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Articles by J Zhang
Total Records ( 39 ) for J Zhang
  S Liu , K Kang , J Zhang , Q Ouyang , Z Zhou , S Tian and M. Xing
 

A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-like protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1–153 and bp 395–547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.

  J Zhang , Y Yang and J. Wu
 

PES1, the human homolog of zebrafish pescadillo, is a nucleolar protein that is essential for cell proliferation. We report herein that a nucleolar marker protein B23 physically interacts with PES1 and is involved in the nucleolar localization of PES1. In vivo interaction between B23 and PES1 was verified by co-immunoprecipitation of endogenous B23 and PES1 proteins, and they showed cellular co-localizations under both normal and actinomycin D-induced stress conditions. Furthermore, we mapped their interaction domains via in vitro pull-down assays. When B23 was knocked down by RNA interference, there appeared an increased nucleoplasmic distribution of PES1. Our results support a previous hypothesis that B23 might be a nucleolar hub protein for protein targeting to the nucleolus, and shed light on the nucleolar localization mechanism of PES1. The physical interaction between B23 and PES1 implies that they may participate in ribosome biogenesis in a protein complex.

  L Guo , W Ying , J Zhang , Y Yuan , X Qian , J Wang , X Yang and F. He
 

Mutations in the TSC1 and TSC2 genes lead to tuberous sclerosis complex (TSC), which is characterized clinically by mental retardation, epilepsy, and benign tumors affecting multiple tissues. Numerous components of the TSC protein complex remain uncharacterized. Here we report the purification of the TSC1 complex under physiological conditions using a proteomic strategy. We purified the TSC1 protein complex using a tandem affinity purification method and identified a protein complex containing 139 components. Two known binding proteins of TSC1 (TSC2 and DOCK7) were identified along with other new potential partners, which cover reported and novel TSC1 functional categories. Bioinformatics and biochemical methods were used to evaluate the observed protein–protein interactions. A comparative analysis with a published expression proteomics/genomics study of TSC1 revealed more than 20 common candidates that might be functionally relevant. The data set provides new directions in which to expand our knowledge of the functions of TSC1 and the mechanisms of TSC. The results are highly reliable, which is reflected by the identification of a few reported partners of TSC1 and many TSC1/2-regulated proteins. Interestingly, many new functional categories were identified, such as DNA repair, which provide novel hints to the function of TSC1. Moreover, a few neuronal disease-related proteins that might regulate the normal functions of neurons were identified. Thus, the results suggest that many of the new interactions should be biologically significance. It will be interesting to further investigate the regulatory mechanisms of these components.

  X Ren , J Zhang , X Gong , X Niu , X Zhang , P Chen and X. Zhang
 

The kidney is formed from two tissue populations derived from the intermediate mesoderm, the ureteric bud, and the metanephric mesenchyme. Metanephric mesenchyme is a pluripotent renal stem population, and conversion of renal mesenchyme into epithelia depends on the ureteric bud in vivo and in vitro. Embryonic stem (ES) cells have been induced to differentiate into a broad spectrum of specialized cell types in vitro, including hematopoietic, pancreatic, and neuronal cells. Such ES-derived cells can provide a valuable source of progenitor cells. However, whether ES cells can be stimulated by factors secreted from the fetal renal cells to differentiate into renal precursor cells in vitro has not been reported. In this study, we showed that murine ES cells can give rise to embryoid bodies in the absence of leukemia inhibitory factor. Culture conditions were optimized [6 days, 10 ng/ml activin and 10–7 M retinoic acid (RA)] to generate maximal mesoderm populations specifically expressing Pax2 and brachyury. Results showed that 72% of the cells were brachyury positive by fluorescent activated cell sorter on Day 6 of EB cell differentiation. Conditioned medium collected from cultures of ureteric bud cells from renal cells of a 13-day-old fetus was added to the culture medium. Mesoderm cells were cultured for up to 10 days before showing expression of renal markers, initiation of nephrogenesis (WT-1 and Pax2), and terminally differentiated renal cell types (POD-1 and E-cadherin). This study suggests that ES cells pre-treated by RA and activin can interact with secreted molecules of the fetal renal cells to specifically differentiate into renal precursor cells. Our results provide an experimental basis for the development of in vitro assays to steer differentiation of ES cells toward renal lineages.

