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Articles by J Li
Total Records ( 36 ) for J Li
  B Huang , W Qin , B Zhao , Y Shi , C Yao , J Li , H Xiao and Y. Jin

MicroRNAs (miRNAs), which are a newly identified class of small single-stranded non-coding RNAs, regulate their target genes via post-transcriptional pathway. It has been proved that miRNAs play important roles in many biological processes. To better understand miRNA function on type 2 diabetes, we used an oligonucleotide microarray to monitor miRNA expression profiles of Goto–Kakizaki (GK) and Wistar rats' skeletal muscle. It was found that seven miRNAs were down-expressed and two miRNAs were over-expressed in the muscle of GK rats. Among them, miR-24 showed the most prominent change. p38 MAPK, which is a direct target of miR-24, also showed expression difference. All the data give a clue that miR-24 might be associated with diabetes through down-regulation of p38 MAPK.

  Y Wu , W Xu , G Huang , S Gong , J Li , Y Qin and X. Li

Arabinogalactan proteins (AGPs) are a large family of highly glycosylated of hydroxyproline-rich glycoproteins that play important roles in plant growth, development, and signal transduction. A cDNA encoding a putative classical AGP named GhH6L was isolated from cotton fiber cDNA libraries, and the deduced protein contains 17 copies of repetitive motif of X–Y–proline–proline–proline (where X is serine or alanine and Y is threonine or serine). Northern blotting analysis and quantitative RT–PCR results showed that it was preferentially expressed in 10 days post-anthesis (dpa) fibers and was also developmentally regulated. A promoter fragment was isolated from cotton (Gossypium hirsutum) by genome walking PCR. Expression of β-glucuronidase (GUS) gene under the GhH6L promoter was examined in the transgenic Arabidopsis plants; only petiole and pedicel were stained, no staining was detected in other tissues. Subcellular localization indicated that GhH6L was localized to the plasma membrane and in the cytoplasm. These data further our understanding of GhH6L as well as shed light on functional insight to GhH6L in cotton.

  L Jiang , J Li and L. Song

Bmi-1, a polycomb gene family member, plays an important role in cell cycle regulation, cell immortalization, and cell senescence. Recently, numerous studies have demonstrated that Bmi-1 is involved in the regulation of self-renewal and differentiation of stem cells. However, the molecular mechanism underlying this biological process remains largely unclear. In the present review, we summarized the function of Bmi-1 as a transcriptional regulator of gene expression, with particular reference to stem cells.

  A Qian , S Di , X Gao , W Zhang , Z Tian , J Li , L Hu , P Yang , D Yin and P. Shang

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

  Q Wang , J Li , J Gu , B Huang , Y Zhao , D Zheng , Y Ding and L. Zeng

The green tea constituent, (–)-epigallocatechin-3-gallate (EGCG), has chemopreventive and anticancer effects. This is partially because of the selective ability of EGCG to induce apoptosis and death in cancer cells without affecting normal cells. In the present study, the activity of EGCG against the myeloma cell line, KM3, was examined. Our results demonstrated, for the first time, that the treatment of the KM3 cell line with EGCG inhibits cell proliferation and induces apoptosis, and there is a synergistic effect when EGCG and bortezomib are combined. Further experiments showed that this effect involves the NF-B pathway. EGCG inhibits the expression of the P65 mRNA and P65/pP65 protein, meanwhile it downregulates pIB expression and upregulates IB expression. EGCG also activates caspase-3, -8, cleaved caspase-9, and poly-ADP-ribose polymerase (PARP) and subsequent apoptosis. These findings provided experimental evidence for efficacy of EGCG alone or in combination with bortezomib in multiple myeloma therapy.

  L Chen , S Xu , X Zeng , J Li , W Yin , Y Chen , Z Shao and W. Jin

Chemokine C-X-C motif ligand 12 (CXCL12) is a potent chemotactic and angiogenic factor that has been proposed to play a role in organ-specific metastasis and angiogenic activity in several malignancies. In this study, we found that the overexpression of c-myb could elevate CXCL12 mRNA level and CXCL12 promoter activity in human T47D and MCF-7 breast cancer cells. Chromatin immunoprecipitation assay demonstrated that c-myb could bind to the CXCL12 promoter in the cells transfected with c-myb expression vector. c-myb siRNA attenuated CXCL12 promoter activity and the binding of c-myb to the CXCL12 promoter in T47D and MCF-7 cells. These results indicated that c-myb could activate CXCL12 promoter transcription.

  L Sun , J Li , C Xu , F Yu , H Zhou , L Tang and J. He

A device has been invented for protein crystallization by sandwiching the liquid droplet between two surfaces, in which both hydrophilic and hydrophobic surfaces can be used as crystallization substrates. Comparing with the traditional hanging drop method, it can also reduce the evaporation rate of the liquid droplet and provide a stable environment for the crystal growth. In this work, crystal growth experiments for several proteins, especially on the hydrophilic substrate of mica, have shown the positive effect on crystal growth for improving crystallization conditions and the quality of crystals. The features of this new sandwich method and its mechanism have also been discussed.

