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Articles by Y Li
Total Records ( 60 ) for Y Li
  C Sun , M Xu , Z Xing , Z Wu , Y Li , T Li and M. Zhao
 

Lissencephaly is a severe disease characterized by brain malformation. The main causative gene of lissencephaly is LIS1. Mutation or deletion of LIS1 leads to proliferation and migration deficiency of neurons in brain development. However, little is known about its biological function in embryonic development. In this article, we identified the expression patterns of zebrafish LIS1 gene and investigated its function in embryonic development. We demonstrated that zebrafish consisted of two LIS1 genes, LIS1a and LIS1b. Bioinformatics analysis revealed that LIS1 genes were conserved in evolution both in protein sequences and genomic structures. The expression patterns of zebrafish LIS1a and LIS1b showed that both transcripts were ubiquitously expressed at all embryonic developmental stages and in adult tissues examined. At the protein level, the LIS1 products mainly exist in brain tissue and in embryos at early stages as shown by western blotting analysis. The whole-mount immunostaining data showed that LIS1 proteins were distributed all over the embryos from 1-cell stage to 5 day post-fertilization. Knockdown of LIS1 protein expression through morpholino antisense oligonucleotides resulted in many developmental deficiencies in zebrafish, including brain malformation, circulation abnormality, and body curl. Taken together, our study suggested that zebrafish LIS1 plays a very important role in embryonic development.

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

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

  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.

  J Du , Y Li and X. Zhu
 

CENP-F (also named mitosin) is a multifunctional protein of 350 kDa. In interphase, it is a nuclear protein, whereas in M phase it localizes to the kinetochore, the major microtubule-binding structure on chromosomes essential for chromosome segregation. CENP-F is also critical for myocyte differentiation through the interaction with Rb. It binds to ATF4 and negatively regulates the transcriptional activity of ATF4. It is also important for mitotic progression. Here we show that depletion of CENP-F by RNAi markedly downregulated the methylation of histone H3 at K4 and K9. Consistently, association of HP1 with mitotic chromosomes was largely decreased. These results uncover a novel role of CENP-F in regulation of epigenetic modification on histone H3.

  F Guo , Y Li , Y Liu , J Wang and G. Li
 

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is suggested to be a long (~7 kb) non-coding RNA. MALAT1 is overexpressed in many human carcinomas, but its function remains unknown. To investigate the role of MALAT1 in human cervical cancer progression, we designed and used short hairpin RNA to inhibit MALAT1 expression in CaSki cells and validated its effect on cell proliferation and invasion. Changes in gene expression were analyzed by reverse transcriptase–polymerase chain reaction. Our data demonstrated that MALAT1 was involved in cervical cancer cell growth, cell cycle progression, and invasion through the regulation of gene expression, such as caspase-3, -8, Bax, Bcl-2, and Bcl-xL, suggesting that MALAT1 could have important implications in cervical cancer biology. Our findings illustrate the biological significance of MALAT1 in cervical cancer progression and provide novel evidence that MALAT1 may serve as a therapeutic target in the prevention of human cervical cancer.

  J Du , Y Zhang , Y Liu , Y Li and X. Zhu
 

Cenp-F (also named mitosin) is a 350-kDa human kinetochore protein important for the mitotic progression. It is also a nuclear matrix protein in interphase cells. Here, we showed that overexpression of N-terminal deletion mutants of Cenp-F containing the C-terminal 112 residues induced chromatin condensation into numerous aggregates of varying sizes in interphase nucleus, colocalizing with the exogenous proteins. In situ hybridization using whole chromosome painting probes indicated that the chromatin aggregates were not prematurely condensed individual chromosomes. Neither were they due to apoptosis. We provided evidence showing association of Cenp-F with certain regions of interphase chromatin fibers. Cenp-F associated with the DNA-dependent protein kinase (DNA-PK), a trimeric protein complex critical for genome homeostasis. Moreover, the DNA-PK association activity of Cenp-F mutants correlated with their ability to induce chromatin aggregation. These results imply a role of Cenp-F in organization of interphase chromatin through association and possibly regulation of DNA-PK.

  H Iwakura , H Ariyasu , Y Li , N Kanamoto , M Bando , G Yamada , H Hosoda , K Hosoda , A Shimatsu , K Nakao , K Kangawa and T. Akamizu
 

Ghrelin is a stomach-derived peptide that has growth hormone-stimulating and orexigenic activities. Although there have been several reports of ghrelinoma cases, only a few cases have elevated circulating ghrelin levels, hampering the investigation of pathophysiological features of ghrelinoma and chronic effects of ghrelin excess. Furthermore, standard transgenic technique has resulted in desacyl ghrelin production only because of the limited tissue expression of ghrelin O-acyltransferase, which mediates acylation of ghrelin. Accordingly, we attempted to create ghrelin promoter SV40 T-antigen transgenic (GP-Tag Tg) mice, in which ghrelin-producing cells continued to proliferate and finally developed into ghrelinoma. Adult GP-Tag Tg mice showed elevated plasma ghrelin levels with preserved physiological regulation. Adult GP-Tag Tg mice with increased plasma ghrelin levels exhibited elevated IGF-I levels despite poor nutrition. Although basal growth hormone levels were not changed, those after growth hormone-releasing hormone injection tended to be higher. These results indicate that chronic elevation of ghrelin activates GH-IGF-I axis. In addition, GP-Tag Tg mice demonstrated glucose intolerance. Insulin secretion by glucose tolerance tests was significantly attenuated in GP-Tag Tg, whereas insulin sensitivity determined by insulin tolerance tests was preserved, indicating that chronic elevation of ghrelin suppresses insulin secretion and leads to glucose intorelance. Thus, we successfully generated a Tg model of ghrelinoma, which is a good tool to investigate chronic effects of ghrelin excess. Moreover, their characteristic features could be a hint on ghrelinoma.

  J Xu , S Stanislaus , N Chinookoswong , Y. Y Lau , T Hager , J Patel , H Ge , J Weiszmann , S. C Lu , M Graham , J Busby , R Hecht , Y. S Li , Y Li , R Lindberg and M. M. Veniant
 

Recombinant fibroblast growth factor (FGF)21 has antihyperglycemic, antihyperlipidemic, and antiobesity effects in diabetic rodent and monkey models. Previous studies were confined to measuring steady-state effects of FGF21 following subchronic or chronic administration. The present study focuses on the kinetics of biological actions of FGF21 following a single injection and on the associated physiological and cellular mechanisms underlying FGF21 actions. We show that FGF21 resulted in rapid decline of blood glucose levels and immediate improvement of glucose tolerance and insulin sensitivity in two animal models of insulin resistance (ob/ob and DIO mice). In ob/ob mice, FGF21 led to a 40–60% decrease in blood glucose, insulin, and amylin levels within 1 h after injection, and the maximal effects were sustained for more than 6 h despite the 1- to 2-h half-life of FGF21. In DIO mice, FGF21 reduced fasting blood glucose and insulin levels and improved glucose tolerance and insulin sensitivity within 3 h of treatment. The acute improvement of glucose metabolism was associated with a 30% reduction of hepatic glucose production and an increase in peripheral glucose turnover. FGF21 appeared to have no direct effect on ex vivo pancreatic islet insulin or glucagon secretion. However, it rapidly induced typical FGF signaling in liver and adipose tissues and in several hepatoma-derived cell lines and differentiated adipocytes. FGF21 was able to inhibit glucose release from H4IIE hepatoma cells and stimulate glucose uptake in 3T3-L1 adipocytes. We conclude that the acute glucose-lowering and insulin-sensitizing effects of FGF21 are potentially associated with its metabolic actions in liver and adipose tissues.

