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Articles by X Liu
Total Records ( 52 ) for X Liu
  L Zhu , J Wang , J Mu , H Wang , C Zhang , X Liu , X Yan , L Dai and D. Ma

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and C-terminal of hTFPI-2 (hTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SP-Sepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, and circular dichroism (CD) experiment results implied that hTFPI-2/KD3C contained small contents of -helix and β-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauche-trans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced β-strands in hTFPI-2/KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.

  J Yang , X Liu , J Yu , L Sheng , Y Shi , Z Li , Y Hu , J Xue , L Wu , Y Liang , J Xia and D. Liang

Gene therapy has emerged as a promising approach for the lethal disorder of Duchenne muscular dystrophy (DMD). Using a novel non-viral delivery system, the human ribosomal DNA (hrDNA) targeting vector, we targeted a minidystrophin-GFP fusion gene into the hrDNA locus of HT1080 cells with a high site-specific integrated efficiency of 10–5, in which the transgene could express efficiently and continuously. The minidystrophin-GFP fusion protein was easily found to localize on the plasma membrane of HT1080 cells, indicating its possible physiologic performance. Our findings showed that the hrDNA-targeting vector might be highly useful for DMD gene therapy study.

  X Liu , X Zhang and I. Lee

Fluid shear stress (FSS) is widely explored regarding its influence on osteoblasts. In vitro studies have shown that the cytoskeleton is very important in cellular responses to FSS. However, morphological changes, which would reflect the cytoskeleton changes as well as other cellular responses, were rarely quantitatively studied in the past years. Therefore, FSS-induced morphological changes in osteoblasts were quantified in this study. Real-time rapid morphological responses were observed by exposing osteoblasts to FSS with magnitude of 1.2, 1.6, and 1.9 Pa for 1 h. Afterward, osteoblast actin cytoskeleton was labeled with rhodamine phalloidin and observed using fluorescence microscopy. The results showed that 1.6 and 1.9 Pa FFS resulted in significant cellular elongation and reorientation along the direction of fluid flow. Besides, along with the enhancement of FSS magnitude, cytoskeleton aggregated more remarkably. Furthermore, extracellular Ca2+-depleted fluid flow was also used to stimulate osteoblasts for 1 h with magnitude of 1.6 and 1.9 Pa. No morphological change was observed after removing extracellular calcium. Our study suggested that the level of FSS from 1.2 to 1.9 Pa is capable of influencing cellular morphology, and extracellular calcium might play a role in osteoblasts' response to FSS stimulation.

  D Song , X Liu , R Liu , L Yang , J Zuo and W. Liu

Connexin 43 (Cx43), known to be the main protein building blocks of gap junctions and hemichannels in mammalian heart, plays an important role in cardiocytes proliferation. Gap junctional intercellular communication has been suggested to be necessary for cellular proliferation and differentiation. However, the effect of Cx43 hemichannel on cardiocytes proliferation and the mechanism remain unclear. In this study, rat heart cell line H9c2 was used. The Cx43 location, the proliferation rate and hemichannel activity of H9c2 cells and Wnt-3a+-H9c2 cells were investigated and the changes of intracellular ATP and [Ca2+] were determined. Results showed that the inhibited hemichannel induced by 18β-glycyrrhetinic acid (GA) evoked intracellular ATP and [Ca2+] increase and enhanced H9c2 cell proliferation. Wnt-3a+-H9c2 cells displayed enhanced hemichannel activity and proliferation rate. Inhibited hemichannel of Wnt-3a+-H9c2 cells induced by 18β-GA decreased intracellular ATP, increased [Ca2+], and enhanced the proliferation of H9c2 cells. This study validated the role of hemichannel in H9c2 cell proliferation regulation, and showed a mechanism involved in the regulation of H9c2 cell proliferation. The proliferation could be enhanced by Cx43 hemichannel-mediated ATP release accompanying intracellular [Ca2+] change. However, different changes of ATP were observed in Wnt-3a+-H9c2 cells. These findings provided new insights into the molecular mechanisms of proliferation regulation in H9c2 cells and the effect of Wnt-3a on intracellular ATP.

  X Liu and R. Feng

The aberrant activation of c-Src regulates multiple functions during tumor progression. This study was conducted to investigate the role of c-Src suppression in epithelial to mesenchymal transition (EMT) process in human breast carcinoma cells. c-Src suppression by PP2 (a Src-family kinase inhibitor) or small interfering RNA (siRNA) was carried out in MCF-7 and MDA-MB-231 cells. Cell migration was analyzed by wound-healing assay. The transcription and protein levels of EMT markers and transcription factors were evaluated by reverse transcription-PCR and Western blot analysis, respectively. The changed cell morphology was photographed by light microscope. c-Src suppression by PP2 or siRNA reversed the mesenchymal-like phenotype in MDA-MB-231 cells. E-cadherin was upregulated in MCF-7 and MDA-MB-231 cells after c-Src suppression, whereas vimentin was downregulated in MDA-MB-231 cells. Slug and SIP1 were downregulated after c-Src suppression in MCF-7 and MDA-MB-231 cells, whereas Twist was unchanged. These results suggest that c-Src suppression by PP2 or siRNA may inhibit EMT through regulation of different transcription factors in breast carcinoma cells that have different metastatic potential.