  J Li , A Dai , R Hu , L Zhu and S. Tan

Oxidative stress is one of the major pathogenesis of chronic obstructive pulmonary disease (COPD). -Glutamylcysteine synthetase (-GCS) is one of the paramount antioxidant enzymes in COPD. Peroxisome proliferator-activated receptor-gamma (PPAR) is a ligand-activated transcription factor, which is activated by specific ligands such as rosiglitazone (RGZ), exerting multiple biological effects. PPAR coactivator-1 (PGC-1) is a PPAR coactivator, which binds to PPAR by induction of PPAR ligands, co-activating PPAR target genes. Growing evidence has suggested that PPAR/PGC-1 can regulate multiple antioxidant genes. However, the effect of PPAR/PGC-1 on -GCS during the development of COPD remains unclear. Here, we measured the expression levels of PPAR, PGC-1 and -GCS, -GCS activity and reactive oxygen species (ROS) contents in lungs of rats treated by cigarette smoke (CS) + lipopolysaccharide (LPS) and CS + LPS + RGZ, as well as lungs of patients suffered from COPD. Compared with lungs from CS + LPS-treated rats, lungs of RGZ-treated rats demonstrated markedly lower ROS contents, and remarkable increase of -GCS activity and increase of the expression levels of PPAR, PGC-1, and -GCS. Furthermore, compared with controls, expression levels of PPAR, PGC-1, and -GCS significantly increased in the lungs of mild COPD patients, and progressively decreased in lungs of patients with moderate and severe COPD. -GCS protein was positively correlated with FEV1%. PPAR and PGC-1 proteins were positively correlated with -GCS activity and mRNA level. In conclusion, -GCS showed compensatory upregulation in the early stage of COPD, which progressively decompensate with increasing COPD severity. The activation of the PPAR/PGC-1 pathway may protect against COPD progression by upregulating -GCS and relieving oxidative stress.

  C Bian , F Zhang , F Wang , Z Ling , M Luo , H Wu , Y Sun , J Li , B Li , J Zhu , L Tang , Y Zhou , Q Shi , Y Ji , L Tian , G Lin , Y Fan , N Wang and B. Sun

DNA immunization is an efficient method for high-affinity monoclonal antibody generation. Here, we describe the generation of several high-quality monoclonal antibodies (mAbs) against retinol-binding protein 4 (RBP4), an important marker for kidney abnormality and dysfunction, with a combination method of DNA priming and protein boost. The mAbs generated could bind to RBP4 with high sensitivity and using these mAbs, an immunocolloidal gold fast test strip was constructed. The strip can give a result in <5 min and is very sensitive with a detection limit of about 1 ng/ml. A small-scale clinical test revealed that the result of this strip was well in accordance with that of an enzyme-labeled immunosorbent assay kit currently available on the market. Consequently, it could be useful for more convenient and faster RBP4 determination in the clinic.

  Q Li , J Li and J. Ren

Diabetic heart disease contributes to the high mortality in diabetics, although effective clinical management is lacking. The protease inhibitor 5-[5-(2-nitrophenyl) furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid (UCF-101) was reported to protect the hearts against ischemic injury. This study examined the role of UCF-101 on streptozotocin (STZ)-induced diabetic heart defect. Vehicle or UCF-101 was administrated to STZ diabetic mice, and cardiomyocyte mechanical properties were analyzed. UCF-101 reduced STZ-induced hyperglycemia and alleviated STZ-induced aberration in cardiomyocyte contractile mechanics. Diabetes dramatically decreased AMPK phosphorylation at Thr172 of catalytic -subunit, which was restored by UCF-101. Neither diabetes nor UCF-101 affected the expression of HtrA2/Omi and XIAP or caspase-3 activity. The AMPK activator resveratrol mimicked the UCF-101-induced beneficial effect against diabetic cardiac dysfunction. Mechanical properties in cardiomyocytes from the AMPK-kinase-dead (KD) mice displayed markedly impaired contractile function reminiscent of diabetes. STZ injection in AMPK-KD mice failed to elicit any additional cardiomyocyte contractile defect. UCF-101 significantly downregulated the AMPK-degrading enzymes PP2A and PP2C, the effect of which was mimicked by resveratrol. Taken together, these results indicate that UCF-101 protects against STZ-induced cardiac dysfunction, possibly through AMPK signaling.