  Y Li , T. P. J Solomon , J. M Haus , G. M Saidel , M. E Cabrera and J. P. Kirwan
 

Identifying the mechanisms by which insulin regulates glucose metabolism in skeletal muscle is critical to understanding the etiology of insulin resistance and type 2 diabetes. Our knowledge of these mechanisms is limited by the difficulty of obtaining in vivo intracellular data. To quantitatively distinguish significant transport and metabolic mechanisms from limited experimental data, we developed a physiologically based, multiscale mathematical model of cellular metabolic dynamics in skeletal muscle. The model describes mass transport and metabolic processes including distinctive processes of the cytosol and mitochondria. The model simulated skeletal muscle metabolic responses to insulin corresponding to human hyperinsulinemic-euglycemic clamp studies. Insulin-mediated rate of glucose disposal was the primary model input. For model validation, simulations were compared with experimental data: intracellular metabolite concentrations and patterns of glucose disposal. Model variations were simulated to investigate three alternative mechanisms to explain insulin enhancements: Model 1 (M.1), simple mass action; M.2, insulin-mediated activation of key metabolic enzymes (i.e., hexokinase, glycogen synthase, pyruvate dehydrogenase); or M.3, parallel activation by a phenomenological insulin-mediated intracellular signal that modifies reaction rate coefficients. These simulations indicated that models M.1 and M.2 were not sufficient to explain the experimentally measured metabolic responses. However, by application of mechanism M.3, the model predicts metabolite concentration changes and glucose partitioning patterns consistent with experimental data. The reaction rate fluxes quantified by this detailed model of insulin/glucose metabolism provide information that can be used to evaluate the development of type 2 diabetes.

  M Arejian , Y Li and M. B. Anand Srivastava
 

We have earlier shown that the treatment of A10 vascular smooth muscle cells with S-nitroso-N-acetyl-penicillamine (SNAP); nitric oxide donor (NO) for 24 h decreased the expression of natriuretic peptide receptor C (NPR-C) and adenylyl cyclase signaling. The present study was undertaken to examine the implication of different signaling mechanisms in a NO-induced response. The treatment of A10 vascular smooth muscle cells with SNAP decreased the expression of NPR-C and Gi proteins in a time-dependent manner. The expression of Gi proteins was decreased at 6 h, whereas the expression of NPR-C was attenuated at 2 h. The NPR-C-mediated inhibition of adenylyl cyclase was attenuated (~50%) after 2 h of treatment and was completely abolished after 6 h of treatment. The decreased expression of NPR-C and NPR-C-mediated attenuation of adenylyl cyclase after 2 h of treatment was reversed to control levels by PD-98059, a MEK inhibitor. SNAP also modulated the ERK1/2 phosphorylation in a time-dependent manner; an increase was observed up to 2 h, and, thereafter, the ERK1/2 phosphorylation was decreased. On the other hand, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823 inhibitor of soluble guanylyl cyclase and protein kinase G, respectively, and Mn(III)tetrakis(4-benzoic acid)porphyrin, a scavenger of peroxynitrite, were unable to restore the SNAP-induced decreased expression of NPR-C protein and increased ERK1/2 phosphorylation to control levels. However, the decreased levels of phosphorylated ERK1/2 and Gi proteins were restored to control levels by 8-bromo-cAMP. These results indicate that a temporal relationship follows between a NO-induced decreased expression of NPR-C and Gi proteins. The decreased expression of NPR-C is mediated through cGMP-independent but MAPK-dependent pathway, whereas NO-induced decreased levels of cAMP may contribute to the decreased activation of MAPK and thereby decreased the expression of Gi proteins.

  A Olschewski , R Schnoebel Ehehalt , Y Li , B Tang , M. E Brau and M. Wolff
 

BACKGROUND: Spinal sensitization and facilitatory processes in dorsal horn neurons after nerve injury alter spinal outflow leading to enhanced pain perception and chronic pain syndromes. Clinically used Na+ channel blockers at doses which do not block conduction can relieve such chronic pain. Although much attention has been paid to their effect upon afferents, less work has been done with their effect on the excitability of central sensory neurons. Thus, we investigated the effects of the Na+ channel blockers mexiletine and lidocaine on sensory spinal dorsal horn neurons.

METHODS: Patch-clamp recordings were directly performed in visualized neurons of the substantia gelatinosa in the spinal cord of young rats to investigate the effect of mexiletine and lidocaine in different types of dorsal horn neurons (tonically firing, adapting-firing, and single spike neurons).

RESULTS: All three different types of neurons responded dose-dependently to mexiletine and lidocaine. Both local anesthetics reversibly inhibited Na+ and K+ currents. The half-maximal inhibitory concentration for Na+ conductance block was 89 ± 2 or 54 ± 6 µM and for delayed-rectifier K+ conductance block was 582 ± 36 or 398 ± 14 µM for lidocaine and mexiletine, respectively. The inhibition of Na+ and K+ currents consecutively altered the properties of single action potentials and reduced the firing rate of tonically firing and adapting-firing neurons.

CONCLUSIONS: In clinically relevant concentrations, lidocaine and mexiletine reduced the excitability of sensory dorsal horn neurons via a blockade of Na+ and K+ channels. Our work confirms that, in addition to the peripheral effects of lidocaine and mexiletine, modulation of voltage-gated ion channels in the central nervous system contributes to the antinociceptive effects of these drugs used in pain therapy.

  J. P Metlay , E Lautenbach , Y Li , J Shults and P. H. Edelstein
 

Background  The introduction of a pneumococcal conjugate vaccine has been associated with a shift in the serotypes responsible for bacteremic pneumococcal disease. We examined recent trends in serotypes responsible for disease and current risk factors among adults.

Methods  Data were obtained from 48 acute care hospitals in the 5-county region surrounding Philadelphia, Pennsylvania, from October 1, 2002, through September 30, 2008, on all hospitalized adult patients with community-acquired bacteremic pneumococcal disease. Isolates were serotyped and patient characteristics were compared with data from a household survey of the adult population in the region.

Results  During the study period, the annual rate of disease due to vaccine serotypes declined by 29% per year, but the rate of disease due to nonvaccine serotypes increased 13% per year, yielding an overall 7% increase in the annual rate of disease among adults. Advanced age was a risk factor for infection with nonvaccine serotypes compared with vaccine serotypes. Comparing all patients with the source population, African Americans were at increased risk of infection, and the presence of additional children in the home was associated with decreased risk of disease. Smoking, advanced age, and diabetes mellitus remained important risk factors in adults.

Conclusions  New serotypes are replacing the serotypes covered in the conjugate vaccine. While some risk factors for pneumococcal disease remain unchanged, the observation that exposure to children in the home is associated with lower risk of disease suggests that the changing epidemiology of pneumococcal disease may be altering the dominant modes of transmission in the community.

  L. A Goguen , C. I Chapuy , Y Li , S. D Zhao and D. J. Annino
 

Objectives  To determine the incidence of postchemoradiotherapy (post-CRT) neck dissection (ND) complications; to ascertain whether timing (<12 vs ≥12 weeks) from CRT to ND or other factors are associated with increased complications; and to determine whether ND timing influences disease control or survival.

Design  Ten-year retrospective analysis.

Setting  Tertiary care center.

Patients  One hundred five patients with head and neck cancer undergoing ND after CRT.