  Q Liu , Z Dai , Z Liu , X Liu , C Tang , Z Wang , G Yi , L Liu , Z Jiang , Y Yang and Z. Yuan

It has been reported that oxidized low-density lipoprotein (Ox-LDL) can increase the expression of adipophilin. However, the detailed mechanisms are not fully understood. The aim of this study was to investigate the mechanism of Ox-LDL on adipophilin expression and the intracellular lipid droplet accumulation. A mouse macrophage-like cell line, RAW264.7, was used throughout, and it was found that Ox-LDL induced adipophilin expression in a dose-dependent manner. Moreover, Ox-LDL induced peroxisome proliferator-activated receptor- (PPAR) expression and PPAR-specific inhibitor T0070907 abrogated Ox-LDL-induced adipophilin expression, but specific agonist GW1929 not. Furthermore, Ox-LDL induced phosphorylation of ERK1/2, and ERK1/2-specific inhibition by PD98059 suppressed the Ox-LDL-induced PPAR and adipophilin expression. The results showed that ERK1/2 or PPAR-specific inhibition decreased the amounts of intracellular lipid droplets. Meanwhile, the PPAR-specific agonist increased intracellular lipid droplets. These results suggested that Ox-LDL-induced increase in adipophilin level via ERK1/2 activation is one of the mechanisms of inducing greater amounts of intracellular lipid droplets in RAW264.7 cells, which indicated that adipophilin is involved in atherosclerotic progression.

  X Nie , X Liu , H Wang and J. Chen

Phenotypic switching in Candida albicans spontaneously generates different cellular morphologies. The reversible switching between white and opaque phenotypes is regulated by multiple regulators including Efg1 and Wor1. In mating-type-like locus (MTL) homozygous cells, the Efg1 functions as a repressor, whereas the Wor1 acts as an activator in white–opaque switching. We presented evidence that switching between white and opaque in efg1/efg1 mutant is regulated by ambient pH. In pH 6.8 media, the efg1/efg1 mutant cells exhibited opaque form, but shifted to white form in pH 4.5 media. The pH-dependent morphological switching is not blocked by further deletion of WOR1 in the efg1/efg1 mutant. Correlated with the phenotype, the opaque-phase-specific gene OP4 was induced in efg1/efg1 mutant cells when cultured in pH 6.8 media, and was repressed in pH 4.5 media. Consistently, the MTLa efg1/efg1 mutant cells could mate efficiently with MTL cells in pH 6.8 media, but poorly in pH 4.5 media. Ectopic expression of the Rim101-405 allele in the efg1/efg1 mutant helped to bypass the pH restriction on white–opaque switching and show opaque form in both neutral and acidic media. We proposed that relief of the Efg1 repression enables C. albicans to undergo white–opaque switching in pH-dependent regulation mediated by Rim101-signaling pathway.

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

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

  B Lu , N Congdon , X Liu , K Choi , D. S. C Lam , M Zhang , M Zheng , Z Zhou , L Li , A Sharma and Y. Song

Objective  To study the associations between near work, outdoor activity, and myopia among children attending secondary school in rural China.

Methods  Among a random cluster sample of 1892 children in Xichang, China, subjects with an uncorrected acuity of 6/12 or less in either eye (n = 984) and a 25% sample of children with normal vision (n = 248) underwent measurement of refractive error. Subjects were administered a questionnaire on parental education, time spent outdoors, and weekly time spent engaged in and preferred working distance for a variety of near-work activities.

Results  Among 1232 children with refraction data, 998 (81.0%) completed the near-work survey. Their mean age was 14.6 years (SD, 0.8 years), 55.6% were girls, and 83.1% had myopia of –0.5 diopters or less (more myopia) in both eyes. Time and diopter-hours spent on near activities did not differ between children with and without myopia. In regression models, time spent on near activities and time outdoors were unassociated with myopia, adjusting for age, sex, and parental education.

Conclusions  These and other recent results raise some doubts about the association between near work and myopia. Additional efforts to identify other environmental factors associated with myopia risk and that may be amenable to intervention are warranted.

  M Zhang , N Congdon , L Li , Y Song , K Choi , Y Wang , Z Zhou , X Liu , A Sharma , W Chen and D. S. C. Lam

Objective  To study the effect of myopia and spectacle wear on bicycle-related injuries in rural Chinese students. Myopia is common among Chinese students but few studies have examined its effect on daily activities.

Methods  Data on visual acuity, refractive error, current spectacle wear, and history of bicycle use and accidents during the past 3 years were sought from 1891 students undergoing eye examinations in rural Guangdong province.

Results  Refractive and accident data were available for 1539 participants (81.3%), among whom the mean age was 14.6 years, 52.5% were girls, 26.8% wore glasses, and 12.9% had myopia of less than –4 diopters in both eyes. More than 90% relied on bicycles to get to school daily. A total of 2931 accidents were reported by 423 participants, with 68 requiring medical attention. Male sex (odds ratio, 1.55; P < .001) and spectacle wear (odds ratio, 1.38; P = .04) were associated with a higher risk of accident, but habitual visual acuity and myopia were unassociated with the crash risk, after adjusting for age, sex, time spent riding, and risky riding behaviors.

Conclusion  These results may be consistent with data on motor vehicle accidents implicating peripheral vision (potentially compromised by spectacle wear) more strongly than central visual acuity in mediating crash risk.

  F Li , P Yang , X Liu , C Wang , S Hou and A. Kijlstra

Objectives  To analyze the expression and potential role of interleukin (IL) 21 in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.

Methods  Blood samples were obtained from patients with VKH disease and from healthy control subjects. Serum IL-21 level and IL-21 messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) were determined by enzyme-linked immunosorbent assay and by reverse transcriptase–polymerase chain reaction, respectively. Interleukin 17 and interferon levels in the supernatants of PBMCs and CD4+ T cells cultured with anti-CD3 and anti-CD28 antibodies in the presence or absence of recombinant IL-21 were detected by enzyme-linked immunosorbent assay.

