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Articles by X Li
Total Records ( 50 ) for X Li
  D Zheng , X Li , H Xu , X Lu , Y Hu and W. Fan
 

Docetaxel (Doc) has extraordinary activities against a variety of solid tumors. However, the clinical efficacy of Doc is limited due to its poor solubility, low selective distribution, fast elimination in vivo, etc. In the present study, Doc was incorporated into the core-shell structure of nanoparticles prepared based on our previous work. The obtained docetaxel-loaded nanoparticles (DOCNP) were characterized with various biophysical methodologies, and its antitumor efficacy against malignant melanoma was evaluated both in vitro and in vivo. Our results indicated that Doc could be incorporated into the nanoparticles with high encapsulation efficiency (>90%). The incorporated Doc can be released from DOCNP in a sustained manner. In vitro cytotoxicity studies indicated that DOCNP could effectively kill B16 cells and show a dose- and time-dependent efficacy. Furthermore, intratumoral administration revealed that DOCNP has significantly higher antitumor effect and lower toxicity to normal cells and tissues than free Doc. These results suggest that DOCNP may be a promising drug delivery system in therapy for malignant melanoma.

  W Yuan , J Guo , X Li , Z Zou , G Chen , J Sun , T Wang and D. Lu
 

It has been reported that phospholipase C-1 (PLC-1) plays an important protective role in hydrogen peroxide (H2O2)-induced pheochromocytoma (PC) 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-1 activation remain to be identified. The present study was designed to examine the roles of PLC-1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PC12 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was significantly increased. We concluded that PLC-1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.

  H Zhao , Y Wang , Y Wu , X Li , G Yang , X Ma , R Zhao and H. Liu
 

Hyperlipidemia is regarded as an independent risk factor in the development of ischemic heart disease, and it can increase the myocardial susceptibility to ischemia/reperfusion (I/R) injury. Ischemic postconditioning (Postcon) has been demonstrated to attenuate the myocardial injury induced by I/R in normal conditions. But the effect of ischemic Postcon on hyperlipidemic animals is unknown. Hypoxia inducible factor-1 (HIF-1) has been demonstrated to play a central role in the cardioprotection by preconditioning, which is one of the protective strategies except for Postcon. The aim of this study was to determine whether Postcon could reduce myocardial injury in hyperlipidemic animals and to assess whether HIF-1 was involved in Postcon mechanisms. Male Wistar rats underwent the left anterior descending coronary occlusion for 30 min followed by 180 min of reperfusion with or without Postcon after fed with high fat diet or normal diet for 8 weeks. The detrimental indices induced by the I/R insult included infarct size, plasma creatine kinase activity and caspase-3 activity. Results showed that hyperlipidemia remarkably enhanced the myocardial injury induced by I/R, while Postcon significantly decreased the myocardial injury in both normolipidemic and hyperlipidemic rats. Moreover, both hyperlipidemia and I/R promoted the HIF-1 expression. Most importantly, we have for the first time demonstrated that Postcon further induced a significant increase in HIF-1 protein level not only in normolipidemic but also in hyperlipidemic conditions. Thus, Postcon reduces the myocardial injury induced by I/R in normal and hyperlipidemic animals, and HIF-1 upregulation may involve in the Postcon-mediated cardioprotective mechanisms.

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

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

  Q Wang , M Liu , X Li , L Chen and H. Tang
 

Kazrin has recently been identified as a functional protein that is involved in cell–cell junctions and in signal transduction. Here, we identified a new isoform, Kazrin F, which is 518 aa in length and has 97 aa unique at the N-terminus. Knockdown of Kazrin F using siRNA caused cell apoptosis and a marked decrease in cell viability measured by MTT and TUNEL assays. Co-immunoprecipitation analysis revealed that Kazrin F interacts with ARC (apoptosis repressor with caspase recruitment domain) and Bax (Bcl-2-associated X protein). Co-localization of Kazrin F with ARC and Bax in the cytoplasm was determined by immunofluorescence analysis. These results suggested that Kazrin F might play an important role in regulating cellular apoptosis by interacting with ARC and Bax.

  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.

  X Jin , H Mei , X Li , Y Ma , A. h Zeng , Y Wang , X Lu , F Chu , Q Wu and J. Zhu
 

We studied the apoptosis-inducing properties of the antimicrobial peptide cecropin of Musca domestica in human hepatocellular carcinoma cell line BEL-7402 and its underlying mechanism. Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells and the human normal liver cells were determined by the MTT assay, and the cell viability was determined by trypan blue dye exclusion assay. The apoptotic tumor cells treated with cecropin were examined by transmission electron microscopy and terminal-deoxynucleotidyl transferase mediated nick end labeling. The apoptosis rate was measured by flow cytometry (FCM) with PI/Annexin-V double staining. Western blot analysis and RT-PCR were used to determine the expression levels of proteins involved in apoptosis, such as Fas, Fas-L, caspase-8, and caspase-3. The experimental results showed that Musca domestica cecropin inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells in dose-dependent and time-dependent manners, without affecting the proliferation of normal liver cells. FCM showed that the cell apoptosis rates were 5.1 ± 0.11%, 8.1 ± 0.04%, and 10.9 ± 0.15% after the treating with 100 µM cecropin for 24, 48, and 72 h, respectively. The rates of apoptosis were 5.4 ± 0.14% and 8.0 ± 0.13% after the treating with 25 and 50 µM cecropin for 72 h, respectively. Western blot analysis and RT-PCR showed that the apoptosis-related molecules including Fas, Fas-L, caspase-8 and caspase-3 were activated. This study showed that the antimicrobial peptide cecropin-inducing apoptosis in human hepatocellular carcinoma BEL-7402 cells, which might be associated with upregulation of Fas, Fas-L, and caspase-8 and caspase-3 and triggering extrinsic apoptotic pathway.

  X Fan , Y Liu , J Jiang , Z Ma , H Wu , T Liu , M Liu , X Li and H. Tang
 

MicroRNAs (miRNAs) are emerging as a class of small regulated RNAs, and the alterations of miRNAs are implicated in the initiation and progression of human cancers. Our study shows that inhibition of miR-20a in OVCAR3 ovarian cancer cell line could suppress, whereas overexpression of miR-20a could enhance cell long-term proliferation and invasion. We also confirmed amyloid precursor protein (APP) as a direct target gene of miR-20a. Furthermore, suppression of APP expression could also promote ovarian cancer cell proliferation and invasion, which is consistent with the results of miR-20a overexpression. Therefore, we concluded that the regulation of APP is an important mechanism for miR-20a to promote proliferation and invasion in ovarian cancer cells.