  J. Q Gu , D. F Wang , X. G Yan , W. L Zhong , J Zhang , B Fan and S. Ikuyama
 

Excessive accumulation of lipids in macrophages results in formation of foam cells and is a hallmark of atherosclerosis. The PAT family of proteins has been implicated in this process, but details of their involvement in foam cell formation have not been fully elucidated. One of dominant members of the PAT proteins, perilipin 3 (TIP47), is likely to be involved in such a regulatory mechanism. In this study, we demonstrated that the Toll-like receptor 9 (TLR9)-mediated pathway stimulates perilipin 3 expression and accumulation of lipids, especially triglycerides, in macrophages. Oligodeoxynucleotide (ODN) 1826, a ligand of TLR9, significantly enhanced perilipin 3 expression in RAW264.7 cells, and chloroquine, a TLR9 inhibitor, almost completely inhibited ODN1826-induced perilipin 3 expression. The inhibitors of c-jun NH2-terminal kinase and PI 3-kinase suppressed the level of perilipin 3 mRNA induced by ODN1826. ODN1826 induced the expression of IL-1 and IFNβ, both of which increased perilipin 3 expression. Antibodies against these cytokines suppressed the ODN1826-induced perilipin 3 mRNA levels. These results suggest that the expression of perilipin 3 in macrophages is in part regulated through the TLR9-mediated mechanism. Furthermore, ODN1826 increased intracellular lipid accumulation in the presence of oxLDL, which was reduced by perilipin 3 siRNA. Perilipin 3 expression was not stimulated by oxLDL. Depletion of perilipin 3 by siRNA specifically reduced triglyceride content in the cells but not cholesterol content, indicating that perilipin 3 is involved mainly in triglyceride accumulation. In conclusion, the TLR9-mediated pathway facilitates foam cell formation in part through increased expression of perilipin 3.

  J Zhang , S Xiao and L. Zhou
  Objective

The authors examined the prevalence and distribution of mental disorders in rural Chinese 15–34 years of age who committed suicide. They hypothesized that mental illness is a risk factor for suicide in this population and that the prevalence of mental illness is lower in females than in males.

Method

In this case-control psychological autopsy study, face-to-face interviews were conducted to collect information from proxy informants for 392 suicide victims and 416 living comparison subjects. Five categories of DSM-IV mental disorders (mood disorders, schizophrenia and other psychotic disorders, substance use disorders, anxiety disorders, and other axis I disorders) at the time of death or interview were assessed using the Chinese version of the Structured Clinical Interview for DSM-IV. Sociodemographic variables, social support, and life events were also assessed.

Results

The prevalence of current mental illness was 48.0% for suicide victims and 3.8% for comparison subjects. Among suicide victims, mental illness was more prevalent in males than in females (55.1% compared with 39.3%). A strong association between mental illness and suicide was observed after adjustment for sociodemographic characteristics. Other risk factors included having a lower education level, not being currently married, having a lower level of social support, and having a history of recent and long-term life events. Additive interactions were observed between mental illness and lower level of social support.

Conclusions

Although mental illness is a strong risk factor for suicide, it is less prevalent among rural Chinese young people who committed suicide, particularly females, in comparison with other populations in China and in the West.

  Z Jia , X Huang , Q Wu , T Zhang , S Lui , J Zhang , N Amatya , W Kuang , R. C. K Chan , G. J Kemp , A Mechelli and Q. Gong
  Objective:

Suicide is a major social and public health problem, but its neurobiology in major depressive disorder is poorly understood. The purpose of this study was to use magnetic resonance diffusion tensor imaging to characterize abnormalities of white matter integrity in major depressive disorder patients with and without a history of suicide attempts.

Method:

Participants were 52 patients with major depressive disorder, with (N=16) and without (N=36) a history of suicide attempts, and 52 healthy comparison subjects matched for age, gender, education, and ethnicity. Diffusion tensor imaging in a 3.0 Tesla magnetic resonance scanner was performed. Whole-brain voxel-based analysis was used to compare fractional anisotropy across the three groups and analyze the correlation with symptom severity. A region-of-interest analysis was applied to the bilateral hippocampus, thalamus, and lentiform nucleus

Results:

Fractional anisotropy was decreased in the left anterior limb of the internal capsule in suicide attempters relative to both nonattempters and healthy comparison subjects, in the right frontal lobe relative to comparison subjects only, and in the right lentiform nucleus relative to nonattempters only. There was no significant correlation with symptom severity.