Results  The results showed a significantly increased serum IL-21 level, as well as higher IL-21 mRNA expression by PBMCs, in patients having chronic or recurrent active VKH disease compared with patients having inactive VKH disease and with controls. In vitro experiments showed that recombinant IL-21 significantly increased IL-17 production by PBMCs and by CD4+ T cells from patients and from controls. However, recombinant IL-21 did not affect interferon expression by PBMCs or by CD4+ T cells.

Conclusion  Interleukin 21 may be involved in the pathogenesis of chronic or recurrent VKH disease, possibly by promoting IL-17 secretion.

Clinical Relevance  Findings from the present study suggest that IL-21 may be a potential target in the development of therapy for VKH disease.

  X Liu , Y Han , D Yuen and B. Ma

Motivation: The bottom-up tandem mass spectrometry (MS/MS) is regularly used in proteomics nowadays for identifying proteins from a sequence database. De novo sequencing software is also available for sequencing novel peptides with relatively short sequence lengths. However, automated sequencing of novel proteins from MS/MS remains a challenging problem.

Results: Very often, although the target protein is novel, it has a homologous protein included in a known database. When this happens, we propose a novel algorithm and automated software tool, named Champs, for sequencing the complete protein from MS/MS data of a few enzymatic digestions of the purified protein. Validation with two standard proteins showed that our automated method yields >99% sequence coverage and 100% sequence accuracy on these two proteins. Our method is useful to sequence novel proteins or ‘re-sequence’ a protein that has mutations comparing with the database protein sequence.

  X Liu

To evaluate the feasibility, acceptability and effects of a Tai Chi and Qigong medical exercise programme that aimed to improve indicators of metabolic syndrome and glycaemic control in adults with raised blood glucose levels.

Design, setting, and participants:

A single-group pre-post trial of 11 participants (3 men and 8 women; age 42–65 years) with raised blood glucose levels conducted from August to November 2005 at a university in Australia.


Participants attended Tai Chi and Qigong exercise training for 1–1.5 h 3 times per week for 12 weeks and were encouraged to practise the exercises at home.

Main outcome measures:

Indicators of metabolic syndrome (body mass index, waist circumference, blood pressure, fasting blood glucose, triglycerides, HDL-cholesterol) and glucose control (HbA1c, fasting insulin and insulin resistance).


There was good adherence and high acceptability for the group-based programme. Significant improvements were seen in four of the seven indicators of metabolic syndrome including body mass index (mean difference –1.05 (95% CI –1.48 to –0.63), p<0.001), waist circumference (–2.80 cm (95% CI –4.97 to –0.62), p<0.05) and both systolic (–11.64 mm Hg (95% CI –19.46 to –3.51), p<0.01) and diastolic blood pressure (–9.73 mm Hg (95% CI –13.58 to –5.88), p<0.001). There were also small improvements in HbA1c (–0.32% (95% CI –0.49% to –0.15%), p<0.01), fasting insulin (–9.93 pmol/l (95% CI –19.93 to 0.07), p = 0.051) and insulin resistance (–0.53 (95% CI –0.97 to –0.09), p<0.05).


The programme was shown to be feasible and acceptable and the findings suggest that it may be helpful for the control of indicators of metabolic syndrome and glycaemic control. Larger controlled studies are needed to confirm these promising results.

  X Liu , Y. D Miller , N. W Burton and W. J. Brown

To evaluate the feasibility, acceptability and effects of a Tai Chi and Qigong exercise programme in adults with elevated blood glucose.

Design, Setting, and Participants

A single group pre–post feasibility trial with 11 participants (3 male and 8 female; aged 42–65 years) with elevated blood glucose.


Participants attended Tai Chi and Qigong exercise training for 1 to 1.5 h, 3 times per week for 12 weeks, and were encouraged to practise the exercises at home.

Main Outcome Measures

Indicators of metabolic syndrome (body mass index (BMI), waist circumference, blood pressure, fasting blood glucose, triglycerides, HDL-cholesterol); glucose control (HbA1c, fasting insulin and insulin resistance (HOMA)); health-related quality of life; stress and depressive symptoms.


There was good adherence and high acceptability. There were significant improvements in four of the seven indicators of metabolic syndrome including BMI (mean difference –1.05, p<0.001), waist circumference (–2.80 cm, p<0.05), and systolic (–11.64 mm Hg, p<0.01) and diastolic blood pressure (–9.73 mm Hg, p<0.001), as well as in HbA1c (–0.32%, p<0.01), insulin resistance (–0.53, p<0.05), stress (–2.27, p<0.05), depressive symptoms (–3.60, p<0.05), and the SF-36 mental health summary score (5.13, p<0.05) and subscales for general health (19.00, p<0.01), mental health (10.55, p<0.01) and vitality (23.18, p<0.05).


The programme was feasible and acceptable and participants showed improvements in metabolic and psychological variables. A larger controlled trial is now needed to confirm these promising preliminary results.

