequency is reduced dramatically in mer3 mutants and the remaining chiasmata distribute randomly among different pollen mother cells, implying possible coexistence of two kinds of crossover in rice. Immunocytological analyses reveal that MER3 only exists as foci in prophase I meiocytes. In addition, MER3 does not colocalize with PAIR2 at the beginning of prophase I, but locates on one end of PAIR2 fragments at later stages, whereas MER3 foci merely locate on one end of REC8 fragments when signals start to be seen in early prophase I. The normal loading of PAIR2 and REC8 in mer3...

  Y Gu , X Liang , W Wu , M Liu , S Song , L Cheng , L Bo , C Xiong , X Wang , X Liu , L Peng and K. Yao
 

Context: Hormonal male contraceptive regimens effectively and reversibly suppress sperm production, but there are few large-scale efficacy studies.

Objective: The safety, contraceptive efficacy, reversibility, and feasibility of injectable testosterone undecanoate (TU) in tea seed oil as a hormonal male contraceptive was assessed.

Design: This was a multicenter, phase III, contraceptive efficacy clinical trial.

Participants: A total of 1045 healthy fertile Chinese men were recruited throughout China into the study.

Intervention(s): Injections of 500 mg TU were administered monthly for 30 months. A definition of severe oligozoospermia (≤1 x 106/ml) was used as a criterion of spermatogenic suppression and as the threshold for entering the contraceptive efficacy phase.

Main Outcome Measure(s): The primary outcome was pregnancy rate in the partner. Other outcomes include: semen parameters, testis volumes, reproductive hormone levels, and safety laboratory tests.

Results: Forty-three participants (4.8%) did not achieve azoospermia or severe oligozoospermia within the 6-month suppression phase. A total of 855 participants entered into the efficacy phase, and 733 participants completed monthly TU treatment and follow-up. There were nine pregnancies in 1554.1 person-years of exposure in the 24-month efficacy phase for a cumulative contraceptive failure rate of 1.1 per 100 men. The combined method failure rate was 6.1%, comprising 4.8% with inadequate suppression and 1.3% with postsuppression sperm rebound. No serious adverse events were reported. Spermatogenesis returned to the normal fertile reference range in all but two participants.

Conclusions: Monthly injection of 500 mg TU provides safe, effective, reversible, and reliable contraception in a high proportion of healthy fertile Chinese men.

  X Wang , Q Li , X Niu , H Chen , L Xu and C. Qi
 

Sspg1d, one of endopolygalacturonases, is an important fungal effector secreted by the necrotrophic fungus Sclerotinia sclerotiorum during early infection. Using sspg1d as bait, a small C2 domain protein (designated as IPG-1) was identified by yeast two-hybrid screening of a canola cDNA library. Deletion analysis confirmed that the C-terminus of IPG-1 is responsible for its interaction with sspg1d in the yeast two-hybrid assay. The sspg1d/IPG-1 interaction was further confirmed in plant cells by a biomolecular fluorescence complementation (BiFC) assay. A transient expression assay showed that the IPG-1–GFP fusion protein was targeted to the plasma membrane and nucleus in onion epidermal cells. Following treatment with a Ca2+ ionophore, it was distributed throughout the cytosol. Real-time PCR assay demonstrated that IPG-1 was highly induced by Sclerotinia sclerotiorum in canola leaves and stems. Southern blot analysis indicated the presence of about five homologues of IPG-1 in the canola genome. Two additional members of the IPG-1gene family were isolated by RT-PCR. Their sequence similarity with IPG-1 is as high as 95%. However, they did not interact with sspg1d in the yeast two-hybrid assay. Possible roles of IPG-1 and its association with sspg1d in the defence signalling pathway were discussed.

  X Wang , W Song , Z Yang , Y Wang , Z Tang and C. Xu
 

The endosperm in plants is a major source of human nutrition and industrial raw material. The genetic study of endosperm poses a great challenge due to its complex genetic composition and unique physical and developmental properties. In this note, we shall revisit 2 classic mating designs—North Carolina Design III (NCIII) and triple test cross (TTC)—and demonstrate their efficiency in detecting quantitative trait loci underlying endosperm traits.