  M Wang , J. J Wang , J Li , K Park , X Qian , J. x Ma and S. X. Zhang

We previously reported that circulating levels of pigment epithelium-derived factor (PEDF), a newly identified adipokine, are increased in patients with type 2 diabetes, correlating with body mass index. However, the role of PEDF in adipogenesis remains elusive. In the present study, we have investigated the effects and mechanisms of PEDF on adipocyte differentiation in 3T3-L1 preadipocytes. Differentiation of 3T3-L1 preadipocytes was induced in the presence or absence of human recombinant PEDF protein. The effects of PEDF on adipogenic gene expression, mitotic clonal expansion (MCE), and MAPK activation were investigated. Physiological concentrations of human PEDF protein inhibited adipocyte differentiation, evidenced by decreased lipid accumulation, downregulation of adipocyte markers, and inhibition of master adipogenic transcription factors such as C/EBP- and PPAR. The antiadipogenic effects of PEDF were observed only when PEDF was added to the cells on day 0, but not on day 3 during differentiation, suggesting that PEDF targets some early adipogenic events. Similarly, overexpression of PEDF by adenovirus attenuated adipocyte differentiation. Further studies revealed that PEDF, or U-0126, a specific MAPK/ERK inhibitor, sequentially inhibited the early activation of ERK and MCE. Moreover, PEDF attenuated expression and the phosphorylation of C/EBP-β at Thr188, an essential step for transcriptional activation of C/EBP-β. In addition, PEDF expression was decreased significantly in the first 24 h during adipocyte differentiation, suggesting that downregulation of PEDF may be essential for the initiation of MCE and adipogenesis. We conclude that PEDF inhibits adipogenesis in 3T3-L1 preadipocytes partially because of inhibition of the MAPK/ERK signaling pathway and MCE.

  W Niu , P. J Bilan , S Ishikura , J. D Schertzer , A Contreras Ferrat , Z Fu , J Liu , S Boguslavsky , K. P Foley , Z Liu , J Li , G Chu , T Panakkezhum , G. D Lopaschuk , S Lavandero , Z Yao and A. Klip

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C2C12 myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 β-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.

  F Liu , J Li , D. M Wang , J. C Liu and Y. N. Huang

We investigated serial changes of circulation platelet activation markers in 40 patients undergoing carotid artery stenting under the protection of dual antiplatelet therapy and filter devices. Monocyte-platelet aggregates and PAC-1 (a marker specific for activated glycoprotein IIb/IIIa) analyzed by flow cytometry were determined in patients with symptomatic stenosis undergoing elective carotid artery stenting. Blood samples were obtained immediately before stent implantation and 0.5 hours, 18 hours, and 6 days after the procedure, respectively. All patients were already on dual antiplatelet therapy of aspirin and clopidogrel before carotid artery stenting, and all were stented with embolic protection devices. Both circulation monocyte-platelet aggregates and PAC-1 did not change significantly at the various time points after the procedure. Serial changes of monocyte-platelet aggregates and PAC-1 analyzed by flow cytometry fail to indicate the occurrence of platelet activation after carotid artery stenting under the treatment with dual antiplatelet therapy before carotid artery stenting and the application of embolic protection devices during the procedure.

  E Tai , L. A Pollack , J Townsend , J Li , C. B Steele and L. C. Richardson

Objective  To examine differences in non-Hodgkin lymphoma (NHL) survival between young adults and children/adolescents.

Design  Survival analysis using 13 Surveillance, Epidemiology, and End Results registries.

Setting  Cancer survival information from population-based cancer registries from 1992 through 2001.

Participants  A total of 2442 cases of NHL among children/adolescents (aged 0-19 years) and young adults (aged 20-29 years).

Main Exposure  Differences in NHL survival between young adults and children.

Main Outcome Measures  Comparison of 5-year survival by constructing Kaplan-Meier survival curves and modeling 5-year survival with multivariate Cox proportional hazards.

Results  Young adults were more likely to die compared with children/adolescents (hazard ratio = 2.06; 95% confidence interval, 1.65-2.56) even after accounting for NHL subtype and stage at diagnosis. Persons diagnosed with stage III disease (hazard ratio = 1.71; 95% confidence interval, 1.20-2.46) and stage IV disease (hazard ratio = 3.19; 95% confidence interval, 2.47-4.13) were more likely to die compared with persons diagnosed with stage I disease.

Conclusions  Being a young adult at diagnosis and having a higher stage of disease at diagnosis were associated with higher risk of death from NHL. Increasing survival with NHL is dependent on receiving appropriate cancer therapy. Therefore, efforts to address survival should include improving enrollment in clinical trials as well as increasing access to care.

  J Li and J. Kim

Our inability to distinguish between low-grade prostate cancers that pose no threat and those that can kill compels newly diagnosed early prostate cancer patients to make decisions that may negatively affect their lives needlessly for years afterward. To reliably stratify patients into different risk categories and apply appropriate treatment, we need a better molecular understanding of prostate cancer progression. Androgen ablation therapy and 5- reductase inhibitors reduce dihydrotestosterone levels and increase apoptosis. Because of the differing biological potentials of tumor cells, however, these treatments may, in some cases, worsen outcome by selecting for or inducing adaptation of stronger androgen receptor signaling pathways. Reduced dihydrotestosterone also may be associated with altered survival pathways. Complicating treatment effects further, molecular adaptation may be accelerated by interactions between epithelial and stromal cells. The hypothesis that early prostate cancer cells with differing biological potential may respond differently to finasteride treatment is worth testing. Ongoing studies using a systems biology approach in a preoperative prostate cancer setting are testing this hypothesis toward developing more-rational clinical interventions.