  T. M Pini , M. R Griffin , C. L Roumie , M. M Huizinga , J. H Fowke , R Greevy , X Liu and H. J. Murff

Thiazolidinediones (TZD) have been shown to down-regulate prostate-specific antigen (PSA) levels in prostate cancer cell lines and decrease PSA velocity among prostate cancer patients; however, the effect of TZDs on serum PSA levels among men with diabetes at risk for prostate cancer is unknown. We conducted a retrospective cohort study of veterans receiving care for diabetes between 1999 and 2005 to determine if TZD use affects PSA levels in veterans at risk for prostate cancer. Eligible patients were male, ≥45 years old, taking at least one oral antidiabetic medication, and with two or more recorded PSA values. Patients with a prior history of prostate cancer or prostatectomy were excluded. Of the 13,791 patients included in the adjusted analysis, 2,016 (14.6%) were prescribed a TZD. No effect of cumulative TZD dose on change in PSA was detected (P = 0.26). Increased TZD exposure was not associated with a change in PSA, suggesting that TZD treatment for diabetes is unlikely to affect prostate cancer detection. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1937–8)

  Z Cao , A Kozielski , X Liu , Y Wang , D Vardeman and B. Giovanella

To find a more effective and less toxic chemotherapeutic agent, we have successfully prepared crystalline camptothecin-20(S)-O-propionate hydrate (CZ48) by reacting camptothecin with propionic anhydride using concentrated sulfuric acid as catalyst. The biological effectiveness of this new anticancer agent was evaluated by using xenografts of human cancers in nude mice as the testing models. The extensive treatment of 21 human tumors with various dose levels of CZ48 has shown that this agent is highly effective against many different human tumors tested with a striking lack of toxicity. Of the 21 human tumor lines tested, 9 regressed, 5 were <10% of the control, 3 were <20%, and 2 were <40%. Two tumors did not respond. The total response rate was 90% (19 of 21). No toxicity was observed in mice. The effective doses required to achieve the positive response varied from 100 to 1,000 mg/kg/d depending on the tumors. The maximum tolerated dose was not reached because of the nontoxic nature of the drug in mice. Thus, this compound has a much wider therapeutic index compared with that of the existing anticancer drugs currently in use. [Cancer Res 2009;69(11):4742–9]

  S. M Mense , B Singh , F Remotti , X Liu and H. K. Bhat

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17β-estradiol (E2), the antioxidant vitamin C, the estrogen metabolic inhibitor -naphthoflavone (ANF), or cotreated with E2 + vitamin C or E2 + ANF for up to 8 months. E2 (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E2, E2 + vitamin C and E2 + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E2-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F2 formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E2 and decreased formation of the highly carcinogenic 4-OHE2. These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E2 metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

  C Yang , D Ji , E. J Weinstein , E Choy , F. J Hornicek , K. B Wood , X Liu , H Mankin and Z. Duan

Osteosarcoma is the most common primary malignant bone tumor affecting children and adolescents. The majority of patients are treated by surgery and chemotherapy but have limited alternative therapeutic options. Kinases play an important role in the growth and survival of tumor cells. We aim to identify specific kinases to be vital in the survival of osteosarcoma cells and thus may be a key target in creating novel anticancer therapies. A lentiviral short hairpin RNA kinase library, screened osteosarcoma cells, identified kinase minibrain-related kinase (Mirk) (Dyrk1B) as a potential target. Knockdown Mirk expression could inhibit cell growth and induce apoptosis. Chemically synthetic small interfering RNA knockdown and complementary DNA rescue assay further confirmed the results from the decrease of Mirk gene expression. The relationship between Mirk gene expression and the clinical characteristics of patients with osteosarcoma was investigated using tissue microarray and immunohistochemistry analysis. The data indicate that the overall survival rate of patients with Mirk high staining (high levels of Mirk protein expression) is significantly shorter than those with Mirk low staining and moderate staining. This highlights Mirk’s potential to serve as a promising target for molecular therapy in the treatment of osteosarcoma.

  Y Cheng , X Liu , J Yang , Y Lin , D. Z Xu , Q Lu , E. A Deitch , Y Huo , E. S Delphin and C. Zhang

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. Recently, we have found that microRNA (miRNA) miR-145 is the most abundant miRNA in normal vascular walls and in freshly isolated VSMCs; however, the role of miR-145 in VSMC phenotypic modulation and vascular diseases is currently unknown. Here we find that miR-145 is selectively expressed in VSMCs of the vascular wall and its expression is significantly downregulated in the vascular walls with neointimal lesion formation and in cultured dedifferentiated VSMCs. More importantly, both in cultured rat VSMCs in vitro and in balloon-injured rat carotid arteries in vivo, we demonstrate that the noncoding RNA miR-145 is a novel phenotypic marker and a novel phenotypic modulator of VSMCs. VSMC differentiation marker genes such as SM -actin, calponin, and SM-MHC are upregulated by premiR-145 or adenovirus expressing miR-145 (Ad-miR-145) but are downregulated by the miR-145 inhibitor 2'OMe-miR-145. We have further identified that miR-145-mediated phenotypic modulation of VSMCs is through its target gene KLF5 and its downstream signaling molecule, myocardin. Finally, restoration of miR-145 in balloon-injured arteries via Ad-miR-145 inhibits neointimal growth. We conclude that miR-145 is a novel VSMC phenotypic marker and modulator that is able of controlling vascular neointimal lesion formation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.