Conclusions:

Decreased fractional anisotropy in the left anterior limb of the internal capsule appears to characterize patients with major depressive disorder who have a history of attempting suicide. Longitudinal studies are required to validate this as a potential marker that may inform the development of strategies for reducing suicide.

  R Chowdhary , J Zhang and J. S. Liu
 

Motivation: Protein–protein interaction (PPI) extraction from published biological articles has attracted much attention because of the importance of protein interactions in biological processes. Despite significant progress, mining PPIs from literatures still rely heavily on time- and resource-consuming manual annotations.

Results: In this study, we developed a novel methodology based on Bayesian networks (BNs) for extracting PPI triplets (a PPI triplet consists of two protein names and the corresponding interaction word) from unstructured text. The method achieved an overall accuracy of 87% on a cross-validation test using manually annotated dataset. We also showed, through extracting PPI triplets from a large number of PubMed abstracts, that our method was able to complement human annotations to extract large number of new PPIs from literature.

Availability: Programs/scripts we developed/used in the study are available at http://stat.fsu.edu/~jinfeng/datasets/Bio-SI-programs-Bayesian-chowdhary-zhang-liu.zip

Contact: jliu@stat.harvard.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

  Y Zhang , J Zhang , L Wang , E Quealy , B. D Gary , R. C Reynolds , G. A Piazza and J. Lu
 

Nonsteroidal anti-inflammatory drugs including sulindac are well documented to be highly effective for cancer chemoprevention. However, their cyclooxygenase (COX)-inhibitory activities cause severe gastrointestinal, renal, and cardiovascular toxicities, limiting their chronic use. Recent studies suggest that COX-independent mechanisms may be responsible for the chemopreventive benefits of nonsteroidal anti-inflammatory drugs and support the potential for the development of a novel generation of sulindac derivatives lacking COX inhibition for cancer chemoprevention. A prototypic sulindac derivative with a N,N-dimethylammonium substitution called sulindac sulfide amide (SSA) was recently identified to be devoid of COX-inhibitory activity yet displays much more potent tumor cell growth-inhibitory activity in vitro compared with sulindac sulfide. In this study, we investigated the androgen receptor (AR) signaling pathway as a potential target for its COX-independent antineoplastic mechanism and evaluated its chemopreventive efficacy against prostate carcinogenesis using the transgenic adenocarcinoma of mouse prostate model. The results showed that SSA significantly suppressed the growth of human and mouse prostate cancer cells expressing AR in strong association with G1 arrest, and decreased AR level and AR-dependent transactivation. Dietary SSA consumption dramatically attenuated prostatic growth and suppressed AR-dependent glandular epithelial lesion progression through repressing cell proliferation in the transgenic adenocarcinoma of mouse prostate mice, whereas it did not significantly affect neuroendocrine carcinoma growth. Overall, the results suggest that SSA may be a chemopreventive candidate against prostate glandular epithelial carcinogenesis. Cancer Prev Res; 3(7); 885–95. ©2010 AACR.