  M Guha , J Plescia , I Leav , J Li , L. R Languino and D. C. Altieri

Endogenous tumor suppression provides a barrier against oncogenesis, but the molecular requirements of this process are not well understood. Here, we show that the dual specificity phosphatase PTEN, a gene almost universally altered in human tumors, silences the expression of survivin, an essential regulator of cell division and apoptosis in cancer. This pathway is independent of p53, involves active repression of survivin gene transcription, and is mediated by direct occupancy of the survivin promoter by FOXO1 and FOXO3a factors. Conditional deletion of PTEN in the mouse prostate causes deregulated induction of survivin before full-blown transformation in vivo, whereas expression of survivin and PTEN is inversely correlated in cancer patients. Therefore, silencing the survivin gene is an essential requirement of endogenous PTEN tumor suppression. [Cancer Res 2009;69(12):4954–8]

  Y Wang , J Li , Y Cui , T Li , K. M Ng , H Geng , H Li , X. s Shu , W Liu , B Luo , Q Zhang , T. S. K Mok , W Zheng , X Qiu , G Srivastava , J Yu , J. J.Y Sung , A. T.C Chan , D Ma , Q Tao and W. Han

Closely located at the tumor suppressor locus 16q22.1, CKLF-like MARVEL transmembrane domain-containing member 3 and 4 (CMTM3 and CMTM4) encode two CMTM family proteins, which link chemokines and the transmembrane-4 superfamily. In contrast to the broad expression of both CMTM3 and CMTM4 in normal human adult tissues, only CMTM3 is silenced or down-regulated in common carcinoma (gastric, breast, nasopharyngeal, esophageal, and colon) cell lines and primary tumors. CMTM3 methylation was not detected in normal epithelial cell lines and tissues, with weak methylation present in only 5 of 35 (14%) gastric cancer adjacent normal tissues. Furthermore, immunohistochemistry showed that CMTM3 protein was absent in 12 of 35 (34%) gastric and 1 of 2 colorectal tumors, which was well correlated with its methylation status. The silencing of CMTM3 is due to aberrant promoter CpG methylation that could be reversed by pharmacologic demethylation. Ectopic expression of CMTM3 strongly suppressed the colony formation of carcinoma cell lines. In addition, CMTM3 inhibited tumor cell growth and induced apoptosis with caspase-3 activation. Thus, CMTM3 exerts tumor-suppressive functions in tumor cells, with frequent epigenetic inactivation by promoter CpG methylation in common carcinomas. [Cancer Res 2009;69(12):5194–201]

  H Wang , A Zhao , L Chen , X Zhong , J Liao , M Gao , M Cai , D. H Lee , J Li , D Chowdhury , Y. g Yang , G. P Pfeifer , Y Yen and X. Xu

Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.

  K. A Sloan , H. A Marquez , J Li , Y Cao , A Hinds , C. J O'Hara , S Kathuria , M. I Ramirez , M. C Williams and H. Kathuria

Caveolin-1 protein has been called a ‘conditional tumor suppressor’ because it can either suppress or enhance tumor progression depending on cellular context. Caveolin-1 levels are dynamic in non-small-cell lung cancer, with increased levels in metastatic tumor cells. We have shown previously that transactivation of an erythroblastosis virus-transforming sequence (ETS) cis-element enhances caveolin-1 expression in a murine lung epithelial cell line. Based on high sequence homology between the murine and human caveolin-1 promoters, we proposed that ETS proteins might regulate caveolin-1 expression in human lung tumorigenesis. We confirm that caveolin-1 is not detected in well-differentiated primary lung tumors. Polyoma virus enhancer activator 3 (PEA3), a pro-metastatic ETS protein in breast cancer, is expressed at low levels in well-differentiated tumors and high levels in poorly differentiated tumors. Conversely, Net, a known ETS repressor, is expressed at high levels in the nucleus of well-differentiated primary tumor cells. In tumor cells in metastatic lymph node sites, caveolin-1 and PEA3 are highly expressed, whereas Net is now expressed in the cytoplasm. We studied transcriptional regulation of caveolin-1 in two human lung cancer cell lines, Calu-1 (high caveolin-1 expressing) and NCI-H23 (low caveolin-1 expressing). Chromatin immunoprecipitation-binding assays and small interfering RNA experiments show that PEA3 is a transcriptional activator in Calu-1 cells and that Net is a transcriptional repressor in NCI-H23 cells. These results suggest that Net may suppress caveolin-1 transcription in primary lung tumors and that PEA3 may activate caveolin-1 transcription in metastatic lymph nodes.

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

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

  L Wang , J Zheng , Y Du , Y Huang , J Li , B Liu , C. j Liu , Y Zhu , Y Gao , Q Xu , W Kong and X. Wang

Rational: Vascular smooth muscle cells (VSMCs) switching from a contractile/differentiated to a synthetic/dedifferentiated phenotype has an essential role in atherosclerosis, postangioplastic restenosis and hypertension. However, how normal VSMCs maintain the differentiated state is less understood.

Objective: We aimed to indentify the effect of cartilage oligomeric matrix protein (COMP), a normal vascular extracellular matrix, on modulation of VSMCs phenotype.