Main Outcome Measures  Complications and survival variables compared between groups undergoing ND less than 12 weeks (less-than-12-weeks ND group) and 12 weeks or more (12-weeks-or-more ND group) after CRT.

Results  Sixty-seven NDs were performed less than 12 weeks and 38 were performed 12 weeks or more after CRT. Patient characteristics, treatment, and ND pathology results were comparable between the 2 ND groups. The incidence of complications between the less-than-12-weeks and the 12-weeks-or-more ND groups included major wound complications in 8 of 67 (11.9%) vs 1 of 38 (2.6%; P = .15), minor wound complications in 11 of 67 (16.4%) vs 4 of 38 (10.5%; P = .56), airway complications in 7 of 67 (10.4%) vs 2 of 38 (5.3%; P = .48), and systemic complications in 9 of 67 (13.4%) vs 2 of 38 (5.3%; P = .32). The number of patients with at least 1 complication was significantly smaller in the 12-weeks-or-more ND group (P = .04). Multivariate analysis showed that radical ND was significantly associated with an increased number of complications, and higher radiation doses approached significance (P = .05). Induction chemotherapy was associated with fewer wound complications (P = .01). There were no significant differences in overall survival (P = .82), progression-free survival (P = .77), or regional relapse (P = .54) between groups. Positive ND findings were associated with diminished progression-free and overall survival.

Conclusion  These findings indicate that ND can be safely performed 12 weeks or more after CRT without adversely affecting surgical complications or survival variables.

  R Li , C Yu , Y Li , T. W Lam , S. M Yiu , K Kristiansen and J. Wang
 

Summary: SOAP2 is a significantly improved version of the short oligonucleotide alignment program that both reduces computer memory usage and increases alignment speed at an unprecedented rate. We used a Burrows Wheeler Transformation (BWT) compression index to substitute the seed strategy for indexing the reference sequence in the main memory. We tested it on the whole human genome and found that this new algorithm reduced memory usage from 14.7 to 5.4 GB and improved alignment speed by 20–30 times. SOAP2 is compatible with both single- and paired-end reads. Additionally, this tool now supports multiple text and compressed file formats. A consensus builder has also been developed for consensus assembly and SNP detection from alignment of short reads on a reference genome.

  J. E Spiegel , A Vasudevan , Y Li and P. E. Hess
  Background

Previous studies evaluating stiff epidural catheters found that the three-holed design provided superior labour analgesia compared with an end-holed design. This was believed due to improved medication distribution. Recently, flexible epidural catheters with both designs have been shown to be superior to the stiff epidural catheters. We investigated the success of labour analgesia comparing the flexible three-holed with the flexible end-holed epidural catheter.

Methods

This was a prospective, single-blinded randomized study. We enrolled 500 parturients in active labour. The primary outcome was complete relief of labour pain assessed at 30 min. We also assessed the occurrence of paresthesias, intravascular and intrathecal placement, catheter replacement, and treatment of breakthrough pain during labour. Comparisons were made using Pearson's 2, with significance determined at the 0.05 level.

Results

Four hundred and ninety-three subjects completed the study. Initial analgesia was similar (complete labour analgesia: end-holed=85% vs 80% 95% CI of difference: 13% to –3%; P=NS). The incidence of paresthesia was similar (end-holed=3.6% vs 5.3%; P=NS). There was one intrathecal and three intravascular catheters in the three-holed group and two intravascular catheters in the end-holed group. The number of supplemental boluses and catheter replacements required during labour was similar between the groups.

Conclusions

There were no differences in the initial analgesia success rate, complications, or labour analgesia between end-hole vs multi-hole flexible epidural catheters.

  C Han , S. D Dib Hajj , Z Lin , Y Li , E. M Eastman , L Tyrrell , X Cao , Y Yang and S. G. Waxman
 

Inherited erythromelalgia (IEM), an autosomal dominant disorder characterized by severe burning pain in response to mild warmth, has been shown to be caused by gain-of-function mutations of sodium channel Nav1.7 which is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Almost all physiologically characterized cases of IEM have been associated with onset in early childhood. Here, we report the voltage-clamp and current-clamp analysis of a new Nav1.7 mutation, Q10R, in a patient with clinical onset of erythromelalgia in the second decade. We show that the mutation in this patient hyperpolarizes activation by only –5.3 mV, a smaller shift than seen with early-onset erythromelalgia mutations, but similar to that of I136V, another mutation that is linked to delayed-onset IEM. Using current-clamp, we show that the expression of Q10R induces hyperexcitability in DRG neurons, but produces an increase in excitability that is smaller than the change produced by I848T, an early-onset erythromelalgia mutation. Our analysis suggests a genotype–phenotype relationship at three levels (clinical, cellular and molecular/ion channel), with mutations that produce smaller effects on sodium channel activation being associated with a smaller degree of DRG neuron excitability and later onset of clinical signs.

  G. A Piazza , A. B Keeton , H. N Tinsley , B. D Gary , J. D Whitt , B Mathew , J Thaiparambil , L Coward , G Gorman , Y Li , B Sani , J. V Hobrath , Y. Y Maxuitenko and R. C. Reynolds
 

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 µmol/L compared with 73 to 85 µmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

  H. N Tinsley , B. D Gary , J Thaiparambil , N Li , W Lu , Y Li , Y. Y Maxuitenko , A. B Keeton and G. A. Piazza
 

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity, but toxicity resulting from cyclooxygenase (COX) inhibition limits their clinical use for chemoprevention. Studies suggest that the mechanism may be COX independent, although alternative targets have not been well defined. Here, we show that the NSAID sulindac sulfide (SS) inhibits cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) activity in colon tumor cell lysates at concentrations that inhibit colon tumor cell growth in vitro and in vivo. A series of chemically diverse NSAIDs also inhibited cGMP hydrolysis at concentrations that correlate with their potency to inhibit colon tumor cell growth, whereas no correlation was observed with COX-2 inhibition. Consistent with its selectivity for inhibiting cGMP hydrolysis compared with cyclic AMP hydrolysis, SS inhibited the cGMP-specific PDE5 isozyme and increased cGMP levels in colon tumor cells. Of numerous PDE isozyme–specific inhibitors evaluated, only the PDE5-selective inhibitor MY5445 inhibited colon tumor cell growth. The effects of SS and MY5445 on cell growth were associated with inhibition of β-catenin–mediated transcriptional activity to suppress the synthesis of cyclin D and survivin, which regulate tumor cell proliferation and apoptosis, respectively. SS had minimal effects on cGMP PDE activity in normal colonocytes, which displayed reduced sensitivity to SS and did not express PDE5. PDE5 was found to be overexpressed in colon tumor cell lines as well as in colon adenomas and adenocarcinomas compared with normal colonic mucosa. These results suggest that PDE5 inhibition, cGMP elevation, and inhibition of β-catenin transcriptional activity may contribute to the chemopreventive properties of certain NSAIDs. Cancer Prev Res; 3(10); 1303–13. ©2010 AACR.