  J Zhang , L Wang , L. B Anderson , B Witthuhn , Y Xu and J. Lu
 

Because the Selenium (Se) and Vitamin E Cancer Prevention Trial (SELECT) failed to show the efficacy of selenomethionine for prostate cancer prevention, there is a critical need to identify safe and efficacious Se forms for future trials. We have recently shown significant preventive benefit of methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) in the transgenic adenocarcinoma mouse prostate (TRAMP) model by oral administration. The present work applied iTRAQ proteomic approach to profile protein changes of the TRAMP prostate and to characterize their modulation by MSeA and MSeC to identify their potential molecular targets. Dorsolateral prostates from wild-type mice at 18 weeks of age and TRAMP mice treated with water (control), MSeA, or MSeC (3 mg Se/kg) from 8 to 18 weeks of age were pooled (9-10 mice per group) and subjected to protein extraction, followed by protein denaturation, reduction, and alkylation. After tryptic digestion, the peptides were labeled with iTRAQ reagents, mixed together, and analyzed by two-dimensional liquid chromatography/tandem mass spectrometry. Of 342 proteins identified with >95% confidence, the expression of 75 proteins was significantly different between TRAMP and wild-type mice. MSeA mainly affected proteins related to prostate functional differentiation, androgen receptor signaling, protein (mis)folding, and endoplasmic reticulum–stress responses, whereas MSeC affected proteins involved in phase II detoxification or cytoprotection, and in stromal cells. Although MSeA and MSeC are presumed precursors of methylselenol and were equally effective against the TRAMP model, their distinct affected protein profiles suggest biological differences in their molecular targets outweigh similarities. Cancer Prev Res; 3(8); 994–1006. ©2010 AACR.

  C Gourley , A. J.W Paige , K. J Taylor , C Ward , B Kuske , J Zhang , M Sun , S Janczar , D. J Harrison , M Muir , J. F Smyth and H. Gabra
 

The WW domain–containing oxidoreductase (WWOX) gene is located at FRA16D, a common fragile site involved in human cancer. Targeted deletion of Wwox in mice causes increased spontaneous tumor incidence, confirming that WWOX is a bona fide tumor suppressor gene. We show that stable transfection of WWOX into human PEO1 ovarian cancer cells, containing homozygous WWOX deletion, abolishes in vivo tumorigenicity, but this does not correlate with alteration of in vitro growth. Rather, WWOX restoration in PEO1, or WWOX overexpression in SKOV3 ovarian cancer cells, results in reduced attachment and migration on fibronectin, an extracellular matrix component linked to peritoneal metastasis. Conversely, siRNA-mediated knockdown of endogenous WWOX in A2780 ovarian cancer cells increases adhesion to fibronectin. In addition, whereas there is no WWOX-dependent difference in cell death in adherent cells, WWOX-transfected cells in suspension culture display a proapoptotic phenotype. We further show that WWOX expression reduces membranous integrin 3 protein but not integrin 3 mRNA levels, and that adhesion of PEO1 cells to fibronectin is predominantly mediated through integrin 3. We therefore propose that WWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix and by inducing apoptosis in detached cells. Consistent with this, the suppression of PEO1 tumorigenicity by WWOX can be partially overcome by implanting these tumor cells in Matrigel. These data suggest a possible role for the loss of WWOX in the peritoneal dissemination of human ovarian cancer cells. [Cancer Res 2009;69(11):4835–42]

  J Zhang , M Sattler , G Tonon , C Grabher , S Lababidi , A Zimmerhackl , M. S Raab , S Vallet , Y Zhou , M. A Cartron , T Hideshima , Y. T Tai , D Chauhan , K. C Anderson and K. Podar
 

Bone marrow angiogenesis is associated with multiple myeloma (MM) progression. Here, we report high constitutive hypoxia-inducible factor-1 (Hif-1) expression in MM cells, which is associated with oncogenic c-Myc. A drug screen for anti-MM agents that decrease Hif-1 and c-Myc levels identified a variety of compounds, including bortezomib, lenalidomide, enzastaurin, and adaphostin. Functionally, based on transient knockdowns and overexpression, our data delineate a c-Myc/Hif-1–dependent pathway mediating vascular endothelial growth factor production and secretion. The antiangiogenic activity of our tool compound, adaphostin, was subsequently shown in a zebrafish model and translated into a preclinical in vitro and in vivo model of MM in the bone marrow milieu. Our data, therefore, identify Hif-1 as a novel molecular target in MM and add another facet to anti-MM drug activity. [Cancer Res 2009;69(12):5082–90]

  Y Dong , B Lu , X Zhang , J Zhang , L Lai , D Li , Y Wu , Y Song , J Luo , X Pang , Z Yi and M. Liu
 