  C Zhang , Y Tan , W Guo , C Li , S Ji , X Li and L. Cai
 

Renal protection against diabetes-induced pathogenic injuries by multiple exposures to low-dose radiation (LDR) was investigated to develop a novel approach to the prevention of renal disease for diabetic subjects. C57BL/6J mice were given multiple low-dose streptozotocin (STZ; 60 x 6 mg/kg) to produce a type 1 diabetes. Two weeks after diabetes onset, some of diabetic mice and age-matched nondiabetic mice were exposed whole body to 25 mGy X-rays every other day for 2, 4, 8, 12, and 16 wk. Diabetes caused a significant renal dysfunction, shown by time-dependent increase in urinary microalbumin (Malb) and decrease in urinary creatinine (Cre), and pathological changes, shown by significant increases in renal structural changes and PAS-positive staining. However, diabetes-induced renal dysfunction and pathological changes were significantly, albeit partially, attenuated by multiple exposures to LDR. Furthermore, LDR protection against diabetes-induced renal dysfunction and pathological changes was associated with a significant suppression of diabetes-increased systemic and renal inflammation, shown by significant increases in serum and renal TNF, ICAM-1, IL-18, MCP-1, and PAI-1 contents. To further explore the mechanism by which LDR prevents diabetes-induced renal pathological changes, renal oxidative damage was examined by Western blotting and immunohistochemical staining for 3-nitrotyrosine and 4-hydroxynonenal. Significant increase in oxidative damage was observed in diabetic mice, but not diabetic mice, with LDR. Renal fibrosis, examined by Western blotting of connective tissue growth factor and Masson's trichrome staining, was also evident in the kidneys of diabetic mice but not diabetic mice with LDR. These results suggest that multiple exposures to LDR significantly suppress diabetes-induced systemic and renal inflammatory response and renal oxidative damage, resulting in a prevention of the renal dysfunction and fibrosis.

  O. G Chepurny , G. G Kelley , I Dzhura , C. A Leech , M. W Roe , E Dzhura , X Li , F Schwede , H. G Genieser and G. G. Holz
 

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 µM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca2+]i that reflected both Ca2+ influx and intracellular Ca2+ mobilization in islet β-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 µM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 µM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca2+-dependent exocytosis.

  Q Li , X Li and B. Stanton
 

Aims: To review the patterns, contexts and impacts of alcohol use associated with commercial sex reported in the global literature. Methods: We identified peer-reviewed English-language articles from 1980 to 2008 reporting alcohol consumption among female sex workers (FSWs) or male clients. We retrieved 70 articles describing 76 studies, in which 64 were quantitative (52 for FSWs, 12 for male clients) and 12 qualitative. Results: Studies increased over the past three decades, with geographic concentration of the research in Asia and North America. Alcohol use was prevalent among FSWs and clients. Integrating quantitative and qualitative studies, multilevel contexts of alcohol use in the sex work environment were identified, including workplace and occupation-related use, the use of alcohol to facilitate the transition into and practice of commercial sex among both FSWs and male clients, and self-medication among FSWs. Alcohol use was associated with adverse physical health, illicit drug use, mental health problems, and victimization of sexual violence, although its associations with HIV/sexually transmitted infections and unprotected sex among FSWs were inconclusive. Conclusions: Alcohol use in the context of commercial sex is prevalent, harmful among FSWs and male clients, but under-researched. Research in this area in more diverse settings and with standardized measures is required. The review underscores the importance of integrated intervention for alcohol use and related problems in multilevel contexts and with multiple components in order to effectively reduce alcohol use and its harmful effects among FSWs and their clients.

  J Ji , X Shu , X Li , K Sundquist , J Sundquist and K. Hemminki
 

Background: Sarcoidosis patients show dysregulated immune function, which may be related to subsequent cancer. We examined here the overall and specific cancer risks among Swedish subjects who had been hospitalized for sarcoidosis.

Methods: A sarcoidosis research database was created by identifying hospitalized sarcoidosis patients from the Swedish Hospital Discharge Register and by linking them with the Cancer Registry. Standardized incidence ratios (SIRs) were calculated for cancers in sarcoidosis patients compared with subjects without sarcoidosis.

Results: A total of 10 037 patients were hospitalized for sarcoidosis during years 1964–2004. Among them 1045 patients developed subsequent cancer, giving an overall SIR of 1.40 and 1.18 for cancer diagnosed later than 1 year of follow-up. A significant excess was noted for skin (squamous cell), kidney and nonthyroid endocrine tumors and additionally for non-Hodgkin's lymphoma and leukemia. Patients with multiple hospitalizations showed high risks.

Conclusions: A 40% overall excess incidence of cancer was noted among sarcoidosis patients, but the increase was confined mainly to the first year after hospitalization. However, the increased risks of skin cancer and non-Hodgkin's lymphoma and leukemia, especially for those with multiple hospitalizations or hospitalized at old age, call for clinical attention.

  J Kong , X Li , Y Wang , W Sun and J. Zhang
 

Objective  To assess the impact of digital problem-based learning (PBL) cases on student learning in ophthalmology courses.

Methods  Ninety students were randomly divided into 3 classes (30 students per class). The first class studied under a didactic model. The other 2 classes were divided into 6 groups (10 students per group) and received PBL teaching; 3 groups studied via cases presented in digital form and the others studied via paper-form cases. The results of theoretical and case analysis examinations were analyzed using the 2 test. Student performance on the interval practice was analyzed using the Kruskal-Wallis test. Questionnaires were used to evaluate student and facilitator perceptions.

Results  Students in the digital groups exhibited better performance in the practice procedures according to tutorial evaluations compared with the other groups (P < .05). The 2 PBL classes had significantly higher mean results of theoretical and case analysis examinations (P < .001), but there was no significant difference between the 2 PBL classes. Ninety-three percent of students in the digital groups (vs 73% in the paper groups) noted that the cases greatly stimulated their interest.

Conclusions  Introducing PBL into ophthalmology could improve educational quality and effectiveness. Digital PBL cases stimulate interest and motivate students to further improve diagnosis and problem-handling skills.

  K He , X Li , J Zhou , X. W Deng , H Zhao and J. Luo
 

Summary:NTAP is designed to analyze ChIP-chip data generated by the NimbleGen tiling array platform and to accomplish various pattern recognition tasks that are useful especially for epigenetic studies. The modular design of NTAP makes the data processing highly customizable. Users can either use NTAP to perform the full process of NimbleGen tiling array data analysis, or choose post-processing modules in NTAP to analyze pre-processed epigenetic data generated by other platforms. The output of NTAP can be saved in standard GFF format files and visualized in GBrowse.

  M. K Karmakar , X Li , A. M. H Ho , W. H Kwok and P. T. Chui
  Background

Current methods of locating the epidural space rely on surface anatomical landmarks and loss-of-resistance (LOR). We are not aware of any data describing real-time ultrasound (US)-guided epidural access in adults.