  Z. D Gu , L. Y Shen , H Wang , X. M Chen , Y Li , T Ning and K. N. Chen
 

Homeobox genes are known to be classic examples of the intimate relationship between embryogenesis and tumorigenesis. Here, we investigated whether inhibition of HOXA13, a member of the homeobox genes, was sufficient to affect the proliferation of esophageal cancer cells in vitro and in vivo, and studied the association between HOXA13 expression and survival of patients with esophageal squamous cell carcinoma (ESCC). HOXA13 expression was permanently knocked down using an RNA interference technique, and cell strain with stable knockdown of HOXA13 protein was established. Colony formation assay showed that the number of colonies in HOXA13 protein–deficient cells was significantly less than that of control cells (P < 0.01). Tumor growth in nude mice showed that the weight and volume of tumors from the HOXA13 knockdown cells was significantly less than that from the control cells (P < 0.01). Then, HOXA13 expression in ESCC specimens and paired noncancerous mucosa was detected by immunohistochemistry, and overexpression of HOXA13 was found to be more pronounced in ESCCs than paired noncancerous mucosa (P < 0.05). Furthermore, the association of HOXA13 expression and disease-free survival time was analyzed in 155 ESCC cases. The median survival time of patients expressing HOXA13 was significantly shorter than HOXA13-negative patients (P = 0.0006). Multivariate analysis indicated that tumor-node-metastasis (TNM) stage and HOXA13 expression were independent predictors of disease-free survival time of patients with ESCC. Our results showed that HOXA13 expression enhanced tumor growth in vitro and in vivo, and was a negative independent predictor of disease-free survival of patients with ESCC. [Cancer Res 2009;69(12):4969–73]

  A Bhattacharya , L Tang , Y Li , F Geng , J. D Paonessa , S. C Chen , M. K.K Wong and Y. Zhang
 

Bladder cancer is one of the common human cancers and also has a very high recurrence rate. There is a great need for agents capable of inhibiting bladder cancer development and recurrence. Here, we report that allyl isothiocyanate (AITC), an ingredient of many common cruciferous vegetables, potently inhibited the proliferation of bladder carcinoma cell lines in vitro [half maximal inhibitory concentration (IC50) of 2.7–3.3 µM], which was associated with profound G2/M arrest and apoptosis. In contrast, AITC was markedly less toxic to normal human bladder epithelial cells (IC50 of 69.4 µM). AITC was then evaluated in two rat bladder cancer models in vivo (an orthotopic model and a subcutaneous model). The orthotopic model closely mimics human bladder cancer development and recurrence. We show that a low oral dose of AITC (1 mg/kg) significantly inhibited the development and muscle invasion of the orthotopic bladder cancers but was ineffective against the subcutaneous xenografts of the same cancer cells in the same animals. This differential effect was explained by our finding that urinary levels of AITC equivalent were two to three orders of magnitude higher than that in the plasma and that its levels in the orthotopic cancer tissues were also three orders of magnitude higher than that in the subcutaneous cancer tissues. Moreover, we show that AITC is a multi-targeted agent against bladder cancer. In conclusion, AITC is selectively delivered to bladder cancer tissue through urinary excretion and potently inhibits bladder cancer development and invasion.

  B Liu , D Chen , L Yang , Y Li , X Ling , L Liu , W Ji , Y Wei , J Wang , Q Wei , L Wang and J. Lu
 

Mitogen-activated protein kinase kinase 4 (MKK4) is a critical mediator of stress-activated protein kinase signals that regulate apoptosis, inflammations and tumorigenesis. Several polymorphisms have been identified in the MKK4 gene. We hypothesized that genetic variants in the MKK4 promoter may alter its expression and thus cancer risk. In a case–control study of 1056 lung cancer cases and 1056 sex and age frequency-matched cancer-free controls, we genotyped two common polymorphisms in the MKK4 promoter region (–1304T>G and –1044A>T) with the Taqman assay, and we found that compared with the most common –1304TT genotype, carriers of –1304G variant genotypes had a decreased risk of lung cancer [odds ratio (OR) = 0.74; 95% confidence interval (CI) = 0.61–0.90 for TG, and OR = 0.62; 95% CI = 0.41–0.94 for GG] in an allele dose–response manner (adjusted Ptrend = 0.0005). Further stratification analysis showed that the protective role of the –1304G variant allele was more evident in low or normal body mass index (BMI) but restrained in the overweighters and that the –1304G variant genotypes interacted with BMI in reducing cancer risk (adjusted Pinteraction = 0.003). Moreover, the luciferase assay showed that the G allele in the promoter significantly increased the transcription activity of the MKK4 gene in vitro and that the MKK4 protein expression levels of the G variant carriers was significantly higher in tumor tissues than those of the –1304TT genotype. However, no significant association was observed between the –1044A>T polymorphism and risk of lung cancer. Our data suggest that the functional –1304G variant in the MKK4 promoter contributes to a decreased risk of lung cancer by increasing the promoter activity and that the G variant may be a marker for susceptibility to lung cancer.

  A Bhattacharya , Y Li , K. L Wade , J. D Paonessa , J. W Fahey and Y. Zhang
 

Allyl isothiocyanate (AITC), which occurs in many common cruciferous vegetables, was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in rats. The present investigation was designed to test the hypothesis that AITC-containing cruciferous vegetables also inhibit bladder cancer development. We focused on an AITC-rich mustard seed powder (MSP-1). AITC was stably stored as its glucosinolate precursor (sinigrin) in MSP-1. Upon addition of water, however, sinigrin was readily hydrolyzed by the accompanying endogenous myrosinase. This myrosinase was also required for full conversion of sinigrin to AITC in vivo, but the matrix of MSP-1 had no effect on AITC bioavailability. Sinigrin itself was not bioactive, whereas hydrated MSP-1 caused apoptosis and G2/M phase arrest in bladder cancer cell lines in vitro. Comparison between hydrated MSP-1 and pure sinigrin with added myrosinase suggested that the anticancer effect of MSP-1 was derived principally, if not entirely, from the AITC generated from sinigrin. In an orthotopic rat bladder cancer model, oral MSP-1 at 71.5 mg/kg (sinigrin dose of 9 µmol/kg) inhibited bladder cancer growth by 34.5% (P < 0.05) and blocked muscle invasion by 100%. Moreover, the anticancer activity was associated with significant modulation of key cancer therapeutic targets, including vascular endothelial growth factor, cyclin B1 and caspase 3. On an equimolar basis, the anticancer activity of AITC delivered as MSP-1 appears to be more robust than that of pure AITC. MSP-1 is thus an attractive delivery vehicle for AITC and it strongly inhibits bladder cancer development and progression.

  Y Wang , Y Huang , K. S.L Lam , Y Li , W. T Wong , H Ye , C. W Lau , P. M Vanhoutte and A. Xu
  Aims

Endothelial dysfunction is a key event that links obesity, diabetes, hypertension, and cardiovascular diseases. The aim of the present study was to examine the protective effect of the alkaloid drug berberine against hyperglycemia-induced cellular injury and endothelial dysfunction.

Methods and results

In both cultured endothelial cells and blood vessels isolated from rat aorta, berberine concentration dependently enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and promoted the association of eNOS with heat shock protein 90 (HSP90), leading to an increased production of nitric oxide. Furthermore, berberine attenuated high glucose-induced generation of reactive oxygen species, cellular apoptosis, nuclear factor-B activation, and expression of adhesion molecules, thus suppressing monocyte attachment to endothelial cells. In mouse aortic rings, berberine elicited endothelium-dependent vasodilatations and alleviated high glucose-mediated endothelial dysfunction. All these beneficial effects of berberine on the endothelium were abolished by either pharmacological inhibition of adenosine monophosphate-activated protein kinase (AMPK) or adenovirus-mediated overexpression of a dominant negative version of AMPK.

Conclusion

Berberine protects against endothelial injury and enhances the endothelium-dependent vasodilatation, which is mediated in part through activation of the AMPK signalling cascade. Berberine or its derivatives may be useful for the treatment and/or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease.