Methods and Results: We demonstrated that COMP was associated positively with the expression of VSMC differentiation marker genes during phenotype transition. Knockdown of COMP by small interfering (si)RNA favored dedifferentiation. Conversely, adenoviral overexpression of COMP markedly suppressed platelet-derived growth factor-BB-elicited VSMC dedifferentiation, characterized by altered VSMC morphology, actin fiber organization, focal adhesion assembly, and the expression of phenotype-dependent markers. Whereas 7 integrin coimmunoprecipitated with COMP in normal rat VSMCs and vessels, neutralizing antibody or siRNA against 7 integrin inhibited VSMC adhesion to COMP, which indicated that 7β1 integrin is a potential receptor for COMP. As well, blocking or interference by siRNA of 7 integrin completely abolished the effect of COMP on conserving the contractile phenotype. In accordance, ectopic adenoviral overexpression of COMP greatly retarded VSMC phenotype switching, rescued contractility of carotid artery ring, and inhibited neointima formation in balloon-injured rats.

Conclusions: Our data suggest that COMP is essential for maintaining a VSMC contractile phenotype and the protective effects of COMP are mainly mediated through interaction with 7β1 integrin. Investigations to identify the factors affecting the expression and integrity of COMP may provide a novel therapeutic target for vascular disorders.

  J Naylor , J Li , C. J Milligan , F Zeng , P Sukumar , B Hou , A Sedo , N Yuldasheva , Y Majeed , D Beri , S Jiang , V. A. L Seymour , L McKeown , B Kumar , C Harteneck , D O'Regan , S. B Wheatcroft , M. T Kearney , C Jones , K. E Porter and D. J. Beech

Rationale: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in β cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system.

Objective: To determine the relevance and regulation of TRPM3 in vascular biology.

Methods and Results: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. β-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity.

Conclusions: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

  J Li , H Huang , L Sun , M Yang , C Pan , W Chen , D Wu , Z Lin , C Zeng , Y Yao , P Zhang and E. Song

Purpose: We aim to examine miR-21 expression in tongue squamous cell carcinomas (TSCC) and correlate it with patient clinical status, and to investigate its contribution to TSCC cell growth, apoptosis, and tumorigenesis.

Experimental Design: MicroRNA profiling was done in 10 cases of TSCC with microarray. MiR-21 overexpression was quantitated with quantitative reverse transcription-PCR in 103 patients, and correlated to the pathoclinical status of the patients. Immunohistochemistry was used to examine the expression of TPM1 and PTEN, and terminal deoxynucleotidyl transferase–mediated dUTP labeling to evaluate apoptosis. Moreover, miR-21 antisense oligonucleotide (ASO) was transfected in SCC-15 and CAL27 cell lines, and tumor cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adherent colony formation, and soft agar assay, whereas apoptosis was determined by Annexin V assay, cytochrome c release, and caspase 3 assay. Tumorigenesis was evaluated by xenografting SCC-15 cells in nude mice.

Results: MiR-21 is overexpressed in TSCC relative to adjacent normal tissues. The level of miR-21 is reversely correlated with TPM1 and PTEN expression and apoptosis of cancer cells. Multivariate analysis showed that miR-21 expression is an independent prognostic factor indicating poor survival. Inhibiting miR-21 with ASO in TSCC cell lines reduces survival and anchorage-independent growth, and induces apoptosis in TSCC cell lines. Simultaneous silencing of TPM1 with siRNA only partially recapitulates the effect of miR-21 ASO. Furthermore, repeated injection of miR-21 ASO suppresses tumor formation in nude mice by reducing cell proliferation and inducing apoptosis.

Conclusions: miR-21 is an independent prognostic indicator for TSCC, and may play a role in TSCC development by inhibiting cancer cell apoptosis partly via TPM1 silencing.

  C. A Milbury , J Li and G. M. Makrigiorgos

Background: Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature–PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples.

Methods: Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6–8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced.

Results: Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of approximately 2% to 10%, whereas COLD-PCR/HRM exhibited limits from approximately 0.1% to 1% mutant-to-wild-type ratio. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons.

Conclusions: COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown low-level mutations in clinical samples.

  J Li , W Dunn , A Breaud , D Elliott , L. J Sokoll and W. Clarke

We evaluated the analytical performance of 4 cystatin C assays (Siemens N Latex on BNII, Roche Tina-quant on Cobas c501, Genzyme on Cobas c501, and Tosoh ST AIA-PACK on Tosoh AIA-600II) according to guidelines published by the Clinical and Laboratory Standards Institute.


We evaluated total imprecision, limit of detection, and limit of quantification for each assay using patient serum pools and linearity/recovery using serial dilutions of a patient serum pool with cystatin C–free serum. We compared patients (n = 102) using the Siemens assay as a comparison method.