Cucurbitacin E (CuE, -elaterin), a tetracyclic triterpenes compound from folk traditional Chinese medicine plants, has been shown to inhibit cancer cell growth, inflammatory response and bilirubin–albumin binding. However, the effects of CuE on tumor angiogenesis and its potential molecular mechanism are still unknown. Here, we demonstrated that CuE significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration and tubulogenesis in vitro and blocked angiogenesis in chick embryo chorioallantoic membrane assay and mouse corneal angiogenesis model in vivo. Furthermore, we found that CuE remarkably induced HUVEC apoptosis, inhibited tumor angiogenesis and suppressed human prostate tumor growth in xenograft tumor model. Finally, we showed that CuE blocked vascular endothelial growth factor receptor (VEGFR) 2-mediated Janus kinase (Jak) 2–signal transducer and activator of transcription (STAT) 3 signaling pathway in endothelial cells and suppressed the downstream protein kinases, such as extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Therefore, our studies provided the first evidence that CuE inhibited tumor angiogenesis by inhibiting VEGFR2-mediated Jak–STAT3 and mitogen-activated protein kinases signaling pathways and CuE is a potential candidate in angiogenesis-related disease therapy.

  Q Lian , Y Zhang , J Zhang , H. K Zhang , X Wu , F. F. Y Lam , S Kang , J. C Xia , W. H Lai , K. W Au , Y. Y Chow , C. W Siu , C. N Lee and H. F. Tse
 

Background— Aging and aging-related disorders impair the survival and differentiation potential of bone marrow mesenchymal stem cells (MSCs) and limit their therapeutic efficacy. Induced pluripotent stem cells (iPSCs) may provide an alternative source of functional MSCs for tissue repair. This study aimed to generate and characterize human iPSC-derived MSCs and to investigate their biological function for the treatment of limb ischemia.

Methods and Results— Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24 and CD105+ sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes. Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms.

Conclusions— Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an "off-the-shelf" format for the treatment of tissue ischemia.

  J Sun , K Hartvigsen , M. Y Chou , Y Zhang , G. K Sukhova , J Zhang , M Lopez Ilasaca , C. J Diehl , N Yakov , D Harats , J George , J. L Witztum , P Libby , H Ploegh and G. P. Shi
 

Background— Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis.

Methods and Results— In low-density lipoprotein receptor–deficient (Ldlr–/–) mice, CD74 deficiency (Ldlr–/–Cd74–/–) significantly reduced atherosclerosis and CD25+-activated T cells in the atheromata. Although Ldlr–/–Cd74–/– mice had decreased levels of plasma immunoglobulin (Ig) G1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably as a result of impaired antigen-presenting cell function, Ldlr–/–Cd74–/– mice showed higher levels of anti–MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr–/–Cd74–/– mice had lower levels of all anti–MDA-LDL Ig isotypes compared with Ldlr–/– mice. As anticipated, only Ldlr–/– splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 immunization enhanced atherogenesis in Ldlr–/– mice, but Ldlr–/– Cd74–/– mice remained protected. Compared with Ldlr–/– mice, Ldlr–/–Cd74–/– mice had higher anti–MDA-LDL autoantibody titers, fewer lesion CD25+-activated T cells, impaired release of Th1/Th2 cytokines from antigen-presenting cells after heat shock protein-65 stimulation, and reduced levels of all plasma anti–heat shock protein-65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL– or heat shock protein-65–immunized mice showed increased percentages of autoantibody-producing marginal zone B and B-1 cells in Ldlr–/–Cd74–/– mice compared with Ldlr–/– mice.

Conclusions— Invariant chain deficiency in Ldlr–/– mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, an unexpected increase in the number of innate-like peripheral B-1 cell populations occurred, resulting in increased IgM/IgG3 titers to the oxidation-specific epitopes.

  Z Fu , M Wang , M Gucek , J Zhang , J Wu , L Jiang , R. E Monticone , B Khazan , R Telljohann , J Mattison , S Sheng , R. N Cole , G Spinetti , G Pintus , L Liu , F. D Kolodgie , R Virmani , H Spurgeon , D. K Ingram , A. D Everett , E. G Lakatta and J. E. Van Eyk
 

Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis.

  M. P Cole , T. K Rudolph , N. K.H Khoo , U. N Motanya , F Golin Bisello , J. W Wertz , F. J Schopfer , V Rudolph , S. R Woodcock , S Bolisetty , M. S Ali , J Zhang , Y. E Chen , A Agarwal , B. A Freeman and P. M. Bauer
 

Rationale: Fatty acid nitroalkenes are endogenously generated electrophilic byproducts of nitric oxide and nitrite-dependent oxidative inflammatory reactions. Existing evidence indicates nitroalkenes support posttranslational protein modifications and transcriptional activation that promote the resolution of inflammation.