Methods

We evaluated the feasibility of performing real-time US-guided paramedian epidural access with the epidural needle inserted in the plane of the US beam in 15 adults who were undergoing groin or lower limb surgery under an epidural or combined spinal–epidural anaesthesia.

Results

The epidural space was successfully identified in 14 of 15 (93.3%) patients in 1 (1–3) attempt using the technique described. There was a failure to locate the epidural space in one elderly man. In 8 of 15 (53.3%) patients, studied neuraxial changes, that is, anterior displacement of the posterior dura and widening of the posterior epidural space, were seen immediately after entry of the Tuohy needle and expulsion of the pressurized saline from the LOR syringe into the epidural space at the level of needle insertion. Compression of the thecal sac was also seen in two of these patients. There were no inadvertent dural punctures or complications directly related to the technique described. Anaesthesia adequate for surgery developed in all patients after the initial spinal or epidural injection and recovery from the epidural or spinal anaesthesia was also uneventful.

Conclusions

We have demonstrated the successful use of real-time US guidance in combination with LOR to saline for paramedian epidural access with the epidural needle inserted in the plane of the US beam.

  N Bhatnagar , X Li , Y Chen , X Zhou , S. H Garrett and B. Guo
 

Butyrate is an inhibitor of histone deacetylase (HDAC) and has been extensively evaluated as a chemoprevention agent for colon cancer. We recently showed that mutations in the adenomatous polyposis coli (APC) gene confer resistance to HDAC inhibitor–induced apoptosis in colon cancers. Here, we show that APC mutation rendered colon cancer cells resistant to butyrate-induced apoptosis due to the failure of butyrate to down-regulate survivin in these cells. Another cancer-preventive agent, 3,3'-diindolylmethane (DIM), was identified to be able to down-regulate survivin in colon cancers expressing mutant APC. DIM inhibited survivin mRNA expression and promoted survivin protein degradation through inhibition of p34cdc2-cyclin B1–mediated survivin Thr34 phosphorylation. Pretreatment with DIM enhanced butyrate-induced apoptosis in colon cancer cells expressing mutant APC. DIM/butyrate combination treatment induced the expression of proapoptotic Bax and Bak proteins, triggered Bax dimerization/activation, and caused release of cytochrome c and Smac proteins from mitochondria. Whereas overexpression of survivin blocked DIM/butyrate–induced apoptosis, knocking down of survivin by small interfering RNA increased butyrate-induced apoptosis in colon cancer cells. We further showed that DIM was able to down-regulate survivin and enhance the effects of butyrate in apoptosis induction and prevention of familial adenomatous polyposis in APCmin/+ mice. Thus, the combination of DIM and butyrate is potentially an effective strategy for the prevention of colon cancer.

  L. M. F Merlo , N. A Shah , X Li , P. L Blount , T. L Vaughan , B. J Reid and C. C. Maley
 

Neoplastic progression is an evolutionary process driven by the generation of clonal diversity and natural selection on that diversity within a neoplasm. We hypothesized that clonal diversity is associated with risk of progression to cancer. We obtained molecular data from a cohort of 239 participants with Barrett's esophagus, including microsatellite shifts and loss of heterozygosity, DNA content tetraploidy and aneuploidy, methylation, and sequence mutations. Using these data, we tested all major diversity measurement methods, including genetic divergence and entropy-based measures, to determine which measures are correlated with risk of progression to esophageal adenocarcinoma. We also tested whether the use of different sets of loci and alterations to define clones (e.g., selectively advantageous versus evolutionarily neutral) improved the predictive value of the diversity indices. All diversity measures were strong and highly significant predictors of progression (Cox proportional hazards model, P < 0.001). The type of alterations evaluated had little effect on the predictive value of most of the diversity measures. In summary, diversity measures are robust predictors of progression to cancer in this cohort. Cancer Prev Res; 3(11); 1388–97. ©2010 AACR.

  X Li and H. H. Tai
 

Previous studies implicate that activation of thromboxane A2 receptor (TP) induced cell proliferation and transformation in several cell lines. We report here that the activation of TP by its agonist, [1S-[1, 2 (Z), 3β (1E, 3S*), 4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo [2.2.1] hept-2-yl]-5-heptenoic acid (I-BOP), induced Nurr1 expression and stimulated proliferation of human lung cancer cells. Nurr1, an orphan nuclear receptor in the nuclear receptor subfamily 4A subfamily, has been implicated in cell proliferation, differentiation and apoptosis. I-BOP markedly induced Nurr1 messenger RNA and protein levels as compared with other subfamily members, Nur77 and Nor-1. The signaling pathways of I-BOP-induced Nurr1 expression were examined by using various inhibitors of signaling molecules. The induction of Nurr1 expression by I-BOP appeared to be mediated through protein kinase A (PKA)/cAMP response element binding (CREB), protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways and not related to epidermal growth factor receptor and prostaglandin E2 pathways. Transcriptional activation of Nurr1 gene by I-BOP was further investigated at the promoter level in H157 cells. 5'-Deletion analysis, site-directed mutagenesis and luciferase reporter assay demonstrated that Nurr1 expression was induced by I-BOP in a PKA/CREB-dependent manner. Further studies have revealed that Nurr1 may mediate cyclin D1 expression and I-BOP-induced cell proliferation in H157 cells since small interfering RNA of Nurr1 blocked I-BOP-induced cyclin D1 expression and cell proliferation and also decreased cell growth rate. These results provide strong evidence that Nurr1 plays a significant role in cell proliferation and may mediate TP agonist-induced proliferation in lung cancer cells.

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

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

  Z Hu , X Li , X Qu , Y He , B. Z Ring , E Song and L. Su
 

A few genetic polymorphisms of TP53 are known to have a significant effect on cancer susceptibility. Intron 3 16 bp duplication polymorphism of TP53 has been reported to be associated with breast cancer, colorectal cancer, lung cancer and other cancers, but the reported results remain inconclusive. The present study, a meta-analysis including a total of 9801 cases and 10 391 controls from 26 studies, revealed that the 16 bp insertion (Ins) allele is significantly associated with an increased cancer risk in overall analysis [Ins/Ins + deletion (Del)/Ins versus Del/Del: odds ratio (OR) = 1.14, 95% confidence interval (CI) = 1.02–1.27, P = 0.02; Ins/Ins versus Del/Del: OR = 1.35, 95% CI = 1.11–1.63, P = 0.002; Del/Ins versus Del/Del: OR = 1.10, 95% CI = 0.98–1.23, P = 0.11.), particularly in breast cancer subgroup (Ins/Ins + Del/Ins versus Del/Del: OR = 1.16, 95% CI = 1.03–1.31, P = 0.02; Ins/Ins versus Del/Del: OR = 1.81, 95% CI = 1.30–2.52, P < 0.001; Del/Ins versus Del/Del: OR = 1.10, 95% CI = 0.97–1.25, P = 0.13). The relative risks to the colorectal and lung cancers increased but their association power was relatively weak, which may result from a limited number of studies of these two cancer types. These results suggest that intron 3 16 bp duplication polymorphism of TP53 is potentially an important and clinically relevant genetic marker contributing to cancer susceptibility.