  X Liu , O Vilenski , J Kwan , S Apparsundaram and R. Weikert

It is a commonly accepted hypothesis that central nervous system (CNS) activity is determined by the unbound brain drug concentration. However, limited experimental data are available in the literature to support this hypothesis. The objective of this study was to test this hypothesis by examining the relationship between in vitro binding affinity (KI) and in vivo activity quantified as the drug concentration occupying 50% of the transporters (OC50) for 18 serotonin (SERT) and dopamine transporter (DAT) inhibitors. In vivo rat OC50 was determined by autoradiography using [3H]N,N-dimethyl-2,2-amino-4-cyanophenylthiobenzylamine and [3H](–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) as the ligands to assess SERT and DAT occupancy, respectively. The unbound brain concentrations were calculated from total brain concentrations and the unbound brain fraction, which was determined by the brain homogenate method. The in vivo total brain SERT and DAT OC50 values (mean ± S.D.) were 408 ± 368- and 410 ± 395-fold greater than the KI values, respectively. In contrast, the in vivo unbound brain SERT and DAT OC50 values were only 3.3 ± 2.1- and 4.1 ± 4.0-fold different from the KI values. Therefore, prediction of the biophase drug concentration by using the unbound brain concentration rather than the total brain concentration results in an approximately 100-fold improvement for the accuracy. In the present study, a 10-fold improvement was also observed by using the unbound plasma concentration rather than the total plasma concentration to predict the biophase concentration in the brain. This study supports the hypothesis that CNS activity is more accurately determined by the unbound brain drug concentration and not by the total brain drug concentration.

  X Liu , O Vilenski , J Kwan , S Apparsundaram and R. Weikert

It is a commonly accepted hypothesis that central nervous system (CNS) activity is determined by the unbound brain drug concentration. However, limited experimental data are available in the literature to support this hypothesis. The objective of this study was to test this hypothesis by examining the relationship between in vitro binding affinity (KI) and in vivo activity quantified as the drug concentration occupying 50% of the transporters (OC50) for 18 serotonin (SERT) and dopamine transporter (DAT) inhibitors. In vivo rat OC50 was determined by autoradiography using [3H]N,N-dimethyl-2,2-amino-4-cyanophenylthiobenzylamine and [3H](–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) as the ligands to assess SERT and DAT occupancy, respectively. The unbound brain concentrations were calculated from total brain concentrations and the unbound brain fraction, which was determined by the brain homogenate method. The in vivo total brain SERT and DAT OC50 values (mean ± S.D.) were 408 ± 368- and 410 ± 395-fold greater than the KI values, respectively. In contrast, the in vivo unbound brain SERT and DAT OC50 values were only 3.3 ± 2.1- and 4.1 ± 4.0-fold different from the KI values. Therefore, prediction of the biophase drug concentration by using the unbound brain concentration rather than the total brain concentration results in an approximately 100-fold improvement for the accuracy. In the present study, a 10-fold improvement was also observed by using the unbound plasma concentration rather than the total plasma concentration to predict the biophase concentration in the brain. This study supports the hypothesis that CNS activity is more accurately determined by the unbound brain drug concentration and not by the total brain drug concentration.

  J. H Quennell , A. C Mulligan , A Tups , X Liu , S. J Phipps , C. J Kemp , A. E Herbison , D. R Grattan and G. M. Anderson

The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 µg leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.

  X Liu , Y. X Fu , T. J Maxwell and E. Boerwinkle

It is known that sequencing error can bias estimation of evolutionary or population genetic parameters. This problem is more prominent in deep resequencing studies because of their large sample size n, and a higher probability of error at each nucleotide site. We propose a new method based on the composite likelihood of the observed SNP configurations to infer population mutation rate = 4Neµ, population exponential growth rate R, and error rate , simultaneously. Using simulation, we show the combined effects of the parameters, , n, , and R on the accuracy of parameter estimation. We compared our maximum composite likelihood estimator (MCLE) of with other estimators that take into account the error. The results show the MCLE performs well when the sample size is large or the error rate is high. Using parametric bootstrap, composite likelihood can also be used as a statistic for testing the model goodness-of-fit of the observed DNA sequences. The MCLE method is applied to sequence data on the ANGPTL4 gene in 1832 African American and 1045 European American individuals.

  M. H Tan , A. J Smith , B Pawlyk , X Xu , X Liu , J. B Bainbridge , M Basche , J McIntosh , H. V Tran , A Nathwani , T Li and R. R. Ali

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are clinically heterogeneous and present as Leber Congenital Amaurosis, the severest form of early-onset retinal dystrophy and milder forms of retinal dystrophies such as juvenile retinitis pigmentosa and dominant cone-rod dystrophy. [Perrault, I., Rozet, J.M., Gerber, S., Ghazi, I., Leowski, C., Ducroq, D., Souied, E., Dufier, J.L., Munnich, A. and Kaplan, J. (1999) Leber congenital amaurosis. Mol. Genet. Metab., 68, 200–208.] Although not yet fully elucidated, AIPL1 is likely to function as a specialized chaperone for rod phosphodiesterase (PDE). We evaluate whether AAV-mediated gene replacement therapy is able to improve photoreceptor function and survival in retinal degeneration associated with AIPL1 defects. We used two mouse models of AIPL1 deficiency simulating three different rates of photoreceptor degeneration. The Aipl1 hypomorphic (h/h) mouse has reduced Aipl1 levels and a relatively slow degeneration. Under light acceleration, the rate of degeneration in the Aipl1 h/h mouse is increased by 2–3-fold. The Aipl1–/– mouse has no functional Aipl1 and has a very rapid retinal degeneration. To treat the different rates of degeneration, two pseudotypes of recombinant adeno-associated virus (AAV) exhibiting different transduction kinetics are used for gene transfer. We demonstrate restoration of cellular function and preservation of photoreceptor cells and retinal function in Aipl1 h/h mice following gene replacement therapy using an AAV2/2 vector and in the light accelerated Aipl1 h/h model and Aipl1–/– mice using an AAV2/8 vector. We have thus established the potential of gene replacement therapy in varying rates of degeneration that reflect the clinical spectrum of disease. This is the first gene replacement study to report long-term rescue of a photoreceptor-specific defect and to demonstrate effective rescue of a rapid photoreceptor degeneration.