All assays had limits of detection and quantification <0.08 and <0.39 mg/L, respectively. Total CVs were generally higher than the manufacturers' claims for all assays. The Roche assay overrecovered cystatin C, particularly at low concentrations (mean recovery 119%, 142% at 0.587 mg/L). Deming regression equations were y = 1.184x + 0.089, Sy|x = 0.246 for Genzyme; y = 0.937x + 0.231, Sy|x = 0.231 for Roche; and y = 1.010x + 0.216, Sy|x = 0.115 for Tosoh. The Genzyme assay appeared to report higher results than the Siemens assay, which is consistent with a higher reference interval specified by the manufacturer.


Although all assays were acceptable for clinical use, their diagnostic performances were not optimal. Limitations include imprecision greater than claimed, overrecovery for the Roche assay on low concentration samples, and differences in results for patient samples. The latter situation requires assay-specific cystatin C–based glomerular filtration rate prediction equations at least until calibration is standardized using the international cystatin C calibrator now being developed.

  F St. Michael , E Vinogradov , C. Q Wenzel , J Li , J C Hoe and J. C Richards

Previous studies on LPS from Neisseria meningitidis strains M992B, the immunotype L6 strain, NMB, the type strain, a candidate LPS vaccine strain 6275z, and an extensively used clinical strain M986 had suggested that the location of the phosphoethanolamine (PEtn) residue was the 7-position of the distal heptose residue (HepII) of the inner-core oligosaccharide (OS). In all cases, this was only established by chemical methods, methylation linkage analyses. In this study, we have used standard NMR techniques to unequivocally show that the PEtn residue is actually located at the 6-position and not at the 7-position of the HepII residue in all of these strains. The 6-PEtn transferase genes were sequenced and their translated amino acid sequences were shown to be greater than 96% identical to that of the Lpt6 transferase from the L4 immunotype strain, which has been shown to transfer PEtn to the 6-position of the distal heptose residue. We discuss the implications of these findings with respect to the immunotyping scheme for the meningococci and in the context of LPS-based vaccine development.

  C. H Dai , J Li , S. B Shi , L. C Yu , L. P Ge and P. Chen

Survivin and livin, which are members of the inhibitor of apoptosis protein family, regulate both programmed cell death and proliferation. Second mitochondria-derived activator of caspase is thought to regulate apoptosis by antagonizing inhibitor of apoptosis protein. These gene expressions are regarded as prognostic markers in some malignancies. However, result in previous studies of the association of these gene expressions with prognosis of patients with non-small cell lung cancer remains contradictory.


Survivin, livin and second mitochondria-derived activator of caspase mRNA was detected by semi-quantitative reverse transcriptase—polymerase chain reaction in surgical resected tumor specimen from 66 non-small cell lung patients who received adjuvant platinum-based chemotherapy.


Results showed that patients with survivin high expression had significantly shorter tumor-free survival (P = 0.012) and overall survival (P = 0.007) than those with survivin low expression. There was a significant association of second mitochondria-derived activator of caspase high expression in non-small cell lung cancer tissue with longer tumor-free survival (P = 0.021) and overall survival (P = 0.0013). However, livin mRNA expression level had no impact on the tumor-free survival and overall survival of the patients. In multivariate analyses, survivin mRNA high expression (P = 0.033 and P = 0.024) and advanced pathologic stage (P = 0.009 and P = 0.008) were the factors which independently predicted a worse tumor-free survival and overall survival.


Our data suggest that assessment of survivin and second mitochondria-derived activator of caspase mRNA expression may be useful for predicting survival in non-small cell lung cancer patients receiving platinum-based chemotherapy after surgical resection and can provide valuable information for deciding better therapy strategy.

  C Guo , J Li , L Myatt , X Zhu and K. Sun

Cytosolic phospholipase A (cPLA2) catalyzes the formation of arachidonic acid in prostaglandin synthesis. In contrast to the well-described down-regulation of cPLA2, up-regulation of cPLA2 by glucocorticoids has been reported in human amnion fibroblasts, which may play a key role in parturition. The mechanisms underlying this paradoxical induction of cPLA2 by glucocorticoids remain largely unknown. Using cultured human amnion fibroblasts, we found that the induction of cPLA2 by cortisol required ongoing transcription and synthesis of at least one other protein. The induction of cPLA2 by cortisol was abolished by mutagenesis of a glucocorticoid response element (GRE) in the promoter. The same GRE was found mediating the classical inhibition of cPLA2 expression by cortisol in human fetal lung fibroblasts (HFL-1). Cortisol increased Gs expression in amnion fibroblasts but not in HFL-1 cells. Inhibition of Gs with NF449 attenuated the phosphorylation of cAMP response element-binding protein-1 (CREB-1) and the induction of cPLA2 by cortisol in amnion fibroblasts. Both glucocorticoid receptor (GR) and CREB-1 were found bound to the GRE upon cortisol stimulation of amnion fibroblasts. The induction of cPLA2 by cortisol was blocked by GR antagonist RU486 or protein kinase A inhibitor H89 or dominant-negative CREB-1. In conclusion, cortisol activates the cAMP/protein kinase A/CREB-1 pathway via Gs induction, and the phosphorylated CREB-1 interacts with GR at the GRE to promote cPLA2 expression in amnion fibroblasts.