Objective: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury.

Methods and Results: The in vivo administration (21 days) of nitro-oleic acid (OA-NO2) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO2 treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals,P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO2 in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO2–induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO2 in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1–/– mice (OA-NO2–treated wild-type versus HO-1–/– mice, P=0.016).

Conclusions: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury.

  G Kang , S. F Giovannone , N Liu , F. Y Liu , J Zhang , S. G Priori and G. I. Fishman
  Rationale:

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca2+ release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking.

Objective:

We sought to determine the frequency and severity of spontaneous Ca2+ release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2R4496C/+ CPVT mutant mice and littermate controls.

Methods and Results:

We crossed RyR2R4496C/+ knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP) and Purkinje cells (EGFP+) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca2+ dynamics recorded by microfluorimetry. Both wild-type and RyR2R4496C/+ mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and decay in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2R4496C/+ mutant Purkinje cells were also most likely to develop spontaneous Ca2+ release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca2+i events, the RyR2R4496C/+ Purkinje cells responded with the most profound abnormalities in intracellular Ca2+ handling, including a significant increase in the frequency of unstimulated Ca2+i events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca2+ release events with flecainide, whereas in RyR2R4496C/+ mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes.

Conclusions:

Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca2+ handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca2+ release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.

  J Zhang , L Villacorta , L Chang , Z Fan , M Hamblin , T Zhu , C. S Chen , M. P Cole , F. J Schopfer , C. X Deng , M. T Garcia Barrio , Y. H Feng , B. A Freeman and Y. E. Chen
  Rationale:

Nitro-oleic acid (OA-NO2) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO2 exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature.

Objective:

The present study sought to investigate the protective role of OA-NO2 in angiotensin (Ang) II–induced hypertension.

Methods and Results:

We show that systemic administration of OA-NO2 results in a sustained reduction of Ang II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO2 significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT1R)-mediated signaling because vascular contraction by other G-protein–coupled receptors is not altered in response to OA-NO2 treatment. From the mechanistic viewpoint, OA-NO2 lowers Ang II–induced hypertension independently of peroxisome proliferation-activated receptor (PPAR) activation. Rather, OA-NO2, but not OA, specifically binds to the AT1R, reduces heterotrimeric G-protein coupling, and inhibits IP3 (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor.

Conclusions:

These results demonstrate that OA-NO2 diminishes the pressor response to Ang II and inhibits AT1R-dependent vasoconstriction, revealing OA-NO2 as a novel antagonist of Ang II–induced hypertension.

  J Zhang , J. Y. F Chang , Y Huang , X Lin , Y Luo , R. J Schwartz , J. F Martin and F. Wang
  Rationale:

Heart valves develop from precursor structures called cardiac cushions, an endothelial-lined cardiac jelly that resides in the inner side of the heart tube. The cushions are then invaded by cells from different sources, undergo a series of complicated and poorly understood remodeling processes, and give rise to valves. Disruption of the fibroblast growth factor (FGF) signaling axis impairs morphogenesis of the outflow tract (OFT). Yet, whether FGF signaling regulates OFT valve formation is unknown.

Objective:

To study how OFT valve formation is regulated and how aberrant cell signaling causes valve defects.

Methods and Results:

By using mouse genetic manipulation, cell lineage tracing, ex vivo heart culture, and molecular biology approaches, we demonstrated that FGF signaling in the OFT myocardium upregulated Bmp4 expression, which then enhanced smooth muscle differentiation of neural crest cells (NCCs) in the cushion. FGF signaling also promoted OFT myocardial cell invasion to the cushion. Disrupting FGF signaling interrupted cushion remodeling with reduced NCCs differentiation into smooth muscle and less cardiomyocyte invasion and resulted in malformed OFT valves.

Conclusions:

The results demonstrate a novel mechanism by which the FGF-BMP signaling axis regulates formation of OFT valve primordia by controlling smooth muscle differentiation of cushion NCCs.