  X Liu , B Wang , X Ma and Y. Guo

Nuclear factor-B (NF-B) activation has been identified in a variety of solid tumors and lymphoid malignancies. The aim of our study was to determine the expression status and clinical significance of NF-B in extranodal natural killer (NK)/T-cell lymphoma, nasal type.


Tumor specimens from 23 patients with previously untreated NK/T-cell lymphoma initially treated with cyclophosphamide, vincristine, doxorubicin and prednisone (CHOP) or CHOP-based chemotherapy were examined by immunohistochemistry for three NF-B subunits (p65, p50 and p52), which are involved in either the canonical or alternative pathway.


None of the cases could be detected with p65 or p50 nuclear staining. On the other hand, 15 (65.2%) cases had p52 nuclear staining, suggesting NF-B activation through the alternative pathway. All major clinical characteristics were balanced between NF-B p52-positive and -negative patients. The objective response rate achieved in NF-B-positive patients was significantly lower than that in negative patients (33.3% vs. 87.5%, P = 0.027). At a median follow-up of 25 months, 8 (53.3%) of 15 NF-B-positive patients had died compared with none of 8 NF-B-negative patients (P = 0.041). In a multivariate analysis, NF-B status and stage were identified to be independent prognostic factors.


Our results suggest that NF-B activation through the alternative pathway is frequently observed in NK/T-cell lymphoma and associated with chemoresistance and poor survival.

  W Yang , S Lv , X Liu , H Liu and F. Hu

T-cell lymphoma invasion and metastasis 1 (Tiam1) specifically activates Rho-like GTPases (e.g. Rac1) and Tiam1–Rac1 pathway affects the migration and invasion of many tumors, such as nasopharyngeal carcinoma, breast cancer and retinoblastoma. However, no studies have yet comprehensively examined the involvement of Tiam1–Rac1 pathway in hepatocellular carcinoma. In this study, we examined the relationship of the up-regulation of Tiam1 and Rac1 with clinicopathological features in patients with hepatocellular carcinoma.


Expression of Tiam1 and Rac1 was assessed in 242 hepatocellular carcinoma tissues and their adjacent normal hepatic tissues by performing immunohistochemistry and was gauged regarding stage, grade and survival.


Immunohistochemistry showed that patients with a high clinical stage hepatocellular carcinoma (III–IV) and -fetoprotein levels had a higher tendency to express Tiam1 and Rac1 on tumor cells than the patients with low pathologic grade hepatocellular carcinoma (I–II) (P = 0.008 and 0.01, respectively) and low -fetoprotein levels (P = 0.006 and 0.002, respectively). In addition, Tiam1 and Rac1 up-regulation was also significantly associated with vascular invasion status (both P = 0.02), intrahepatic metastasis status (P = 0.009 and 0.01, respectively) and histological differentiation (P = 0.008 and 0.009, respectively) of patients with hepatocellular carcinoma. Moreover, post-operative survival analysis indicated that hepatocellular carcinoma patients with strong Tiam1 (P = 0.01) and Rac1 (P = 0.02) expression had shorter disease-specific survival than those with weak expression. Multivariate analysis also showed that Tiam1 and Rac1 overexpression could be two predictors of poor prognosis (P = 0.02 and 0.03, respectively).


The current study demonstrated for the first time that the Tiam1–Rac1 pathway may play a critical role in tumor progression of hepatocellular carcinoma. The expression of Tiam1 and Rac1 can be considered as the two useful indicators for predicting the prognosis of hepatocellular carcinoma.

  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.

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

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

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

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

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

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

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

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

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

  C Tian , J Tan , X Wu , W Ye , X Liu , D Li and H. Yang

To describe the variation in bacterioplankton diversity within a large hypertrophic freshwater lake, as well as changes in the diversity that occurred with time, PCR- (denaturing gradient gel electrophoresis) DGGE was utilized to study water samples collected from Lake Taihu in China. To accomplish this, water samples were collected from different locations and during different months. The trophic status of these sampling sites ranged from eutrophic to hypertrophic. Cluster and multidimensional scaling analyses revealed that the temporal transition in the diversity of the bacterioplankton occurred primarily in response to a cyanobacterial bloom, and that all samples could be divided into normal-bloom, peak-bloom and winter period groups. Spatial differences in the bacterial diversity were also detected among the three sampling sites, with diversity being found to be strongly correlated with the gradient of the trophic status of the three sampling sites. In addition, these temporal and spatial changes could be characterized by several specific DGGE bands. The results were further analyzed by canonical correspondence analysis, which revealed that the bacterioplankton diversity of Lake Taihu was primarily associated with temperature, pH, total nitrogen (TN), total phosphorus (TP) and dissolved oxygen. Of these factors, TN and TP were only shown to be significant influencing factors at Wuxi, which had the highest trophic level.