  J. R Wang , H Hu , G. H Wang , J Li , J. Y Chen and P. Wu

Twelve genes of the PIN family in rice were analyzed for gene and protein structures and an evolutionary relationship with reported AtPINs in Arabidopsis. Four members of PIN1 (designated as OsPIN1a–d), one gene paired with AtPIN2 (OsPIN2), three members of PIN5 (OsPIN5a–c), one gene paired with AtPIN8 (OsPIN8), and three monocot-specific PINs (OsPIN9, OsPIN10a, and b) were identified from the phylogenetic analysis. Tissue-specific expression patterns of nine PIN genes among them were investigated using RT–PCR and GUS reporter. The wide variations in the expression domain in different tissues of the PIN genes were observed. In general, PIN genes are up-regulated by exogenous auxin, while different responses of different PIN genes to other hormones were found.

  B Kang , H Wang , K. H Nam , J Li and J. Li

Brassinosteroids (BRs) are important plant hormones that act synergistically with auxin to regulate a variety of plant developmental and physiological processes. In the past decade, genetic and biochemical studies have revealed a linear signaling pathway that relies on protein phosphorylation to transmit the BR signal into the nucleus, altering expression of hundreds of genes to promote plant growth. We conducted an activation-tagging based suppressor screen to look for Arabidopsis genes that, when overexpressed by inserted 35S enhancer elements, could suppress the dwarf phenotype of a weak BR receptor mutant bri1-301. This screen identified a total of six dominant activation-tagged bri1 suppressors (atbs-Ds). Using a plasmid rescue approach, we discovered that the bri1-301 suppression effect in four atbs-D mutants (atbs3-D to atbs6-D) was caused by overexpression of a YUCCA gene thought to be involved in tryptophan-dependent auxin biosynthesis. Interestingly, the three activation-tagged YUCCA genes belong to the YUCCA IIA subfamily that includes two other members out of 11 known Arabidopsis YUCCA genes. In addition, our molecular studies revealed a T-DNA insertion near a basic helix-loop-helix gene in atbs1-D and a T-DNA insertion in a region carrying a BR biosynthetic gene in atbs2-D. Further studies of these atbs-D mutants could lead to better understanding of the BR signaling process and the BR–auxin interaction.

  M. O Song , J Li and J. H. Freedman

Copper is an essential trace element; however, at supraphysiological levels, it can be extremely toxic. Microarray data from HepG2 cells exposed to 100, 200, 400, and 600 µM copper for 4, 8, 12 and 24 h were generated and analyzed. Principal components, K-means, and hierarchical clustering, interactome, and pathway mapping analyses indicated that these exposure conditions induce physiological and toxicological changes in the HepG2 transcriptome. As a general trend, when the level of toxicity increases, the number and diversity of affected genes, Gene Ontology categories, regulatory pathways, and complexity of interactomes increase. Physiological responses to copper include transition metal ion binding and responses to stress/stimulus, whereas toxicological responses include apoptosis, morphogenesis, and negative regulation of biomolecule metabolism. The global gene expression profile was overlaid onto biomolecular interaction networks and signal transduction cascades using pathway mapping and interactome identification. This analysis indicated that copper modulates signal transduction pathways associated with MAPK, NF-B, death receptor, IGF-I, hypoxia, IL-10, IL-2, IL-6, EGF, Toll-like receptor, protein ubiquitination, xenobiotic metabolism, leukocyte extravasation, complement and coagulation, and sonic hedgehog signaling. These results provide insights into the global and molecular mechanisms regulating the physiological and toxicological responses to metal exposure.

  V. Y Polotsky , V Savransky , S Bevans Fonti , C Reinke , J Li , D. N Grigoryev and L. A. Shimoda

Obstructive sleep apnea may cause vascular inflammation and atherosclerosis, which has been attributed to intermittent hypoxia (IH). Recent data suggest that IH, but not sustained hypoxia (SH), activates proinflammatory genes in HeLa cells. Effects of IH and SH on the gene expression profile in human aortic endothelial cells (HAEC) have not been compared. We perfused media with alternating flow of 16% and 0% O2 (IH) or constant flow of 4% O2 (SH-4%), 8% O2 (SH-8%), or 16% O2 (control) for 8 h. Illumina gene microarrays were performed, with subsequent verification by real-time PCR. Proinflammatory cytokines in the media were measured by ELISA. Both IH and SH-4% upregulated proinflammatory genes, including heat shock protein 90-kDa B1, tumor necrosis factor superfamily member 4, and thrombospondin 1. Among all proinflammatory genes, only IL-8 mRNA showed significantly higher levels of expression (1.78-fold) during IH, compared with SH-4%, but both types of hypoxic exposure elicited striking three- to eightfold increases in IL-8 and IL-6 protein levels in the media. IH and SH-4% also upregulated antioxidant genes, including heme oxygenase-1 and nuclear factor (erythroid-derived 2)-like 2 (NRF2), whereas classical genes regulated by hypoxia-inducible factor 1 (HIF-1), such as endothelin and glucose transporter GLUT1, were not induced. SH-8% induced changes in gene expression and cytokine secretion that were similar to those of IH and SH-4%. In conclusion, short exposures to IH and SH upregulate proinflammatory and antioxidant genes in HAEC and increase secretion of proinflammatory cytokines IL-8 and IL-6 into media in similar fashions.