  X Li , Y Li , L Shan , E Shen and T. Peng
  Aims

Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF- expression, and myocardial dysfunction during LPS stimulation.

Methods and results

In cultured adult rat cardiomyocytes, LPS (1 µg/mL) induced calpain and caspase-3 activity, and up-regulated TNF- expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91phox-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF- expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF- expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively.

Conclusion

gp91phox-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF- expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.

  P Subramanian , E Karshovska , P Reinhard , R. T. A Megens , Z Zhou , S Akhtar , U Schumann , X Li , M van Zandvoort , C Ludin , C Weber and A. Schober
 

Rationale: The chemokine CXCL12 (CXC motif ligand 12) and its receptor CXCR 4 (CXC motif receptor 4) direct the recruitment of smooth muscle progenitor cells (SPCs) in neointima formation after vascular injury. Lysophosphatidic acid (LPA) induces CXCL12 and neointimal accumulation of smooth muscle cells (SMCs) in uninjured arteries. Thus, we hypothesize that LPA may regulate CXCL12-mediated vascular remodelling.

Objectives: We evaluated the role of LPA receptors in initiating CXCL12-dependent vascular repair by SPCs.

Methods and Results: Wire-induced carotid injury was performed in apolipoprotein E–/– mice on western-type diet. LPA receptor expression was studied by immunostaining and quantitative RT-PCR. LPA receptors LPA1 and LPA3 were detected in the media of uninjured arteries and in the injury-induced neointima. LPA3 mRNA was upregulated and LPA1 mRNA downregulated at one week after injury. The LPA1/3 antagonist Ki16425 inhibited neointima formation by 71% and reduced both relative neointimal SMCs and the macrophage content. Additionally, neointimal hypoxia-inducible factor-1 and CXCL12 expression, the injury-induced peripheral stem cell antigen-1 (Sca-1)+/Lin SPC mobilization, and the neointimal recruitment of Sca-1+SMCs were inhibited by Ki16425. In wild type mice, LPA20:4 increased CXCL12 and hypoxia-inducible factor-1 expression in carotid arteries as early as 1 day following short-term endoluminal incubation. LPA20:4-induced SPC mobilization and neointima formation were blocked by Ki16425, LPA1- and LPA3-specific small interfering (si)RNA, and the CXCR4 antagonist POL5551. Ki16425 reduced LPA20:4-mediated neointimal recruitment of SPC as demonstrated by 2-photon microscopy in bone marrow chimeric mice after repopulation with SM22-LacZ transgenic, hematopoietic cells. Moreover, POL5551 decreased the neointimal accumulation of CXCR4+ SMCs.

Conclusions: LPA1 and LPA3 promote neointima formation through activation of CXCL12-mediated mobilization and recruitment of SPCs.

  J Luan , J Yuan , X Li , S Jin , L Yu , M Liao , H Zhang , C Xu , Q He , B Wen , X Zhong , X Chen , H. L.Y Chan , J. J.Y Sung , B Zhou and C. Ding
 

Background: Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy.

Methods: A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results.

Results: The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls.

Conclusions: This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.

  A Lin , J Qian , X Li , X Yu , W Liu , Y Sun , N Chen , C Mei and for the Icodextrin National Multi center Cooperation Group
 

Background and objectives: While peritoneal dialysis with icodextrin is commonly used in patients with poor peritoneal membrane characteristics, the data on the usefulness of this solution in patients with lower transport characteristics are limited. The study was designed to compare icodextrin to glucose in Chinese prevalent peritoneal dialysis patients of different peritoneal transport characteristics (PET) categories.

Design, setting, participants, & measurements: This was a randomized, double-blind, perspective control study. Stable prevalent continuous ambulatory peritoneal dialysis (CAPD) patients were randomized to either 7.5% icodextrin (ICO) or 2.5% glucose (GLU) solution for 4 wk. Peritoneal membrane function was measured to define PET category in baseline. Creatinine clearance (Ccr), urea nitrogen clearance (CBUN), ultrafiltration (UF) during the long night dwell, dialysate, and metabolic biomarkers were measured at baseline, 2, and 4 wk. UF, Ccr, and CBUN were compared among different PET categories.

Results: A total of 201 CAPD patients were enrolled in the study. There were no baseline differences between the groups. Following 2 and 4 wk of therapy, Ccr, CBUN, and UF were all significantly higher in the ICO versus the GLU group. Additionally, switching to ICO resulted in a significant increase in UF in high, high-average, and low-average transporters as compared with baseline. The extent of increased UF was more obvious in higher transporters. Blood cholesterol level in the ICO group decreased significantly than that in the GLU group.

Conclusion: Compared with glucose-based solution, 7.5% icodextrin significantly improved UF and small solute clearance, even in patients with low-average peritoneal transport.

  E. C Lorenz , T. J Vrtiska , J. C Lieske , J. J Dillon , M. D Stegall , X Li , E. J Bergstralh and A. D. Rule
 

Background and objectives: Management of incidental renal artery and kidney abnormalities in patients undergoing computed tomography scans is a clinical challenge because their frequency in healthy subjects has not been precisely estimated. Therefore, the prevalence and management of these abnormalities were determined among a large cohort of potential kidney donors undergoing protocol evaluations.

Design, setting, participants, & measurements: All patients at the Mayo Clinic who underwent computed tomographic angiography and urography as part of their kidney donor evaluation between 2000 and 2008 were identified. Radiographic reports were abstracted for abnormalities of the renal arteries and kidneys. The prevalence of radiographic abnormalities was stratified by age and gender, and the effect on approval for kidney donation was determined.

Results: Among 1957 potential kidney donors, the mean ± SD age was 43 ± 12 years, and 58% were women. The most common abnormalities were kidney stones (11%), focal scarring (3.6%), fibromuscular dysplasia (2.8%), and other renal artery narrowing or atherosclerosis (5.3%). Fibromuscular dysplasia, focal scarring, parenchymal atrophy, and upper tract dilation were more common in women. Renal artery narrowing, focal scarring, and indeterminate masses increased with age. Overall, 25% of potential donors had at least one abnormality. However, these incidental radiographic abnormalities contributed to exclusion from donation in only 6.7% of potential donors.

Conclusions: Incidental radiographic abnormalities of the renal arteries and kidneys are common. The majority of imaging findings are not perceived to be harmful enough to prevent kidney donation, but future studies are needed to determine their clinical relevance.