  X Wang , J Hao , Y Xie , Y Sun , B Hernandez , A. K Yamoah , M Prasad , Q Zhu , J. Q Feng and C. Qin
 

Mutations in FAM20C were recently identified as the cause of lethal osteosclerotic bone dysplasia, which highlighted the important role of this molecule in biomineralization. No systematic studies have been performed to evaluate the expression pattern of this relatively new molecule in the developmental processes of bone and tooth. In the present study, we analyzed in detail the expression profile of FAM20C during osteogenesis and odontogenesis using ISH and IHC approaches. The specimens analyzed were mouse tissues spanning embryonic day 13.5 (E13.5) to postnatal 8 weeks. The earliest presence of FAM20C was observed at E14.5. During osteogenesis, FAM20C mRNA was detected in the chondrocytes and osteoblasts of the long bone, whereas its protein was observed in the extracellular matrix (ECM) of bone and in the cytoplasm of the chondrocytes, osteoblasts, and osteocytes. During odontogenesis, FAM20C mRNA was detected in the ameloblasts, odontoblasts, cementoblasts, and periodontal ligament fibroblasts, whereas its protein was observed in the matrices of dentin, enamel, and alveolar bone and in the cytoplasm of the aforementioned cells. The temporospatial expression profile revealed in this study indicates that FAM20C is an ECM protein that may play an important role in controlling the mineralization of bone and tooth. (J Histochem Cytochem 58:957–967, 2010)

  X Wang , B Qin , G Gao and H. W. Paerl
 

An experiment was conducted with a natural freshwater phytoplankton community from a eutrophic pond to investigate the combined effects of phytoplankton community competitive interactions, nutrient enrichment and zooplankton on Microcystis bloom formation. The pond water initially had a very low concentration of Microcystis, but the total phytoplankton biomass as chlorophyll a reached ~110 µg L–1 in summer. The pond water was incubated outdoors at natural temperature and light with a 2 x 2 factorial manipulation of nutrient (nutrient additions versus no additions) with and without zooplankton. The interaction of a two-phase nutrient addition (a net increase in concentrations of 250.0 µM N and 16.1 µM P each time) and the presence of zooplankton significantly altered phytoplankton community composition. When the water initially contained zooplankton, nitrogen and phosphorus enrichment promoted a surface Microcystis bloom. However, when the zooplankton was removed from the water at the start of the experiment, no surface Microcystis bloom formed, regardless of nutrient additions. Chlorophyta dominated in the absence of zooplankton, when the same nutrient was provided. Our results demonstrate that Microcystis bloom formation in this eutrophic water body at a mean temperature of about 36°C at 14:00 h was closely related to the initial presence of zooplankton and a sufficient supply of nitrogen and phosphorus. We believe this is one of the first demonstrations of zooplankton controlling Microcystis bloom formation in a water body previously free of surface cyanobacterial blooms.

  E Liu , S Cheng , X Wang , D Hu , T Zhang and C. Chu
  Background

Contact investigation is a logical approach to intensified case finding in China. However, currently there are no written national guidelines. The aim of this study is to review the published literature that describes the procedures followed by local level and report the yield for active tuberculosis (TB) cases.

Methods

Studies conducted in China and published between 1997 and 2007 on contact investigation were searched.

Results

Twelve studies were included in the review. There was no standard definition of contact and no study provided details on how to prioritize contacts. Investigation methods vary between each study. The number of contacts investigated per index case ranged from 22.7 to 658 in congregate settings and from 1.5 to 5.8 in household. The yields for active TB ranged from 0 to 11.765% in congregate settings and from 0 to 6.897% in household. The weighted yields for smear-positive index and smear-negative index were 1 and 0.2% respectively in household and 0.5% for pulmonary case index in congregate settings.

Conclusion

There is considerable heterogeneity amongst the methods used and the cases yielded in these studies, and in general the quality of contact investigation is low; therefore, there is a need for China to develop national guidelines on contact investigation.

  Y Li , S Huang , X Wang , D Zhou , K Huang , H Guo , J Fang , C Chen and Q. Liu
 

We report on 2 children with Burkitt's lymphoma accompanied by extensive extranodal involvement treated with chemotherapy and Rituximab in combination with autologous peripheral blood stem cell transplantation (Auto-PBSCT) regimens. No obvious side effects could be seen during the Rituximab therapy. Both children achieved complete remission with no relapse after being followed up for 4.3 and 4 years, respectively. Our limited experience show that Rituximab in combination with chemotherapy and Auto-PBSCT might have better therapeutic effects on Burkitt's lymphoma of children and the side effects of Rituximab therapy is minimal and can be well tolerated.