  Y Tan , Y Li , J Xiao , H Shao , C Ding , G. E Arteel , K. A Webster , J Yan , H Yu , L Cai and X. Li
  Aims

The effects on angiogenesis of a novel CXC chemokine receptor 4 (CXCR4) antagonist, SDF-1βP2G, derived from human stromal cell-derived factor-1β (SDF-1β), were examined in a model of hind limb ischaemia in mice.

Methods and results

The antagonistic activities of SDF-1βP2G against CXCR4 were evaluated in vitro and in vivo and compared with phosphate-buffered saline and AMD3100 (a small bicyclam antagonist of SDF-1). Angiogenesis, muscle regeneration and the expression of pro-angiogenic factors were evaluated in ischaemic gastrocnemius muscles. Distant toxic effects of SDF-1βP2G were evaluated by inflammatory and apoptotic markers. SDF-1βP2G induced CXCR4 internalization and competitively inhibited the chemotaxis of SDF-1β but did not mediate migration, calcium influx, or the phosphorylation of Akt and extracellular signal-regulated kinase in cultured T-lymphoblastic leukaemia cells or H9C2 cells. SDF-1βP2G enhanced blood flow, angiogenesis, and muscle regeneration in ischaemic hind limbs, and the enhancement was significantly better than that of AMD3100. Markers of angiogenesis and progenitor cell migration, including phosphorylated Akt, vascular endothelial growth factor (VEGF), SDF-1 and CXCR4, were up-regulated by SDF-1βP2G and co-localized with CD31-positive cells. Neutralization of VEGF with its specific antibody abolished SDF-1βP2G-induced blood reperfusion and angiogenesis. No apparent inflammatory and apoptotic effects were found in heart, liver, kidneys, and testes after SDF-1βP2G administration.

Conclusion

Our findings indicate that the novel CXCR4 antagonist, SDF-1βP2G, can efficiently enhance ischaemic angiogenesis, blood flow restoration, and muscle regeneration without apparent adverse effects, most likely through a VEGF-dependent pathway.

  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 Wang , J Liu , Y Li , S Wu , J Luo , H Yang , R Subbiah , J Chatham , O Zhelyabovska and Q. Yang
 

Rationale: Peroxisome proliferator-activated receptors (PPARs) (, , and /β) are nuclear hormone receptors and ligand-activated transcription factors that serve as key determinants of myocardial fatty acid metabolism. Long-term cardiomyocyte-restricted PPAR deficiency in mice leads to depressed myocardial fatty acid oxidation, bioenergetics, and premature death with lipotoxic cardiomyopathy.

Objective: To explore the essential role of PPAR in the adult heart.

Methods and Results: We investigated the consequences of inducible short-term PPAR knockout in the adult mouse heart. In addition to a substantial transcriptional downregulation of lipid metabolic proteins, short-term PPAR knockout in the adult mouse heart attenuated cardiac expression of both Cu/Zn superoxide dismutase and manganese superoxide dismutase, leading to increased oxidative damage to the heart. Moreover, expression of key mitochondrial biogenesis determinants such as PPAR coactivator-1 were substantially decreased in the short-term PPAR deficient heart, concomitant with a decreased mitochondrial DNA copy number. Rates of palmitate and glucose oxidation were markedly depressed in cardiomyocytes of PPAR knockout hearts. Consequently, PPAR deficiency in the adult heart led to depressed cardiac performance and cardiac hypertrophy.

Conclusions: PPAR is an essential regulator of cardiac mitochondrial protection and biogenesis and PPAR activation can be a potential therapeutic target for cardiac disorders.

  C. A Peralta , Y Li , C Wassel , S Choudhry , W Palmas , M. F Seldin , N Risch , D Siscovick , D Arnett , B Psaty and M. G. Shlipak
  Background—

Reports show higher prevalence of albuminuria among Hispanics compared with whites. Differences by country of origin or genetic background are unknown.

Methods and Results—

In Multi-Ethnic Study of Atherosclerosis, we studied the associations of both genetic ancestry and country of origin with albumin to creatinine ratio among 1417 Hispanic versus white participants using multivariable linear regression and back transforming beta coefficients into relative difference (%RD, 95% CI). Percentage European, Native American, and African ancestry components for Hispanics were estimated using genetic admixture analysis. The proportions of European, Native American, and African genetic ancestry differed significantly by country of origin (P<0.0001); Mexican/Central Americans had the highest Native American (41±13%), Puerto Ricans had the highest European (61±15%), and Dominicans had the highest African (39±21%) ancestry. Hispanic ethnicity was associated with higher albumin/creatinine ratio compared with whites, but the association varied with the country of origin (adjusted P interaction=0.04). Mexican/Central Americans and Dominicans had higher albumin/creatinine ratio compared with whites after adjustment (RD 19%, 2% to 40% and RD 27%, 1% to 61%), but not Puerto Ricans (RD 8%, –12% to 34%). Higher Native American ancestry was associated with higher albuminuria after age and sex adjustment among all Hispanics (RD 11%, 1% to 21%) but was attenuated after further adjustment. Higher European ancestry was independently associated with lower albumin/creatinine ratio among Puerto Ricans (–21%, –34% to –6%) but not among Mexican/Central Americans and Dominicans.

Conclusions—

Hispanics are a heterogeneous group with varying genetic ancestry. Risks of albuminuria differ across the country of origin groups. These differences may be due, in part, to differences in genetic ancestral components.

  S. D Reed , D. J Whellan , Y Li , J. Y Friedman , S. J Ellis , I. L Pina , S. J Settles , L Davidson Ray , J. L Johnson , L. S Cooper , C. M O'Connor , K. A Schulman and for the HF ACTION Investigators
  Background—

Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training (HF-ACTION) assigned 2331 outpatients with medically stable heart failure to exercise training or usual care. We compared medical resource use and costs incurred by these patients during follow-up.

Methods and Results—

Extensive data on medical resource use and hospital bills were collected throughout the trial for estimates of direct medical costs. Intervention costs were estimated using patient-level trial data, administrative records, and published unit costs. Mean follow-up was 2.5 years. There were 2297 hospitalizations in the exercise group and 2332 in the usual care group (P=0.92). The mean number of inpatient days was 13.6 (standard deviation [SD], 27.0) in the exercise group and 15.0 (SD, 31.4) in the usual care group (P=0.23). Other measures of resource use were similar between groups, except for trends indicating that fewer patients in the exercise group underwent high-cost inpatient procedures. Total direct medical costs per participant were an estimated $50 857 (SD, $81 488) in the exercise group and $56 177 (SD, $92 749) in the usual care group (95% confidence interval for the difference, $–12 755 to $1547; P=0.10). The direct cost of exercise training was an estimated $1006 (SD, $337). Patient time costs were an estimated $5018 (SD, $4600).

Conclusions—

The cost of exercise training was relatively low for the health care system, but patients incurred significant time costs. In this economic evaluation, there was little systematic benefit in terms of overall medical resource use with this intervention.

Clinical Trial Registration—

URL: http://www.clinicaltrials.gov. Unique identifier: NCT00047437.

  C Zhang , L Fu , J Fu , L Hu , H Yang , T. H Rong , Y Li , H Liu , S. B Fu , Y. X Zeng and X. Y. Guan
 

Purpose: Tumor fibroblasts (TF) have been suggested to play an essential role in the complex process of tumor-stroma interactions and tumorigenesis. The aim of the present study was to investigate the specific role of TF in the esophageal cancer microenvironment.

Experimental Design: An Affymetrix expression microarray was used to compare gene expression profiles between six pairs of TFs and normal fibroblasts from esophageal squamous cell carcinoma (ESCC). Differentially expressed genes were identified, and a subset was evaluated by quantitative real-time PCR and immunohistochemistry.