  F. C Fervenza , R. S Abraham , S. B Erickson , M. V Irazabal , A Eirin , U Specks , P. H Nachman , E. J Bergstralh , N Leung , F. G Cosio , M. C Hogan , J. J Dillon , L. J Hickson , X Li , D. C Cattran and for the Mayo Nephrology Collaborative Group
 

Background and objectives: It was postulated that in patients with membranous nephropathy (MN), four weekly doses of Rituximab (RTX) would result in more effective B cell depletion, a higher remission rate, and maintaining the same safety profile compared with patients treated with RTX dosed at 1 g every 2 weeks. This hypothesis was supported by previous pharmacokinetic (PK) analysis showing that RTX levels in the two-dose regimen were 50% lower compared with nonproteinuric patients, which could potentially result in undertreatment.

Design, setting, participants, & measurements: Twenty patients with MN and proteinuria >5 g/24 h received RTX (375 mg/m2 x 4), with re-treatment at 6 months regardless of proteinuria response. PK analysis was conducted simultaneously with immunological analyses of T and B cells to ascertain the effect of RTX on lymphocyte subpopulations.

Results: Baseline proteinuria of 11.9 g/24 h decreased to 4.2 and 2.0 g/24 h at 12 and 24 months, respectively, whereas creatinine clearance increased from 72.4 ml/min per 1.73 m2 at baseline to 88.4 ml/min per 1.73 m2 at 24 months. Of 18 patients who completed 24-month follow-up, 4 are in complete remission, 12 are in partial remission, 1 has a limited response, and 1 patient relapsed. Serum RTX levels were similar to those obtained with two doses of RTX.

Conclusions: Four doses of RTX resulted in more effective B cell depletion, but proteinuria reduction was similar to RTX at 1 g every 2 weeks. Baseline quantification of lymphocyte subpopulations did not predict response to RTX therapy.

  X Li , Y He , C. H Ruiz , M Koenig and M. D. Cameron
 

Dasatinib was approved in 2006 for the treatment of imatinib-resistant chronic myelogenous leukemia and functions primarily through the inhibition of BCR-ABL and Src kinase. Dasatinib is extensively metabolized in humans by CYP3A4. In this study, we report that the bioactivation of dasatinib by CYP3A4 proceeds through a reactive intermediate that leads to CYP3A4 inactivation with KI = 6.3 µM and kinact = 0.034 min–1. The major mechanism of inactivation proceeds through hydroxylation at the para-position of the 2-chloro-6-methylphenyl ring followed by further oxidation, forming a reactive quinone-imine, similar to the reactive intermediates formed by acetaminophen and diclofenac. Formation of a reactive imine-methide was also detected but appears to be a minor pathway. When glutathione was added to human liver microsomal incubations, dasatinib-glutathione adducts were detected. Numerous dasatinib analogs were synthesized in an effort to understand what modifications would block the formation of reactive intermediates during dasatinib metabolism. It is interesting to note that blocking the site of hydroxylation with a methyl group was not effective because a reactive imine-methide was formed, nor was blocking the site with fluorine because the fluorine was removed through an oxidative defluorination mechanism and the reactive quinone-imine was still formed. Numerous analogs are presented that did effectively block the formation of glutathione adducts and prevent the inactivation of CYP3A4.

  X Li , Y He , C. H Ruiz , M Koenig and M. D. Cameron
 

Dasatinib was approved in 2006 for the treatment of imatinib-resistant chronic myelogenous leukemia and functions primarily through the inhibition of BCR-ABL and Src kinase. Dasatinib is extensively metabolized in humans by CYP3A4. In this study, we report that the bioactivation of dasatinib by CYP3A4 proceeds through a reactive intermediate that leads to CYP3A4 inactivation with KI = 6.3 µM and kinact = 0.034 min–1. The major mechanism of inactivation proceeds through hydroxylation at the para-position of the 2-chloro-6-methylphenyl ring followed by further oxidation, forming a reactive quinone-imine, similar to the reactive intermediates formed by acetaminophen and diclofenac. Formation of a reactive imine-methide was also detected but appears to be a minor pathway. When glutathione was added to human liver microsomal incubations, dasatinib-glutathione adducts were detected. Numerous dasatinib analogs were synthesized in an effort to understand what modifications would block the formation of reactive intermediates during dasatinib metabolism. It is interesting to note that blocking the site of hydroxylation with a methyl group was not effective because a reactive imine-methide was formed, nor was blocking the site with fluorine because the fluorine was removed through an oxidative defluorination mechanism and the reactive quinone-imine was still formed. Numerous analogs are presented that did effectively block the formation of glutathione adducts and prevent the inactivation of CYP3A4.

  X Li , J He , W Hu and Z. Yin
 

Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

  Y Yang , X Li , L Cui , M Fu , A. B Rabie and D. Zhang
 

Mechanical stress induces human periodontal ligament (PDL) cells to express an osteoblastic phenotype in vitro. Core binding factor a1 (CBFA1) is a key regulator of osteoblast differentiation. This study was designed to investigate the role of CBFA1 in alveolar bone remodelling, specifically the expression of CBFA1 messenger RNA (mRNA) in human PDL cells under mechanical stress and its up- and downstream relationships with other bone remodelling markers. Cultured human PDL cells were exposed to mechanical stress. The expressions of CBFA1 and alkaline phosphatase (ALP), osteopontin (OPN), osteoprotegrin (OPG), and receptor activator nuclear factor kappa B ligand (RANKL) were detected before and after RNA interference (RNAi) of CBFA1. The data were analysed using a t-test and one-way analysis of variance.

After mechanical stress loading, CBFA1 mRNA expression was raised initially, followed by an increased expression of ALP and RANKL, decreased expression of OPG, and a change in OPN expression. After CBFA1 knock-down in human PDL cells by small hairpin (sh) RNA, the expression of ALP, OPN, OPG, and RANKL also changed. These findings suggest that in the present model system CBFA1 may play an important role in PDL-mediated bone remodelling in response to mechanical stimulation. Mechanical stress: CBFA1–ALP and OPG–PDL homeostasis may be one of the signal transduction pathways of human PDL cell differentiation under mechanical stress without exclusion of the involvement of other pathways.

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

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

  Y Zhang , X Li , J Qi , J Wang , X Liu , H Zhang , S. C Lin and A. Meng
 

The Rho-associated serine/threonine kinases Rock1 and Rock2 play important roles in cell contraction, adhesion, migration, proliferation and apoptosis. Here we report that Rock2 acts as a negative regulator of the TGFβ signaling pathway. Mechanistically, Rock2 binds to and accelerates the lysosomal degradation of TGFβ type I receptors internalized from the cell surface in mammalian cells. The inhibitory effect of Rock2 on TGFβ signaling requires its kinase activity. In zebrafish embryos, injection of rock2a mRNA attenuates the expression of mesodermal markers during late blastulation and blocks the induction of mesoderm by ectopic Nodal signals. By contrast, overexpression of a dominant negative form of zebrafish rock2a, dnrock2a, has an opposite effect on mesoderm induction, suggesting that Rock2 proteins are endogenous inhibitors for mesoderm induction. Thus, our data have unraveled previously unidentified functions of Rock2, in controlling TGFβ signaling as well as in regulating embryonic patterning.