  Y Gan , Y Zhang , D. J DiGirolamo , J Jiang , X Wang , X Cao , K. R Zinn , D. P Carbone , T. L Clemens and S. J. Frank
 

GH promotes longitudinal growth and regulates multiple cellular functions in humans and animals. GH signals by binding to GH receptor (GHR) to activate the tyrosine kinase, Janus kinase 2 (JAK2), and downstream pathways including signal transducer and activator of transcription 5 (STAT5), thereby regulating expression of genes including IGF-I. GH exerts effects both directly and via IGF-I, which signals by activating the IGF-I receptor (IGF-IR). IGF-IR is a cell surface receptor that contains intrinsic tyrosine kinase activity within its intracellular domain. In this study, we examined the potential role of IGF-IR in facilitating GH-induced signal transduction, using mouse primary calvarial osteoblasts with Lox-P sites flanking both IGF-IR alleles. These cells respond to both GH and IGF-I and in vitro infection with an adenovirus that drives expression of Cre recombinase (Ad-Cre) dramatically reduces IGF-IR abundance without affecting the abundance of GHR, JAK2, STAT5, or ERK. Notably, infection with Ad-Cre, but not a control adenovirus, markedly inhibited acute GH-induced STAT5 activity (more than doubling the ED50 and reducing the maximum activity by nearly 50%), while sparing GH-induced ERK activity, and markedly inhibited GH-induced transactivation of a STAT5-dependent luciferase reporter. The effect of Ad-Cre on GH signaling was specific, as platelet-derived growth factor-induced signaling was unaffected by Ad-Cre-mediated reduction of IGF-IR. Ad-Cre-mediated inhibition of GH signaling was reversed by adenoviral reexpression of IGF-IR, but not by infection with an adenovirus that drives expression of a hemagglutination-tagged somatostatin receptor, which drives expression of the unrelated somatostatin receptor, and Ad-Cre infection of nonfloxed osteoblasts did not affect GH signaling. Notably, infection with an adenovirus encoding a C-terminally truncated IGF-IR that lacks the tyrosine kinase domain partially rescued both acute GH-induced STAT5 activity and GH-induced IGF-I gene expression in cells in which endogenous IGF-IR was reduced. These data, in concert with our earlier findings that GH induces a GHR-JAK2-IGF-IR complex, suggest a novel function for IGF-IR. In addition to its role as a key IGF-I signal transducer, this receptor may directly facilitate acute GH signaling. The implications of these findings are discussed.

  Y Soeda , H Tsuneki , H Muranaka , N Mori , S Hosoh , Y Ichihara , S Kagawa , X Wang , N Toyooka , Y Takamura , T Uwano , H Nishijo , T Wada and T. Sasaoka
 

Impairment of insulin and IGF-I signaling in the brain is one of the causes of dementia associated with diabetes mellitus and Alzheimer’s disease. However, the precise pathological processes are largely unknown. In the present study, we found that SH2-containing inositol 5'-phosphatase 2 (SHIP2), a negative regulator of phosphatidylinositol 3,4,5-trisphosphate-mediated signals, is widely expressed in adult mouse brain. When a dominant-negative mutant of SHIP2 was expressed in cultured neurons, insulin signaling was augmented, indicating physiological significance of endogenous SHIP2 in neurons. Interestingly, SHIP2 mRNA and protein expression levels were significantly increased in the brain of type 2 diabetic db/db mice. To investigate the impact of increased expression of SHIP2 in the brain, we further employed transgenic mice overexpressing SHIP2 and found that increased amounts of SHIP2 induced the disruption of insulin/IGF-I signaling through Akt. Neuroprotective effects of insulin and IGF-I were significantly attenuated in cultured cerebellar granule neurons from SHIP2 transgenic mice. Consistently, terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay demonstrated that the number of apoptosis-positive cells was increased in cerebral cortex of the transgenic mice at an elderly age. Furthermore, SHIP2 transgenic mice exhibited impaired memory performance in the Morris water maze, step-through passive avoidance, and novel-object-recognition tests. Importantly, inhibition of SHIP2 ameliorated the impairment of hippocampal synaptic plasticity and memory formation in db/db mice. These results suggest that SHIP2 is a potent negative regulator of insulin/IGF-I actions in the brain, and excess amounts of SHIP2 may be related, at least in part, to brain dysfunction in insulin resistance with type 2 diabetes.