  P Dai , A. K Stewart , F Chebib , A Hsu , J Rozenfeld , D Huang , D Kang , V Lip , H Fang , H Shao , X Liu , F Yu , H Yuan , M Kenna , D. T Miller , Y Shen , W Yang , I Zelikovic , O. S Platt , D Han , S. L Alper and B. L. Wu

Mutations of the human SLC26A4/PDS gene constitute the most common cause of syndromic and nonsyndromic hearing loss. Definition of the SLC26A4 mutation spectrum among different populations with sensorineural hearing loss is important for development of optimal genetic screening services for congenital hearing impairment. We screened for SLC26A4 mutations among Chinese and U.S. subjects with hearing loss, using denaturing HPLC (DHPLC) and direct DNA sequencing. Fifty-two of 55 Chinese subjects with deafness accompanied by enlargement of the vestibular aqueduct (EVA) exhibited at least one mutant SLC26A4 allele, whereas SLC26A4 mutations were found in only 2 of 116 deaf Chinese patients without EVA. The spectrum of SLC26A4 mutations differed among Chinese and U.S. subjects and included 10 previously unreported SLC26A4 variants: 4 in the Chinese population (p.E303Q, p.X329, p.X467, p.X573) and 6 in the U.S. population (p.V250A, p.D266N, p.F354S, p.D697A, p.K715N, p.E737D). Among the seven novel in-frame missense mutations, five encoded SLC26A4 proteins with substantially reduced Cl/anion exchange activity as expressed and measured in Xenopus oocytes, but four of these were sufficiently active to allow study of anion selectivity. The only mutant polypeptide exhibiting complete loss of anion exchange function, p.E303Q, was expressed at or near the oocyte surface at near-wild-type levels. Two variants, p.F354S and p.E737D, displayed selective reduction in relative rate of Cl/HCO3 exchange compared with similarly measured rates of Cl/Cl and Cl/I exchange. Our data show that mutation analysis of the SLC26A4 gene is of high diagnostic yield among subjects with deafness and bilateral EVA in both China and the U.S. However, the pathogenicity of monoallelic SLC26A4 gene variants in patients with hearing loss remains unclear in many instances.

  X Liu and J. R. Ritter

Using a sample of fifty-six companies going public in 1996–2000 in which top executives received allocations of other hot initial public offerings (IPOs) from the bookrunner, a practice known as spinning, we examine the consequences of spinning. The fifty-six IPOs had first-day returns that were, on average, 23% higher than similar IPOs. The profits collected by these executives were only a small fraction of the incremental amount of money left on the table by their companies when they went public. These companies were dramatically less likely to switch investment bankers in a follow-on offer: only 6% of issuers whose executives were spun switched underwriters, whereas 31% of other issuers switched. These findings suggest that the spinning of executives accomplished its goal of affecting corporate decisions.

  R. P Rhoads , R. M Johnson , C. R Rathbone , X Liu , C Temm Grove , S. M Sheehan , J. B Hoying and R. E. Allen

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1 (HIF-1), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1 activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

  X Liu , Y Cheng , J Yang , T. J Krall , Y Huo and C. Zhang

It is well established that vascular smooth muscle cell (VSMC) apoptosis and proliferation are critical cellular events in a variety of human vascular diseases. However, the molecular mechanisms involved in controlling VSMC apoptosis and proliferation are still unclear. In the current study, we have found that programmed cell death 4 (PDCD4) is significantly downregulated in balloon-injured rat carotid arteries in vivo and in platelet-derived growth factor-stimulated VSMCs in vitro. Overexpression of PDCD4 via adenovirus (Ad-PDCD4) increases VSMC apoptosis in an apoptotic model induced by serum deprivation. In contrast, VSMC apoptosis is significantly decreased by knockdown of PDCD4 via its small interfering RNA. In the rat carotid arteries in vivo, VSMC apoptosis is increased by Ad-PDCD4. We have further identified that activator protein 1 is a downstream signaling molecule of PDCD4 that is associated with PDCD4-mediated effects on VSMC apoptosis. In addition, VSMC proliferation was inhibited by overexpression of PDCD4. The current study has identified, for the first time, that PDCD4 is an essential regulator of VSMC apoptosis and proliferation. The downregulation of PDCD4 expression in diseased vascular walls may be responsible for the imbalance of VSMC proliferation and apoptosis. The results indicate that PDCD4 may be a new therapeutic target in proliferative vascular diseases.

  G Twig , X Liu , M Liesa , J. D Wikstrom , A. J. A Molina , G Las , G Yaniv , G Hajnoczky and O. S. Shirihai

Studies in various types of cells find that, on average, each mitochondrion becomes involved in a fusion event every 15 min, depending on the cell type. As most contact events do not result in mitochondrial fusion, it is expected that properties of the individual mitochondrion determine the likelihood of a fusion event. However, apart from membrane potential, the properties that influence the likelihood of entering a fusion event are not known. Here, we tag and track individual mitochondria in H9c2, INS1, and primary β-cells and determine the biophysical properties that increase the likelihood of a fusion event. We found that the probability for fusion is independent of contact duration and organelle dimensions, but it is influenced by organelle motility. Furthermore, the history of a previous fusion event of the individual mitochondrion influenced both the likelihood for a subsequent fusion event, as well as the site on the mitochondrion at which the fusion occurred. These observations unravel the specific properties that distinguish mitochondria that will enter fusion events from the ones that will not. Altogether, these properties may help to elucidate the molecular mechanisms that regulate fusion at the level of the single mitochondrion.