  H Xu , B Zhang , J Li , H Chen , J Tooley and F. K. Ghishan

Sodium/hydrogen exchangers (NHEs) play a major role in Na+ absorption, cell volume regulation, and intracellular pH regulation. Of the nine identified mammalian NHEs, three (NHE2, NHE3, and NHE8) are localized on the apical membrane of epithelial cells in the small intestine and the kidney. Although the regulation of NHE2 and NHE3 expression has been extensively studied in the past decade, little is known about the regulation of NHE8 gene expression under physiological conditions. The current studies were performed to explore the role of epidermal growth factor (EGF) on NHE8 expression during intestinal maturation. Brush-border membrane vesicles (BBMV) were isolated from intestinal epithelia, and Western blot analysis was performed to determine NHE8 protein expression of sucking male rats treated with EGF. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcriptional factors involved in EGF-mediated regulation. Our results showed that intestinal NHE8 mRNA expression was decreased in EGF-treated rats and Caco-2 cells, and NHE8 protein abundance was also decreased in EGF-treated rats. The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by EGF treatment. This could be explained by reduced binding of transcription factor Sp3 on the NHE8 basal promoter region in the presence of EGF. Pretreatment with MEK1/2 inhibitor UO-126 could prevent EGF-mediated inhibition of NHE8 gene expression. In conclusion, this study showed that EGF inhibits NHE8 gene expression through reducing its basal transcription, suggesting an important role of EGF in regulating NHE expression during intestinal maturation.

  F Yokoi , G Yang , J Li , M. P DeAndrade , T Zhou and Y. Li

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder caused by mutations in DYT1 coding for torsinA with ~30% penetrance. Most of the DYT1 dystonia patients exhibit symptoms during childhood and adolescence. On the other hand, DYT1 mutation carriers without symptoms during these periods mostly do not exhibit symptoms later in their life. Little is known about what controls the timing of the onset, a critical issue for DYT1 mutation carriers. DYT11 myoclonus-dystonia is caused by mutations in SGCE coding for -sarcoglycan. Two dystonia patients from a single family with double mutations in DYT1 and SGCE exhibited more severe symptoms. A recent study suggested that torsinA contributes to the quality control of -sarcoglycan. Here, we derived mice carrying mutations in both Dyt1 and Sgce and found that these double mutant mice showed earlier onset of motor deficits in beam-walking test. A novel monoclonal antibody against mouse -sarcoglycan was developed by using Sgce knock-out mice to avoid the immune tolerance. Western blot analysis suggested that functional deficits of torsinA and -sarcoglycan may independently cause motor deficits. Examining additional mutations in other dystonia genes may be beneficial to predict the onset in DYT1 mutation carriers.

  F. J Vizeacoumar , N van Dyk , F S.Vizeacoumar , V Cheung , J Li , Y Sydorskyy , N Case , Z Li , A Datti , C Nislow , B Raught , Z Zhang , B Frey , K Bloom , C Boone and B. J. Andrews

A combination of yeast genetics, synthetic genetic array analysis, and high-throughput screening reveals that sumoylation of Mcm21p promotes disassembly of the mitotic spindle.

  A Dessein , C Chevillard , V Arnaud , X Hou , A. A Hamdoun , H Dessein , H He , S. A Abdelmaboud , X Luo , J Li , A Varoquaux , A Mergani , M Abdelwahed , J Zhou , A Monis , M. G.R Pitta , N Gasmelseed , S Cabantous , Y Zhao , A Prata , C Brandt , N. E Elwali , L Argiro and Y. Li

Abnormal fibrosis occurs during chronic hepatic inflammations and is the principal cause of death in hepatitis C virus and schistosome infections. Hepatic fibrosis (HF) may develop either slowly or rapidly in schistosome-infected subjects. This depends, in part, on a major genetic control exerted by genes of chromosome 6q23. A gene (connective tissue growth factor [CTGF]) is located in that region that encodes a strongly fibrogenic molecule. We show that the single nucleotide polymorphism (SNP) rs9402373 that lies close to CTGF is associated with severe HF (P = 2 x 10–6; odds ratio [OR] = 2.01; confidence interval of OR [CI] = 1.51–2.7) in two Chinese samples, in Sudanese, and in Brazilians infected with either Schistosoma japonicum or S. mansoni. Furthermore, SNP rs12526196, also located close to CTGF, is independently associated with severe fibrosis (P = 6 x 10–4; OR = 1.94; CI = 1.32–2.82) in the Chinese and Sudanese subjects. Both variants affect nuclear factor binding and may alter gene transcription or transcript stability. The identified variants may be valuable markers for the prediction of disease progression, and identify a critical step in the development of HF that could be a target for chemotherapy.

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