Results: About 43% (126 of 292) of known deregulated genes in TFs were associated with cell proliferation, extracellular matrix remodeling, and immune response. Up-regulation of fibroblast growth factor receptor 2 (FGFR2), which showed the most significant change, was detected in all six tested TFs compared with their paired normal fibroblasts. A further study found that FGFR2-positive fibroblasts were only observed inside the tumor tissues and not in tumor-surrounding stromal tissues, suggesting that FGFR2 could be used as a TF-specific marker in ESCC. Moreover, the conditioned medium from TFs was found to be able to promote ESCC tumor cell growth, migration, and invasion in vitro.

Conclusions: Our study provides new candidate genes for the esophageal cancer microenvironment. Based on our results, we hypothesize that FGFR2(+)-TFs might provide cancer cells with a suitable microenvironment via secretion of proteins that could promote cancer development and progression through stimulation of cancer cell proliferation, induction of angiogenesis, inhibition of cell adhesion, enhancement of cell mobility, and promotion of the epithelial-mesenchymal transition.

  Y Li , A. S Ptolemy , L Harmonay , M Kellogg and G. T. Berry
 

Background: The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based assay for GALT enzyme activity measurement.

Method: Our assay used stable isotope-labeled - galactose-1-phosphate ([13C6]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard.

Results: The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) µmol · (g Hgb)–1 · h–1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 µmol · (g Hgb)–1 · h–1 (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent Km of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity.

Conclusions: This LC-MS/MS–based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.

  C Zhang , C Wang , X Chen , C Yang , K Li , J Wang , J Dai , Z Hu , X Zhou , L Chen , Y Zhang , Y Li , H Qiu , J Xing , Z Liang , B Ren , K Zen and C. Y. Zhang
  BACKGROUND:

Sensitive and specific biomarkers for the early detection of esophageal squamous cell carcinoma (ESCC) are urgently needed to reduce the high morbidity and mortality of the disease. The discovery of serum microRNAs (miRNAs) and their unique concentration profiles in patients with various diseases makes them attractive, novel noninvasive biomarkers for tumor diagnosis. In this study, we investigated the serum miRNA profile in ESCC patients to develop a novel diagnostic ESCC biomarker.

METHODS:

Serum samples were taken from 290 ESCC patients and 140 age- and sex-matched controls. Solexa sequencing technology was used for an initial screen of miRNAs in serum samples from 141 patients and 40 controls. A hydrolysis probe–based stem–loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and verification phases to confirm the concentrations of selected miRNAs in serum samples from 149 patients and 100 controls.

RESULTS:

The Solexa sequencing results demonstrated marked upregulation of 25 serum miRNAs in ESCC patients compared with controls. RT-qPCR analysis identified a profile of 7 serum miRNAs (miR-10a, miR-22, miR-100, miR-148b, miR-223, miR-133a, and miR-127-3p) as ESCC biomarkers. The area under the ROC curve for the selected miRNAs ranged from 0.817 to 0.949, significantly higher than for carcinoembryonic antigen (0.549; P < 0.0005). More importantly, this panel of 7 miRNAs clearly distinguished stage I/II ESCC patients from controls.

CONCLUSIONS:

This panel of 7 serum miRNAs holds promise as a novel blood-based biomarker for the diagnosis of ESCC.

  J Dong , Y Li , Z Yang and J. Luo
 

Background and objectives: To explore the correlation between dietary sodium intake and cardiovascular and overall mortality, and then determine whether this correlation can be explained by protein and energy intake paralleled with sodium intake in dialysis patients.

Design, setting, participants, & measurements: This single-center retrospective cohort study enrolled 305 incident patients who started peritoneal dialysis in our unit from July 2002 to February 2007. All patients were followed until death or until being censored in February 2008. Demographic data were collected at baseline. Biochemical, dietary, and nutrition data were examined at baseline and thereafter at regular intervals to calculate the average values throughout the study.

Results: Participants with the highest average sodium intake were more likely to be younger, male, and overweight. Patients in the high tertile of average sodium intake had higher albumin, prealbumin, and lean body mass levels, and more nutrient intakes paralleling with sodium intake. Low average sodium intake independently predicted the increased risk for overall and cardiovascular death after adjusting for recognized confounders. Further adjustment for dietary protein, energy, and other nutrient intakes individually had minimal impact on the association between average sodium intake and overall death, with hazard ratios varying between 0.35 and 0.44, and cardiovascular death, with hazard ratios varying between 0.06 and 0.11.

Conclusions: This study revealed that low dietary sodium intake independently predicts the high overall and cardiovascular mortality in dialysis patients. This correlation could not be entirely explained by deficient protein and energy intake.

  S Cao , J. W Hewett , F Yokoi , J Lu , A. C Buckley , A. J Burdette , P Chen , F. C Nery , Y Li , X. O Breakefield , G. A Caldwell and K. A. Caldwell
  Songsong Cao, Jeffrey W. Hewett, Fumiaki Yokoi, Jun Lu, Amber Clark Buckley, Alexander J. Burdette, Pan Chen, Flavia C. Nery, Yuqing Li, Xandra O. Breakefield, Guy A. Caldwell, and Kim A. Caldwell

Movement disorders represent a significant societal burden for which therapeutic options are limited and focused on treating disease symptomality. Early-onset torsion dystonia (EOTD) is one such disorder characterized by sustained and involuntary muscle contractions that frequently cause repetitive movements or abnormal postures. Transmitted in an autosomal dominant manner with reduced penetrance, EOTD is caused in most cases by the deletion of a glutamic acid (E) in the DYT1 (also known as TOR1A) gene product, torsinA. Although some patients respond well to anticholingerics, therapy is primarily limited to either neurosurgery or chemodenervation. As mutant torsinA (E) expression results in decreased torsinA function, therapeutic strategies directed toward enhancement of wild-type (WT) torsinA activity in patients who are heterozygous for mutant DYT1 may restore normal cellular functionality. Here, we report results from the first-ever screen for candidate small molecule therapeutics for EOTD, using multiple activity-based readouts for torsinA function in Caenorhabditis elegans, subsequent validation in human DYT1 patient fibroblasts, and behavioral rescue in a mouse model of DYT1 dystonia. We exploited the nematode to rapidly discern chemical effectors of torsinA and identified two classes of antibiotics, quinolones and aminopenicillins, which enhance WT torsinA activity in two separate in vivo assays. Representative molecules were assayed in EOTD patient fibroblasts for improvements in torsinA-dependent secretory function, which was improved significantly by ampicillin. Furthermore, a behavioral defect associated with an EOTD mouse knock-in model was also rescued following administration of ampicillin. These combined data indicate that specific small molecules that enhance torsinA activity represent a promising new approach toward therapeutic development for EOTD, and potentially for other diseases involving the processing of mutant proteins.