  Y Takeda , X Liu , M Sumiyoshi , A Matsushima , M Shimohigashi and Y. Shimohigashi

Estrogen-related receptor (ERR), one of the 48 human nuclear receptors, has a fully active conformation with no ligand. We recently demonstrated that ERR binds strongly bisphenol A (BPA), one of the nastiest endocrine disruptors, and thus retaining ERR's high basal constitutive activity. A report that BPA accumulates in the human maternal–fetal placental unit has led us to hypothesize that a large amount of ERR might exist in the human placenta. Here we report evidence that placenta indeed expresses ERR exceptionally strongly. We first ascertained the presence of nine different ERR mRNA variants and the resulting three ERR protein isoforms. By real-time PCR, we estimated the relative amount of ERR mRNA using total RNA extracts from human reproductive tissues. Placenta was found to express ERR extremely highly. Among the three ERR protein isoforms, placenta exclusively expresses the type-1 isoform, which possesses additional 23-mer amino-acid residues at the N-terminus of the ordinary ERR. This N-terminal elongation was found to elevate by approximately 50% the basal constitutive activity of ERR, as evidenced in the luciferase reporter gene assay. The present results suggest that BPA accumulates in the placenta by binding to ERR.

  X Liu , Q Luo , G Zhong , M Rizwan ul Haq and M. Hu

Some chemosensory proteins (CSPs) expressed in insect sensory appendages are thought to be involved in chemical signaling in moths. We cloned and characterized four CSP genes from Plutella xylostella. The deduced amino acid sequences of PxylCSP1, PxylCSP2, PxylCSP3 and PxylCSP4 revealed open reading frames of 152, 128, 126 and 126 amino acids, respectively, with four conserved cysteine residues. The expression patterns of the four PxylCSP genes were further investigated by reverse transcription (RT) PCR and real-time PCR. PxylCSP1 and PxylCSP2 genes were expressed in all the tested tissues with the highest expression level in the antennae and heads (without antennae) whereas PxylCSP3 and PxylCSP4 mRNA were distributed extensively in all the tested tissues without apparent quantitative differences. The transcription levels of these CSP genes depended on sex, age, mating and the genes. Fluorescence quenching with Rhodojaponin-III (R-III) and homology modelling studies indicated that PxylCSP1 was able to bind non-volatile oviposition deterrents, such as R-III. These ubiquitous proteins might have the role of extracting non-volatile compounds (oviposition deterrents or antifeedants) dispersed in the environment and transporting them to their receptor.

  X Liu , A Matsushima , H Okada and Y. Shimohigashi

Bisphenol A (BPA) strongly binds to human estrogen-related receptor (ERR). BPA is an oestrogenic endocrine disruptor that influences various physiological functions at very low doses. BPA functions as an inverse-type antagonist of ERR to retain its high basal constitutive activity by inhibiting the deactivating inverse agonist activity of 4-hydroxytamoxifen (4-OHT). We recently demonstrated that ERR receptor residues Glu275 and Arg316 function as the intrinsic binding site of BPA’s phenol-hydroxyl group. We also determined the chief importance of phenol-hydroxylArg316 hydrogen bonding and the corroborative role of phenol-hydroxylGlu275 hydrogen bonding. However, there appeared to be a distinct difference between the receptor binding modes of BPA and 4-OHT. In the present study, using tritium-labelled or non-labelled BPA and 4-OHT, we evaluated in detail the receptor binding capabilities of wild-type ERR and its mutants with amino acid alterations at positions 275 and 316. Both compounds exhibited a strong binding ability to wild-type ERR due to the hydrogen bonding to Glu275 and Arg316. However, 4-OHT revealed significantly reduced occupancy for both wild-type and mutant receptors. The data obtained suggest that 4-OHT barely binds to ERR due to the strong ability of Glu275 and Arg316 to recruit phenol compounds.

  X Liu , C. O. C Bellamy , M. A Bailey , L. J Mullins , D. R Dunbar , C. J Kenyon , G Brooker , S Kantachuvesiri , K Maratou , A Ashek , A. F Clark , S Fleming and J. J. Mullins

Severe forms of hypertension are characterized by high blood pressure combined with end organ damage. Through the development and refinement of a transgenic rat model of malignant hypertension incorporating the mouse renin gene, we previously identified a quantitative trait locus on chromosome 10, which affects malignant hypertension severity and morbidity. We next generated an inducible malignant hypertensive model where the timing, severity, and duration of hypertension was placed under the control of the researcher, allowing development of and recovery from end organ damage to be investigated. We have now generated novel consomic Lewis and Fischer rat strains with inducible hypertension and additional strains that are reciprocally congenic for the refined chromosome 10 quantitative trait locus. We have captured a modifier of end organ damage within the congenic region and, using a range of bioinformatic, biochemical and molecular biological techniques, have identified angiotensin-converting enzyme as the modifier of hypertension-induced tissue microvascular injury. Reciprocal differences between angiotensin-converting enzyme and the anti-inflammatory tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro in the kidney, a tissue susceptible to end organ damage, suggest a mechanism for the amelioration of hypertension-dependent damage.

  O Penack , O. M Smith , A Cunningham Bussel , X Liu , U Rao , N Yim , I. K Na , A. M Holland , A Ghosh , S. X Lu , R. R Jenq , C Liu , G. F Murphy , K Brandl and M. R.M. van den Brink

Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2–/– hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function.

  O Schulz , E Jaensson , E. K Persson , X Liu , T Worbs , W. W Agace and O. Pabst

Chemokine receptor CX3CR1+ dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103+ DCs in the mucosa, which, through their ability to synthesize retinoic acid (RA), appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103+ DC, CX3CR1+ cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103+ DCs, but not CX3CR1-expressing cells, migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover, CX3CR1+ cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103+ DC. These findings indicate that selectively CD103+ DCs serve classical DC functions and initiate adaptive immune responses in local LNs, whereas CX3CR1+ populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens.

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