  B Peng , L Cao , W Wang , L Xian , D Jiang , J Zhao , Z Zhang , X Wang and L. Yu
 

Matrix metalloproteinase (MMP) 1 and MMP3 are enzymes that degrade the extracellular matrix and have been implicated to play an important role in cancer development. Many studies have been carried out on the association between polymorphisms of MMP1 –1607 1G>2G and MMP3 –1171 5A>6A and cancer risk. However, results from these studies remain inconclusive. Here, we performed a meta-analysis of >38 000 subjects to better assess the purported associations. For MMP1, –1607 2G/2G genotype carriers were found to have an increased risk of colorectal cancer [2G/2G versus 2G/1G + 1G/1G, odds ratio (OR) = 1.48, 95% confidence interval (CI) (1.26–1.74), Pheterogeneity = 0.066, I2 = 49.3%], head and neck cancer [2G/2G versus 2G/1G + 1G/1G, OR = 1.61, 95% CI (1.26–2.07), Pheterogeneity = 0.002, I2 = 64.7%] and renal cancer [2G/2G versus 2G/1G + 1G/1G, OR = 1.82, 95% CI (1.38–2.39), Pheterogeneity = 0.589, I2 = 0.0%] risk. For MMP3, no association was found between –1171 5A>6A polymorphism and cancer risk in the overall group [6A versus 5A, OR = 1.00, 95% CI (0.95–1.05), Pheterogeneity = 0.124, I2 = 24.9%] and individual cancer subgroups, but stratified analysis by smoking status showed that this polymorphism had different effects on smokers and non-smokers under recessive genetic model. In summary, our study suggests that MMP1 –1607 2G may be associated with an increased cancer risk for certain types of cancers, MMP3 –1171 5A>6A may not be a major risk factor for cancer, but it may be modified by certain environmental factors. Future studies with larger sample sizes are warranted to further evaluate these associations in more detail.

  X Wang and Z. Sun
 

Klotho is a recently identified antiaging gene. Brain endothelin-1 (ET1) is important in the regulation of blood pressure (BP). We hypothesized that silence of brain klotho potentiates cold-induced elevation of BP via the endothelin pathway. To silence brain klotho, we constructed adeno-associated virus (AAV) carrying rat klotho small interference hairpin RNA (KL-shRNA). AAV carrying ET1-shRNA was used to silence brain ET1. Scrambled shRNA was used as Control-shRNA. Three groups of male Sprague-Dawley rats (6 rats/group) received KL-shRNA, KL-shRNA plus ET1-shRNA, and Control-shRNA, respectively, via intracerebroventricular injection. BP was monitored daily using a telemetry system. All animals were exposed to a moderate cold environment (5°C) at 12 days after gene delivery. KL-shRNA significantly increased BP by 9 days of exposure to cold, while BP in the Control-shRNA group remained unchanged. ET1-shRNA abolished KL-shRNA-induced elevation of BP during cold exposure. Interestingly, KL-shRNA increased brain ET1 expression and plasma norepinephrine level, suggesting that silencing of brain klotho increased ET1 production and the sympathetic nervous activity. The KL-shRNA-induced increase in sympathetic nervous activity was mediated by ET1 because it could be abolished by silencing of ET1. These results demonstrated that silencing of brain klotho potentiated and expedited cold-induced elevation of BP by upregulation of ET1 and the subsequent activation of the sympathetic nervous system.