  B. A Pallante , S Giovannone , L Fang Yu , J Zhang , N Liu , G Kang , W Dun , P. A Boyden and G. I. Fishman
 

Background— Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes.

Methods and Results— Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express β-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca2+ transients in Cntn2-EGFP+ cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations.

Conclusions— Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.

  M Yu , G. A Smolen , J Zhang , B Wittner , B. J Schott , E Brachtel , S Ramaswamy , S Maheswaran and D. A. Haber
 

Epithelial-to-mesenchymal transition (EMT) plays an important role during normal embryogenesis, and it has been implicated in cancer invasion and metastasis. Here, we report that Ladybird homeobox 1 (LBX1), a developmentally regulated homeobox gene, directs expression of the known EMT inducers ZEB1, ZEB2, Snail1, and transforming growth factor β2 (TGFB2). In mammary epithelial cells, overexpression of LBX1 leads to morphological transformation, expression of mesenchymal markers, enhanced cell migration, increased CD44high/CD24low progenitor cell population, and tumorigenic cooperation with known oncogenes. In human breast cancer, LBX1 is up-regulated in the unfavorable estrogen receptor (ER)/progesterone (PR)/HER2 triple-negative basal-like subtype. Thus, aberrant expression of LBX1 may lead to the activation of a developmentally regulated EMT pathway in human breast cancer.

  G. A Smolen , J Zhang , M. J Zubrowski , E. J Edelman , B Luo , M Yu , L. W Ng , C. M Scherber , B. J Schott , S Ramaswamy , D Irimia , D. E Root and D. A. Haber
 

To define the functional pathways regulating epithelial cell migration, we performed a genome-wide RNAi screen using 55,000 pooled lentiviral shRNAs targeting ~11,000 genes, selecting for transduced cells with increased motility. A stringent validation protocol generated a set of 31 genes representing diverse pathways whose knockdown dramatically enhances cellular migration. Some of these pathways share features of epithelial-to-mesenchymal transition (EMT), and together they implicate key regulators of transcription, cellular signaling, and metabolism, as well as novel modulators of cellular trafficking, such as DLG5. In delineating downstream pathways mediating these migration phenotypes, we observed universal activation of ERKs and a profound dependence on their RSK effectors. Pharmacological inhibition of RSK dramatically suppresses epithelial cell migration induced by knockdown of all 31 genes, suggesting that convergence of diverse migratory pathways on this kinase may provide a therapeutic opportunity in disorders of cell migration, including cancer metastasis.

  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.

  T Fei , K Xia , Z Li , B Zhou , S Zhu , H Chen , J Zhang , Z Chen , H Xiao , J. D. J Han and Y. G. Chen
 

Embryonic stem (ES) cells are under precise control of both intrinsic self-renewal gene regulatory network and extrinsic growth factor-triggered signaling cascades. How external signaling pathways connect to core self-renewal transcriptional circuits is largely unknown. To probe this, we chose BMP signaling, which is previously recognized as a master control for both self-renewal and lineage commitment of murine ES cells. Here, we mapped target gene promoter occupancy of SMAD1/5 and SMAD4 on a genome-wide scale and found that they associate with a large group of developmental regulators that are enriched for H3K27 trimethylation and H3K4 trimethylation bivalent marks and are repressed in the self-renewing state, whereas they are rapidly induced upon differentiation. Smad knockdown experiments further indicate that SMAD-mediated BMP signaling is largely required for differentiation-related processes rather than directly influencing self-renewal. Among the SMAD-associated genes, we further identified Dpysl2 (previously known as Crmp2) and the H3K27 demethylase Kdm6b (previously known as Jmjd3) as BMP4-modulated early neural differentiation regulators. Combined with computational analysis, our results suggest that SMAD-mediated BMP signaling balances self-renewal versus differentiation by modulating a set of developmental regulators.