  X Fang , X Li , B Stanton , Y Hong , L Zhang , G Zhao , J Zhao , X Lin and D. Lin
 

Objective To assess the relationship between parental HIV/AIDS and psychosocial adjustment of children in rural central China. Methods Participants included 296 double AIDS orphans (children who had lost both their parents to AIDS), 459 single orphans (children who had lost one parent to AIDS), 466 vulnerable children who lived with HIV-infected parents, and 404 comparison children who did not experience HIV/AIDS-related illness and death in their families. The measures included depressive symptoms, loneliness, self-esteem, future expectations, hopefulness about the future, and perceived control over the future. Results AIDS orphans and vulnerable children consistently demonstrated poorer psychosocial adjustment than comparison children in the same community. The level of psychosocial adjustment was similar between single orphans and double orphans, but differed by care arrangement among double orphans. Conclusion The findings underscore the urgency and importance of culturally and developmentally appropriate intervention efforts targeting psychosocial problems among children affected by AIDS and call for more exploration of risk and resilience factors, both individual and contextual, affecting the psychosocial wellbeing of these children.

  S Lu , Y. M Xie , X Li , J Luo , X. Q Shi , X Hong , Y. H Pan and X. Ma
 

TH2B, an important testis histone, plays a key role in remodeling chromatin structure during spermatogenesis. We present a detailed study of post-translational modifications (PTMs) of histone TH2B from different developmental stages of sperm cells, using a combination of high performance liquid chromatography, enzymatic Glu-c digestions of peptides, liquid chromatography–mass spectrometry (LC–MS) and LC–MS/MS analysis. The results showed modification patterns of the intact histone TH2B during spermatogenesis. Acetylated TH2B was most abundant in spermatogonia (28.9%) when compared with the spermatocytes (8.3%) and round spermatids (11.2%). Several new PTMs of TH2B were identified. In spermatogonia, spermatocytes and round spermatids, T116 and K117, were modified by phosphorylation and methylation, respectively, forming a novel ‘phospho switch’ site. The identified modification patterns of histone TH2B in spermatogenic cells provides a basis for future studies on histone coding and epigenetic regulation during spermatogenesis.

  M Jiang , Y Ma , C Chen , X Fu , S Yang , X Li , G Yu , Y Mao , Y Xie and Y. Li
 

Androgen signaling plays an important role in many biological processes. Androgen Responsive Gene Database (ARGDB) is devoted to providing integrated knowledge on androgen-controlled genes. Gene records were collected on the basis of PubMed literature collections. More than 6000 abstracts and 950 original publications were manually screened, leading to 1785 human genes, 993 mouse genes, and 583 rat genes finally included in the database. All the collected genes were experimentally proved to be regulated by androgen at the expression level or to contain androgen-responsive regions. For each gene important details of the androgen regulation experiments were collected from references, such as expression change, androgen-responsive sequence, response time, tissue/cell type, experimental method, ligand identity, and androgen amount, which will facilitate further evaluation by researchers. Furthermore, the database was integrated with multiple annotation resources, including National Center for Biotechnology Information, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway, to reveal the biological characteristics and significance of androgen-regulated genes. The ARGDB web site is mainly composed of the Browse, Search, Element Scan, and Submission modules. It is user friendly and freely accessible at http://argdb.fudan.edu.cn. Preliminary analysis of the collected data was performed. Many disease pathways, such as prostate carcinogenesis, were found to be enriched in androgen-regulated genes. The discovered androgen-response motifs were similar to those in previous reports. The analysis results are displayed in the web site. In conclusion, ARGDB provides a unified gateway to storage, retrieval, and update of information on androgen-regulated genes.

  X. j Cai , L Chen , L Li , M Feng , X Li , K Zhang , Y. y Rong , X. b Hu , M. x Zhang , Y Zhang and M. Zhang
 

Adiponectin is an important antiatherogenic adipocytokine that inhibits inflammation, insulin resistance, and oxide stress. Inflammation in the vascular adventitia is a crucial factor in the pathogenesis of atherosclerosis. Adventitial fibroblasts (AFs) can proliferate, divide into myofibroblasts, and migrate to the intima to become a new component of atherosclerotic plaque under inflammation and atherosclerosis. We investigated whether adiponectin might prevent AFs from proliferating, migrating, and transforming into myofibroblasts. Cultured AFs were stimulated with lipopolysaccharide (LPS) in the presence or absence of adiponectin. Methyl thiazolyl tetrazolium assay and migration and scratch-wound assays demonstrated that adiponectin reduced the AF proliferation and migration induced by LPS, respectively, whereas treatment with AdipoR1 small interfering (si) RNA (siAdipoR1), AMP-activated protein kinase (AMPK) siRNA (siAMPK), and an AMPK inhibitor reversed the effect. Immunocytochemistry and Western blot revealed that adiponectin reduced the transition of AFs to myofibroblasts, and treatment with siAdipoR1, siAMPK, and the AMPK inhibitor increased the transition. RT-PCR, Western blotting, and nitric oxide (NO) assay showed that adiponectin reduces induced NO synthase (iNOS) and nitrotyrosine expression and NO and ONOO production induced by LPS. Treatment with siAdipoR1, siAMPK, and the AMPK inhibitor significantly attenuated adiponectin-induced phosphorylation of AMPK and its downstream target acetyl-coenzyme A carboxylase and up-regulated iNOS mRNA and protein expression, which resulted in a marked increase of NO and ONOO production. In apolipoprotein E-deficient mice, immunohistochemistry of treated vascular adventitia showed that both iNOS expression and ONOO production could be reversed with an adenovirus-adiponectin vector. Taken together, these results suggest that adiponectin reduces LPS-induced NO production and nitrosative stress and prevents AFs from proliferating, transforming to myoflbroblasts, and migrating to the intima, thus worsening atherosclerosis, by inhibiting the AdipoR1-AMPK-iNOS pathway in AFs.

  G Yao , M Yin , J Lian , H Tian , L Liu , X Li and F. Sun
 

Many members of the TGF-β superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-β1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-β-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-β1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFβ superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-β1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-β1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-β1. In addition, both miR-224 and TGF-β1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-β1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.