  E Baljinnyam , K Iwatsubo , R Kurotani , X Wang , C Ulucan , M Iwatsubo , D Lagunoff and Y. Ishikawa
 

Melanoma, the most malignant form of human skin cancer, has a poor prognosis due to its strong metastatic ability. It was recently demonstrated that Epac, an effector molecule of cAMP, is involved in regulating cell migration; however, the role of Epac in melanoma cell migration remains unclear. We thus examined whether Epac regulates cell migration and metastasis of melanoma. Epac activation, by either specific agonist or overexpression of Epac, increased melanoma cell migration. Deletion of endogenous Epac with small interfering RNA decreased basal melanoma cell migration. These data suggested a major role of Epac in melanoma cell migration. Epac-induced cell migration was mediated by translocation of syndecan-2, a cell-surface heparan sulfate proteoglycan, to lipid rafts. This syndecan-2 translocation was regulated by tubulin polymerization via the Epac/phosphoinositol-3 kinase pathway. Epac-induced cell migration was also regulated by the production of heparan sulfate, a major extracellular matrix. Epac-induced heparan sulfate production was attributable to the increased expression of N-deacetylase/N-sulfotransferase-1 (NDST-1) accompanied by an increased NDST-1 translation rate. Finally, Epac overexpression enhanced lung colonization of melanoma cells in mice. Taken together, these data indicate that Epac regulates melanoma cell migration/metastasis mostly via syndecan-2 translocation and heparan sulfate production.

  X Wang , S. V McLennan , T. J Allen , T Tsoutsman , C Semsarian and S. M. Twigg
 

Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and -skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures. small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes.

  A. E Awad , V Kandalam , S Chakrabarti , X Wang , J. M Penninger , S. T Davidge , G. Y Oudit and Z. Kassiri
 

Tumor necrosis factor (TNF) is an inflammatory cytokine that is upregulated in a number of cardiomyopathies. Adverse cardiac remodeling and dilation result from degradation of the extracellular matrix by matrix metalloproteinases (MMPs). We investigated whether TNF can directly trigger expression and activation of MMPs in cardiac cells. We compared MMP expression profile and activities between primary cultures of mouse neonatal cardiomyocytes and cardiofibroblasts and in cellular and extracellular compartments. In response to recombinant TNF (rTNF, 20 ng/ml), cardiomyocytes exhibited faster and more pronounced superoxide production compared with cardiofibroblasts, concomitant with increased expression of several MMPs. MMP9 levels increased more rapidly and about twofold more in cardiomyocytes than in cardiofibroblasts. TNF did not induce MMP2 expression. Expression of collagenases (MMP8, MMP12, MMP13, and MMP14) increased significantly, while total collagenase activity increased to a greater degree in conditioned medium of cardiomyocytes than in cardiofibroblasts. rTNF-mediated MMP expression and activation were dependent on superoxide production and were blocked by apocynin, an NADPH oxidase inhibitor. We identified phosphatidylinositol 3-kinase (PI3K) as a key factor in TNF-mediated events since TNF-induced superoxide production, MMP expression, and activity were significantly suppressed in cardiomyocytes and cardiofibroblasts deficient in PI3K. We further demonstrated that the TNF-superoxide-MMP axis of events is in fact activated in heart disease in vivo. Wild-type and TNF–/– mice subjected to cardiac pressure overload revealed that TNF deficiency resulted in reduced superoxide levels, collagenase activities, PI3K activity, and fibrosis leading to attenuated cardiac dilation and dysfunction. Our study demonstrates that TNF triggers expression and activation of MMPs faster and stronger in cardiomyocytes than in cardiofibroblasts in a superoxide-dependent manner and via activation of PI3K, thereby contributing to adverse myocardial remodeling in disease.