  R Li , Y Li , X Fang , H Yang , J Wang , K Kristiansen and J. Wang
 

Next-generation massively parallel sequencing technologies provide ultrahigh throughput at two orders of magnitude lower unit cost than capillary Sanger sequencing technology. One of the key applications of next-generation sequencing is studying genetic variation between individuals using whole-genome or target region resequencing. Here, we have developed a consensus-calling and SNP-detection method for sequencing-by-synthesis Illumina Genome Analyzer technology. We designed this method by carefully considering the data quality, alignment, and experimental errors common to this technology. All of this information was integrated into a single quality score for each base under Bayesian theory to measure the accuracy of consensus calling. We tested this methodology using a large-scale human resequencing data set of 36x coverage and assembled a high-quality nonrepetitive consensus sequence for 92.25% of the diploid autosomes and 88.07% of the haploid X chromosome. Comparison of the consensus sequence with Illumina human 1M BeadChip genotyped alleles from the same DNA sample showed that 98.6% of the 37,933 genotyped alleles on the X chromosome and 98% of 999,981 genotyped alleles on autosomes were covered at 99.97% and 99.84% consistency, respectively. At a low sequencing depth, we used prior probability of dbSNP alleles and were able to improve coverage of the dbSNP sites significantly as compared to that obtained using a nonimputation model. Our analyses demonstrate that our method has a very low false call rate at any sequencing depth and excellent genome coverage at a high sequencing depth.

  M Guo , H Feng , J Zhang , W Wang , Y Wang , Y Li , C Gao , H Chen , Y Feng and Z. G. He
 

Sequence-specific DNA-binding transcription factors have widespread biological significance in the regulation of gene expression. However, in lower prokaryotes and eukaryotic metazoans, it is usually difficult to find transcription regulatory factors that recognize specific target promoters. To address this, we have developed in this study a new bacterial one-hybrid reporter vector system that provides a convenient and rapid strategy to determine the specific interaction between target DNA sequences and their transcription factors. Using this system, we have successfully determined the DNA-binding specificity of the transcription regulator Rv3133c to a previously reported promoter region of the gene Rv2031 in Mycobacterium tuberculosis. In addition, we have tested more than 20 promoter regions of M. tuberculosis genes using this approach to determine if they interact with ~150 putative regulatory proteins. A variety of transcription factors are found to participate in the regulation of stress response and fatty acid metabolism, both of which comprise the core of in vivo-induced genes when M. tuberculosis invades macrophages. Interestingly, among the many new discovered potential transcription factors, the WhiB-like transcriptional factor WhiB3 was identified for the first time to bind with the promoter sequences of most in vivo-induced genes. Therefore, this study offers important data in the dissection of the transcription regulations in M. tuberculosis, and the strategy should be applicable in the study of DNA-binding factors in a wide range of biological organisms.

  H Wang , A Chattopadhyay , Z Li , B Daines , Y Li , C Gao , R Gibbs , K Zhang and R. Chen
 

One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously characterized dac5 or sensE2 mutation. Targeted amplification of genomic regions near these two loci was used to enrich DNA for sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr mutant flies, the underlying mutation was identified as a single-base mutation in the gene Spase18-21. This targeted-genome-sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model organisms such as Drosophila.

  X Lu , J. A Shapiro , C. T Ting , Y Li , C Li , J Xu , H Huang , Y. J Cheng , A. J Greenberg , S. H Li , M. L Wu , Y Shen and C. I. Wu
 

Postmating reproductive isolation is often manifested as hybrid male sterility, for which X-linked genes are overrepresented (the so-called large X effect). In contrast, X-linked genes are significantly under-represented among testis-expressing genes. This seeming contradiction may be germane to the X:autosome imbalance hypothesis on hybrid sterility, in which the X-linked effect is mediated mainly through the misexpression of autosomal genes. In this study, we compared gene expression in fertile and sterile males in the hybrids between two Drosophila species. These hybrid males differ only in a small region of the X chromosome containing the Ods-site homeobox (OdsH) (also known as Odysseus) locus of hybrid sterility. Of genes expressed in the testis, autosomal genes were, indeed, more likely to be misexpressed than X-linked genes under the sterilizing action of OdsH. Since this mechanism of X:autosome interaction is only associated with spermatogenesis, a connection between X:autosome imbalance and the high rate of hybrid male sterility seems plausible.

  Y He , Y Li , Z Peng , H Yu , X Zhang , L Chen , Q Ji , W Chen and R. Wang
 

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A, and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two, or three N-glycans, but was partially inhibited when lacking four, five, and six N-glycans. In all, 2%, 48%, 85%, and 73% of the products from N3457A, N34567A, N134567A, and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

  J Cheng , S Huang , H Yu , Y Li , K Lau and X. Chen
 

Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. 2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida sialyltransferase PmST1. 2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni sialyltransferase CstII. We report here the discovery of the 2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial 2,6-sialyltransferase from Photobacterium damsela (15Pd2,6ST). This is the first time that the 2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that 15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5'-monophosphate, the product of sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcpNP and LacβMU, 2,6-linked sialoside Neu5Ac2,6LacβMU has been obtained in one-step in high yield using the trans-sialidase activity of 15Pd2,6ST. In addition to the 2,6-trans-sialidase activity, 15Pd2,6ST also has 2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial sialyltransferases.

  L Zhang , K Lau , J Cheng , H Yu , Y Li , G Sugiarto , S Huang , L Ding , V Thon , P. G Wang and X. Chen
 

Lewis x (Lex) and sialyl Lewis x (SLex)-containing glycans play important roles in numerous physiological and pathological processes. The key enzyme for the final step formation of these Lewis antigens is 1-3-fucosyltransferase. Here we report molecular cloning and functional expression of a novel Helicobacter hepaticus 1-3-fucosyltransferase (HhFT1) which shows activity towards both non-sialylated and sialylated Type II oligosaccharide acceptor substrates. It is a promising catalyst for enzymatic and chemoenzymatic synthesis of Lex, sialyl Lex and their derivatives. Unlike all other 1-3/4-fucosyltransferases characterized so far which belong to Carbohydrate Active Enzyme (CAZy, http://www.cazy.org/) glycosyltransferase family GT10, the HhFT1 shares protein sequence homology with 1-2-fucosyltransferases and belongs to CAZy glycosyltransferase family GT11. The HhFT1 is thus the first 1-3-fucosyltransferase identified in the GT11 family.

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

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

Methods

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

Results

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

Conclusion

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

  Y Yu , Y Li , L Li , J Lin , C Zheng and L. Zhang
 

Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed -tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of -tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

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

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

  Y Li , L Birnbaumer and C. T. Teng
 

In selected tissues and cell lines, 17β-estradiol (E2) regulates the expression of estrogen-related receptor (ERR), a member of the orphan nuclear receptor family. This effect is thought to be mediated by the estrogen receptor (ER). However in the ER- and ERβ-negative SKBR3 breast cancer cell line, physiological levels of E2 also stimulate ERR expression. Here, we explored the molecular mechanism that mediates estrogen action in ER-negative breast cancer cells. We observed that E2, the ER agonist, as well as the ER antagonists ICI 182,780 and tamoxifen (TAM), a selective ER modulator, stimulate the transcriptional activity of the ERR gene and increase the production of ERR protein in SKBR3 cells. Moreover, the ERR downstream target genes expression and cellular proliferation are also increased. We show further that the G protein-coupled receptor GPR30/GPER-1 (GPER-1) mediates these effects. The GPER-1 specific ligand G-1 mimics the actions of E2, ICI 182,780, and TAM on ERR expression, and changing the levels of GPER-1 mRNA by overexpression or small interfering RNA knockdown affected the expression of ERR accordingly. Utilizing inhibitors, we delineate a different downstream pathway for ER agonist and ER antagonist-triggered signaling through GPER-1. We also find differential histone acetylation and transcription factor recruitment at distinct nucleosomes of the ERR promoter, depending on whether the cells are activated with E2 or with ER antagonists. These findings provide insight into the molecular mechanisms of GPER-1/ERR-mediated signaling and may be relevant to what happens in breast cancer cells escaping inhibitory control by TAM.