  X Li , J Bergelson and C. Chapple
 

Sinapoylmalate is the major sinapate ester found in leaves of Arabidopsis thaliana, where it plays an important role in UV-B protection. Metabolic profiling of rosette leaves from 96 Arabidopsis accessions revealed that the Pna-10 accession accumulates sinapoylglucose instead of sinapoylmalate. This unique leaf sinapate ester profile is similar to that of the previously characterized sinapoylglucose accumulator1 (sng1) mutants. SNG1 encodes sinapoylglucose:malate sinapoyltransferase (SMT), a serine carboxypeptidase-like (SCPL) enzyme that catalyzes the conversion of sinapoylglucose to sinapoylmalate. In the reference Columbia genome, the SNG1 gene is located in a cluster of five SCPL genes on Chromosome II. PCR and sequencing analysis of the same genomic region in the Pna-10 accession revealed a 13-kb deletion that eliminates the SNG1 gene (At2g22990) and the gene encoding sinapoylglucose:anthocyanin sinapoyltransferase (SAT) (At2g23000). In addition to its sinapoylmalate-deficient phenotype, and consistent with the loss of SAT, Pna-10 is unable to accumulate sinapoylated anthocyanins. Interestingly, the Pna-17 accession, collected from the same location as Pna-10, has no such deletion. Further analysis of 135 lines collected from the same location as Pna-10 and Pna-17 revealed that four more lines contain the deletion found in Pna-10 accession, suggesting that either the deletion found in Pna-10 is a recent event that has not yet been eliminated through selection or that sinapoylmalate is dispensable for the growth of Arabidopsis under field conditions.

  H Kimura , X Li , K Torii , T Okada , K Kamiyama , D Mikami , N Takahashi and H. Yoshida
 

Background. Long-term treatment with glucocorticoids (GCs) reportedly exaggerates renal fibrosis in chronic progressive inflammatory kidney disease. GCs induce the gene expression of plasminogen activator inhibitor-1 (PAI-1), a fibrosis enhancer in non-renal cells. Tumour necrosis factor-alpha (TNF-) reduces the gene expression of 11β-hydroxysteroid dehydrogenase (HSD) 2, an inactivator of GCs, and may enhance GC activity. However, the individual and collective effects of adrenal steroids, TNF- and HSD2 status on PAI-1 production are unknown in human proximal renal tubular epithelial cells (HPTECs).

Methods. Confluent HPTECs were treated with adrenal steroids (10 nM to 10 µM) or TNF- (10 ng/ml) for up to 48 h. The mRNA amounts of the target genes were determined by TaqMan quantitative PCR, and the PAI-1 protein amounts were measured by an immunoassay.

Results. Dexamethasone (DXA) maximally increased the amounts of PAI-1 mRNA and protein at 100 nM. Aldosterone (Ald) increased PAI-1 expression dose dependently, but the effect was over 100-fold weaker than that of DXA. The PAI-1-increasing effects of DXA and Ald were abolished completely by U-486, a specific inhibitor of the glucocorticoid receptor (GR) but not by spironolactone, a specific inhibitor of the mineralocorticoid receptor. The effect of DXA was also blocked partially by AG1478 and herbimycin A, tyrosine kinase inhibitors. DXA further increased TNF--stimulated PAI-1 expression via the GR. Although TNF- treatment caused an 80% reduction in the gene expression of HSD2, an inactivator of GCs, HSD2 inhibition did not enhance DXA-induced PAI-1 production.

Conclusions. DXA induces basal and TNF--stimulated PAI-1 expression via the GR pathway, regardless of HSD2 status in HPTECs. Excess GCs may serve as a pro-fibrotic factor in chronic inflammatory kidney diseases.

  M Yuan , Z Chu , X Li , C Xu and S. Wang
 

The fully recessive disease resistance (R) gene xa13, which mediates race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), encodes a plasma membrane protein that differs by one amino acid from that encoded by its dominant (susceptible) allele Xa13. The molecular mechanism of xa13-mediated resistance is largely unknown. Here we show that, compared with its dominant allele, expressional non-reaction of xa13 to Xoo infection, not its protein composition, is the key factor for xa13-mediated resistance. We used the promoter (PXa13) of the dominant Xa13, which was induced by only the incompatible Xoo strain for xa13, to regulate xa13 and xa13Leu49 (a natural recessive allele of xa13) in the rice line IRBB13 carrying xa13. The transgenic plants showed the same level of susceptibility and bacterial growth rate as those of the rice line carrying dominant Xa13, accompanied by the induced accumulation of xa13 or xa13Leu49 proteins. Constitutive expression of dominant XA13 or different xa13 proteins (xa13, xa13Leu49, xa13Ala85 or xa13Val184) in IRBB13 had no effect on Xoo infection in the transgenic plants. These results suggest that race-specific pathogen-induced Xa13 expression is critical for infection. Thus, xa13 stands out from other R genes in that its functions in disease resistance are due to only the loss of pathogen-induced transcriptional motivation caused by natural selection.

  J Jia , C Wang , Z Shi , J Zhao , Y Jia , Z Zhao Hui , X Li , Z Chen and P. Zhu
 

Objective. To explore the therapeutic potential of CD147/HAb18 mAb in the treatment of RA in severe combined immunodeficiency (SCID) mice engrafted with human cartilage and rheumatoid synovium tissue (SCID-HuRAg).

Methods. SCID-HuRAg mice were treated separately with CD147/HAb18 mAb, anti-TNF- mAb or a combination of both. The mice in control group were treated with anti-Japanese encephalitis virus mAb. The volume of engrafts was measured and the number of inflammatory cells and cartilage erosion score were examined. Expression of MMP-2, -3 and -9 was determined by immunohistochemistry. Human inflammatory cytokine levels in mouse sera were assessed using cytometric bead array kit.

Results. The volume of engrafts decreased significantly in SCID-HuRAg mice treated separately with anti-CD147 mAb or anti-TNF- mAb, and in the mice treated with anti-CD147 mAb plus anti-TNF- mAb (P < 0.05). Significant reduction was observed in cartilage erosion score in anti-CD147 treatment group and combined treatment group (P < 0.05). Immunohistochemical analysis showed that expression of MMP-2, -3 and -9 was lower in the anti-CD147 treatment group and combined treatment group than in the control mAb group (P < 0.05). Moreover, the level of TNF-, IL-6 and -8 in CD147 mAb group showed a significant decrease compared with that of the control mAb group (P < 0.05).

Conclusions. CD147/HAb18 mAb can reduce cartilage erosion and synovitis by inhibition of the MMPs and reduction of inflammatory cytokines in SCID-HuRAg mice, which suggests that CD147/HAb18 mAb is a promising treatment option for RA patients.

  F Cao , X Li , S Hiew , H Brady , Y Liu and Y. Dou
 

Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are ~24 nucleotides in length, Dicer-independent, and 2'-O-methylated at the 3' terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll(–/–)) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2(–/–)), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs.