  B Chen , D Guan , Z. J Cui , X Wang and X. Shen
 

To know whether thioredoxin 1 (Trx1) works for an antioxidant defense mechanism in atherosclerosis, the effect of Trx1 on the release of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for recruitment and accumulation of monocytes/macrophages in the intima of artery vessel, was investigated in human endothelial-like EA.hy 926 cells. It was found that overexpression of Trx1 suppressed, whereas knockdown of endogenous Trx1 enhanced, oxidized low-density lipoprotein (oxLDL)-stimulated MCP-1 release and expression in the cells. It was also observed that overexpression of Trx1 suppressed, whereas depletion of endogenous Trx1 greatly promoted, nuclear translocation of c-Jun and the redox factor-1 (Ref-1). Electrophoretic mobility shift assay showed significantly reduced DNA-binding activity of activator protein-1 (AP-1) in Trx1-overexpressing cells but apparently enhanced DNA binding activity of AP-1 in Trx1-knockdown cells, indicating that nuclear Ref-1 rather than Trx1 itself finally dominates the regulation of AP-1 activity, although Trx1 is considered to upregulate AP-1 activity. It was also observed that Trx1 depressed intracellular generation of reactive oxygen species (ROS). Diphenyleneiodonium (DPI), the inhibitor of NADPH oxidase, suppressed MCP-1 secretion, whereas transient expression of Nox1 enhanced transcription of MCP-1 in endothelial cells. Assays with AP-1 and MCP-1 luciferase reporters further demonstrated that transient expression of Trx1 significantly depressed the transcriptional activity of c-Jun/c-Fos and consequent MCP-1 transcription. This study suggests that Trx1 inherently suppresses MCP-1 expression in vascular endothelium and may prevent atherosclerosis by depressing MCP-1 release. Besides the suppression of intracellular ROS generation, the inhibition of nuclear translocation of AP-1 and Ref-1 are mainly responsible for the downregulation of MCP-1 by Trx1.

  A Ramalingam , X Wang , M Gabello , M. C Valenzano , A. P Soler , A Ko , P. J Morin and J. M. Mullin
 

The beneficial effects of caloric restriction in increasing longevity and forestalling age-related diseases are well known. Dietary restriction of methionine also renders similar benefits. We recently showed in a renal epithelial cell culture system that reduction of culture medium methionine by 80% resulted in altered tight junctional (TJ) claudin composition and also improved epithelial barrier function (51). In the current study, we examined the effect of dietary restriction of methionine on TJ barrier function in rat gastrointestinal tissue to see whether this phenomenon also holds true in a tissue model and for a different epithelial cell type. After 28 days on methionine-restricted (MR) diet, rats showed small but significant reductions in the plasma and (intracellular) colonocyte levels of methionine. Colon mucosal sheets from rats on the MR diet showed increased transepithelial electrical resistance with concomitant decrease in paracellular diffusion of 14C-d-mannitol, suggesting improved barrier function relative to rats on control diet. This improved barrier function could not be explained by changes in colon crypt length or frequency. Neither was the colonocyte mitotic index nor the apoptotic frequency altered significantly. However, TJ composition/structure was being altered by the MR diet. RT-PCR and Western blot analysis showed an increase in the abundance of claudin-3 and an apparent change in the posttranslational modification of occludin, data reinforcing a paracellular barrier alteration. Overall, our data suggest that reduction in dietary intake of methionine results in improved epithelial barrier function by inducing altered TJ protein composition.

  K Guo , X Wang , G Gao , C Huang , K. S Elmslie and B. Z. Peterson
 

We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149–1159, 2010). The short 17 amino acid extracellular NH2-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate CaV1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with CaV1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on CaV1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca2+ dynamics in the heart.

  K Corcoran , X Wang and L. Lybarger
 

During endoplasmic reticulum (ER)-associated degradation (ERAD), a relatively small number of ubiquitin ligases (E3) must be capable of ubiquitinating an assortment of substrates diverse in both structure and location (ER lumen, membrane, and/or cytosol). Therefore, mechanisms that operate independently of primary sequence determinants must exist to ensure specificity during this process. Here we provide direct evidence for adapter-mediated substrate recruitment for a virus-encoded ERAD E3 ligase, mK3. Members of an ER membrane protein complex that normally functions during major histocompatibility complex class I biogenesis in the immune system are required for mK3 substrate selection. We demonstrate that heterologous substrates could be ubiquitinated by mK3 if they were recruited by these ER accessory molecules to the proper position relative to the ligase domain of mK3. This mechanism of substrate recruitment by adapter proteins may explain the ability of some E3 ligases, including cellular ERAD E3 ligases, to specifically target the ubiquitination of multiple substrates that are unrelated in sequence.

  X Wang , R. A Herr , M Rabelink , R. C Hoeben , E. J.H.J Wiertz and T. H. Hansen
 

An E2–E3 complex can ubiquitinate substrates via either an isopeptide bond (to a lysine) or an ester bond (to a serine or threonine) and preferentially uses the latter to induce ERAD.

 
 
 
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