  Y Niu , G. Z Wu , R Ye , W. H Lin , Q. M Shi , L. J Xue , X. D Xu , Y Li , Y. G Du and H. W. Xue
 

In order to study Brassica napus fatty acid (FA) metabolism and relevant regulatory networks, a systematic identification of fatty acid (FA) biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization (>8000 EST clones from seed). The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17–21 d after flowering (DAF), whereas FA biosynthesis-related genes were highly expressed primarily at 21 DAF. Hormone (auxin and jasmonate) signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.

  Y Li , W Pan , W Xu , N He , X Chen , H Liu , L Darryl Quarles , H Zhou and Z. Xiao
 

Cleidocranial dysplasia (CCD) is an autosomal dominant bone disease in humans caused by haploinsufficiency of the RUNX2 gene. The RUNX2 has two major isoforms derived from P1 and P2 promoters. Over 90 mutations of RUNX2 have been reported associated with CCD. In our study, DNA samples of nine individuals from three unrelated CCD families were collected and screened for all exons of RUNX2 and 2 kb of P1 and P2 promoters. We identified two point mutations in the RUNX2 gene in Case 1, including a nonsense mutation (c.577C>T) that has been reported previously and a silent substitution (c.240G>A). In vitro studies demonstrated that c.577C>T mutation led to truncated RUNX2 protein production and diminished stimulating effects on mouse osteocalcin promoter activity when compared with full-length Runx2-II and Runx2-I isoforms. These results confirm that loss of function RUNX2 mutation (c.577C>T) in Case 1 family is responsible for its CCD phenotype.

  D Tchapyjnikov , Y Li , T Pisitkun , J. D Hoffert , M. J Yu and M. A. Knepper
 

Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 (Aqp2) and aquaporin-3 (Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool (ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as "transcription factor" proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5'-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5'-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS3 technique. The newly identified proteins have been incorporated into the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).

  S. M Vaingankar , Y Li , A Corti , N Biswas , J Gayen , D. T O'Connor and S. K. Mahata
 

Chromogranin A (CHGA) plays a catalytic role in formation of catecholamine storage vesicles and also serves as precursor to the peptide fragment catestatin, a catecholamine secretory inhibitor whose expression is diminished in the hypertensive individuals. We previously reported the hypertensive, hyperadrenergic phenotype of Chga–/– knockout (KO) mice and rescue by the human ortholog. In the present study, we compare two humanized CHGA mouse models. Into the Chga null background, by bacterial artificial chromosome transgenesis human CHGA transgene has been introduced. Both lines have the complete ~12 kbp CHGA gene integrated stably in the genome but have substantial differences in CHGA expression, as well as consequent sympathochromaffin biochemistry and physiology. A mouse model with longer-insert HumCHGA31 displays integration encompassing not only CHGA but also long human flanking sequences. This is in contrast to mouse model HumCHGA19 with limited flanking human sequence co-integrated. As a consequence, HumCHGA19 mice have normal though diminished pattern of spatial expression of CHGA, and 14-fold lower circulating CHGA, with failure to rescue KO phenotypes to normalcy. In the longer-insert HumCHGA31 mice, catecholamine secretion, exaggerated responses to environmental stress, and hypertension were all alleviated. Promoter regions of the transgenes in both HumCHGA19 and HumCHGA31 display minimal CpG methylation, weighing against differential "position effects" of integration, and thus suggesting that lack of cis elements required for optimal CHGA expression occurs in HumCHGA19 mice. Such "humanized" CHGA mouse models may be useful in probing the physiological consequences of variation in CHGA expression found in humans, with consequences for susceptibility to hypertension and cardiovascular disease.

  J. H Davis , C Aperlo , Y Li , E Kurosawa , Y Lan , K. M Lo and J. S. Huston
 

Bispecific antibodies and asymmetric Fc fusion proteins offer opportunities for important advances in therapeutics. Bivalent IgG depends upon in vivo dimerization of its heavy chains, mediated by homodimeric association of its CH3 domains. We have developed a heterodimeric Fc platform that supports the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) CH3 heterodimers. These derivatives of human IgG and IgA CH3 domains create complementary human SEED CH3 heterodimers that are composed of alternating segments of human IgA and IgG CH3 sequences. The resulting pair of SEED CH3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-CH2-[SEED CH3], that may be genetically linked to one or more fusion partners. This investigation reports on the generation of mono-Fab-Sb and Sb-IL2 monocytokine as models. They were expressed at high levels in NS/0 cells, purified on recombinant protein A resin and were well-behaved in solution. When administered intravenously to mice, Sb pharmacokinetics exhibited the long serum half-life extensions typical of comparable Fc-containing immunofusion and IgG1 controls.

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

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

  G Fan , C Feng , Y Li , C Wang , J Yan , W Li , J Feng , X Shi and Y. Bi
 

Background: We carried out animal experiments based on the orthogonal design L8(27) setting seven factors with two different levels of each and 10 groups of rats. The nutrients tested were tyrosine, glycine, methionine, taurine, ascorbic acid, thiamine and zinc.

Objectives: The objective of this study was to explore the optimal combinations of nutrients for prevention or amelioration of lead-induced learning and memory impairment.

Methods: Rats were supplemented with nutrients by gavage once a day in two experiments: one was simultaneous nutrient supplementation with lead acetate administration (800 mg l–1) for 8 weeks (prophylactic supplementation) and the other was nutrient supplementation for 4 weeks after the cessation of 4 weeks of lead administration (remedial supplementation). Morris water maze was initiated at ninth week. Rats were terminated for assays of levels of Pb in blood, activities of superoxide dismutase (SOD) and nitric oxide synthase (NOS) in hippocampus, levels of nitric oxide (NO) in hippocampus and expressions of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) response element-binding protein messenger RNA in hippocampus.

Results: Results showed that in prophylactic supplementation, methionine, taurine, zinc, ascorbic acid and glycine were the effective preventive factors for decreasing prolonged escape latency, increasing SOD and NOS activities and NO levels in the hippocampus, respectively. On the other hand, in remedial supplementation, taurine was the effective factor for reversing Pb-induced decrease in activities of SOD, NOS and levels of NO.

Conclusions: In conclusion, the optimum combinations of nutrients appear to be methionine, taurine, zinc, ascorbic acid and glycine for the prevention of learning and memory impairment, while taurine and thiamine appear to be the effective factors for reversing Pb neurotoxicity.

  Y Li , J Zeng and Z. G. He
 

Virulence in pathogenic bacteria is due in part to the action of two-component systems. However, in the human pathogen Mycobacterium tuberculosis, the molecular mechanisms underlying these systems are as yet unclear. In this study, MtrA was shown to contain a functional C-terminus and also to have Ca2+ as its preferred cofactor for DNA binding. Further mutation experiments demonstrated that the C-terminus of MtrA was responsible for specific interactions with the target DNA motif and also with its partner protein, MtrB. The physical interaction between MtrA and MtrB inhibited DNA binding by MtrA. These findings yield critical information about the unique regulatory mechanisms of the essential MtrAB two-component system in this pathogen.

  Y Li , S Lim , D Hoffman , P Aspenstrom , H. J Federoff and D. A. Rempe
 

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 (HIF-1). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states.

  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.

  Z Tang , P Arjunan , C Lee , Y Li , A Kumar , X Hou , B Wang , P Wardega , F Zhang , L Dong , Y Zhang , S. Z Zhang , H Ding , R. N Fariss , K. G Becker , J Lennartsson , N Nagai , Y Cao and X. Li
 

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3β (GSK3β) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine–induced Parkinson’s dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC–PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.

 
 
 
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