  L Lindahl , A Bommankanti , X Li , L Hayden , A Jones , M Khan , T Oni and J. M. Zengel
 

RNase MRP is a nucleolar RNA–protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

  A. E Abbot , X Li and B. D. Busconi
  Background:

Rotator cuff tears commonly occur in combination with other shoulder injuries such as superior labral anterior posterior (SLAP) lesions. The incidence of these associated lesions increases with age; however, the management of concomitant SLAP and rotator cuff tears has yet to be convincingly addressed in the literature.

Hypothesis:

Patients over the age of 45 years who have concomitant arthroscopic rotator cuff repair and debridement of their type II SLAP lesions will have improved patient satisfaction and functional outcome compared with those who undergo simultaneous rotator cuff and type II SLAP repair.

Study Design:

Cohort study; Level of evidence, 2.

Methods:

We recruited 48 patients (mean age, 51.9 years; range, 45–60 years) who had concomitant rotator cuff and type II SLAP tears. All underwent arthroscopic rotator cuff repair with subacromial decompression. Patients were randomized intraoperatively into 2 groups: repair versus debridement of their type II SLAP lesions. Ten patients were lost at final follow-up (4 in debridement and 6 in repair group). The outcome was assessed by the Tegner score and University of California at Los Angeles (UCLA) score and clinically to evaluate range of motion (forward elevation/internal rotation/external rotation).

Results:

At 2 years postoperatively, both the debridement and repair groups showed significant improvement in Tegner score, UCLA score, and range of motion. Patients who underwent rotator cuff repair in combination with debridement of their SLAP tears had significantly better overall UCLA scores (34 vs 31; P < .001) and improved function (5.5 vs 3.8; P < .005) and pain relief (9.6 vs 7.7; P < .001) compared with those who underwent simultaneous rotator cuff and SLAP repair. Range of motion in both internal and external rotation was also significantly better in those patients who had SLAP debridement as compared to SLAP repair.

Conclusion:

In patients over the age of 45 years with a minimally retracted rotator cuff tear and associated SLAP lesion, arthroscopic repair of the rotator cuff with combined debridement of the type II SLAP lesion may provide greater patient satisfaction and functional outcome in terms of pain relief and motion.

  A. C Luke , C Stehling , R Stahl , X Li , T Kay , S Takamoto , B Ma , S Majumdar and T. Link
 

Background: There is continuing controversy whether long-distance running results in irreversible articular cartilage damage. New quantitative magnetic resonance imaging (MRI) techniques used at 3.0 T have been developed including T1rho (T1) and T2 relaxation time measurements that detect early cartilage proteoglycan and collagen breakdown.

Hypothesis: Marathon runners will demonstrate T1 and T2 changes in articular cartilage on MRI after a marathon, which are not seen in nonrunners. These changes are reversible.

Study Design: Cohort study; Level of evidence, 2.

Methods: Ten asymptomatic marathon runners had 3-T knee MRI scans 2 weeks before, within 48 hours after, and 10 to 12 weeks after running a marathon. The T1 and T2 MRI sequences in runners were compared with those of 10 age- and gender-matched controls who had MRI performed at baseline and 10 to 12 weeks.

Results: Runners did not demonstrate any gross morphologic MRI changes after running a marathon. Postmarathon studies, however, revealed significantly higher T2 and T1 values in all articular cartilage areas of the knee (P < .01) except the lateral compartment. The T2 values recovered to baseline except in the medial femoral condyle after 3 months. Average T1 values increased after the marathon from 37.0 to 38.9 (P < .001) and remained increased at 3 months.

Conclusion: Runners showed elevated T1 and T2 values after a marathon, suggesting biochemical changes in articular cartilage, T1 values remain elevated after 3 months of reduced activity. The patellofemoral joint and medial compartment of the knee show the highest signal changes, suggesting they are at higher risk for degeneration.

  B Huang , H Wu , N Hao , F Blombach , J van der Oost , X Li , X. C Zhang and Z. Rao
 

GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment.

  X Li , G. S Baillie and M. D. Houslay
 

β-Arrestin plays a key role in regulating β2-adrenoreceptor signaling by interdicting activation of adenylyl cyclase and selectively sequestering cAMP phosphodiesterase-4D5 (PDE4D5) for delivery of an active cAMP degrading system to the site of cAMP synthesis. Here we show that the β-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing β2-adrenoceptors. Reconstitution analyses in β-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a β-arrestin-scaffolded pool of the E3-ubiquitin ligase, Mdm2, mediates PDE4D5 ubiquitination. Critical for this is the ubiquitin-interacting motif located in the extreme C terminus of PDE4D5, which is specific to the PDE4D sub-family. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquitination occurs at Lys-48, Lys-53, and Lys-78, which are located within its isoform-specific N-terminal region, as well as at Lys-140 located within its regulatory UCR1 module. We suggest that mono-ubiquitination at Lys-140 primes PDE4D5 for a subsequent cascade of polyubiquitination occurring within its isoform-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated β-arrestin sub-population that is likely to be protected from Mdm2-mediated ubiquitination due to steric hindrance caused by sequestered PDE4D5. Ubiquitination of PDE4D5 elicits an increase in the fraction of PDE4D5 sequestered by β-arrestin in cells, thereby contributing to the fidelity of PDE4D5-β-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.

  K. E Szulwach , X Li , R. D Smrt , Y Li , Y Luo , L Lin , N. J Santistevan , W Li , X Zhao and P. Jin
 

The microRNA miR-137 represses expression of Ezh2, a histone methyltransferase, which in turn alters the epigenetic architecture of chromatin that is important for regulation of miR-137 levels.

  S Zhu , W Pan , P Shi , H Gao , F Zhao , X Song , Y Liu , L Zhao , X Li , Y Shi and Y. Qian
 

Interleukin 17 (IL-17) plays critical roles in the pathogenesis of various autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE). How the signals triggered by this powerful inflammatory cytokine are controlled to avoid abnormal inflammatory responses is not well understood. In this study, we report that TRAF3 is a receptor proximal negative regulator of IL-17 receptor (IL-17R) signaling. TRAF3 greatly suppressed IL-17–induced NF-B and mitogen-activated protein kinase activation and subsequent production of inflammatory cytokines and chemokines. Mechanistically, the binding of TRAF3 to IL-17R interfered with the formation of the receptor signaling activation complex IL-17R–Act1–TRAF6, resulting in suppression of downstream signaling. TRAF3 markedly inhibited IL-17–induced expression of inflammatory cytokine and chemokine genes in vivo and consequently delayed the onset and greatly reduced the incidence and severity of EAE. Thus, TRAF3 is a negative regulator of IL-17R proximal signaling.

 
 
 
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