  S Maegawa , G Hinkal , H. S Kim , L Shen , L Zhang , J Zhang , N Zhang , S Liang , L. A Donehower and J. P. J. Issa
 

Aberrant methylation of promoter CpG islands in cancer is associated with silencing of tumor-suppressor genes, and age-dependent hypermethylation in normal appearing mucosa may be a risk factor for human colon cancer. It is not known whether this age-related DNA methylation phenomenon is specific to human tissues. We performed comprehensive DNA methylation profiling of promoter regions in aging mouse intestine using methylated CpG island amplification in combination with microarray analysis. By comparing C57BL/6 mice at 3-mo-old versus 35-mo-old for 3627 detectable autosomal genes, we found 774 (21%) that showed increased methylation and 466 (13%) that showed decreased methylation. We used pyrosequencing to quantitatively validate the microarray data and confirmed linear age-related methylation changes for all 12 genomic regions examined. We then examined 11 changed genomic loci for age-related methylation in other tissues. Of these, three of 11 showed similar changes in lung, seven of 11 changed in liver, and six of 11 changed in spleen, though to a lower degree than the changes seen in colon. There was partial conservation between age-related hypermethylation in human and mouse intestines, and Polycomb targets in embryonic stem cells were enriched among the hypermethylated genes. Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.

  H Skovierova , G Larrouy Maumus , J Zhang , D Kaur , N Barilone , J Kordulakova , M Gilleron , S Guadagnini , M Belanova , M. C Prevost , B Gicquel , G Puzo , D Chatterjee , P. J Brennan and M. Jackson
 

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of -1,5-linked d-Araf residues with some -1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with -1,3-branching activity on linear -1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

  J Zhang , C Yu , Z Liao , X Zhang , L You and D. Yu
 

Mn-doped ZnO nanobelts have been synthesized via the vapor phase evaporation method, which exhibited ferromagnetism at room temperature. Electron energy-loss spectroscopy was used to investigate the chemical state of Mn dopants. It revealed that the Mn species had the chemical valence of +2. Meanwhile, the fine structures of the Mn L23 edges indicated that the Mn dopant was located at the center of an oxygen octahedron but not an oxygen tetrahedron. This suggested that the Mn dopants did not substitute on the Zn sites as expected and sub-nanoscale MnO clusters had been formed in the synthesized ZnO nanobelts.

  N Nakamura , Y Shimizu , T Shinkawa , M Nakata , B Bammes , J Zhang and W. Chiu
 

We have developed an automated specimen search algorithm for cryo-electron microscopy imaging of ice-embedded single particles suspended across regularly spaced holes. To maximize the particle visibility under a low electron exposure rate condition, specimen searching is carried out in diffraction mode. However, images in diffraction mode contain significant pincushion distortion, making it difficult to computationally predict the locations of the regularly spaced holes. We have implemented a distortion-correction mechanism to restore the primitive distortion-free image and a correlation-based algorithm to accurately determine the periodicity of the holes. A stage-shift method to optimize positional reproducibility is also implemented. Addition of our algorithms to the JADAS software for automated transmission electron microscopy data acquisition has significantly improved the accuracy of specimen search.

  H Wang , D Zhang , W Wu , J Zhang , D Guo , Q Wang , T Jing , C Xu , X Bian and K. Yang
 

Steroid receptor coactivator-3 (SRC-3) has been reported to be overexpressed in the development and progression of many tumor types. SRC-3 has been detected in several lung cancer cell lines, but its expression and clinical significance in non–small cell lung cancer (NSCLC) remain unclear. In this study, 48 NSCLC tissues were collected and tissue microarrays were performed. The expression of SRC-3 was examined using nickel-intensified IHC. The results showed that of these 48 cases, 18 (37.5%) exhibited high levels of SRC-3 immunoreactivity, 23 (47.9%) exhibited moderate levels of SRC-3 immunoreactivity, and 7 (14.6%) were negative; thus, the total frequency of SRC-3 overexpression was 85.4% (41/48). This SRC-3 overexpression frequency was similar to the overexpression frequency observed for squamous cell carcinoma and adenocarcinoma (82.1% vs 90%) and for metastasis and non-metastasis patients (84.6% vs 85.7%). Data analysis demonstrated a significantly higher overexpression frequency in male patients compared with that in female patients (88.6% vs 76.9%). However, female patients tended to have higher expression levels of SRC-3, as measured by immunoreactivity, than male patients. These results demonstrate a high frequency of SRC-3 overexpression in NSCLC with a gender difference, suggesting that there is a specific role for SRC-3 in the pathogenesis of NSCLC. (J Histochem Cytochem 58:1121–1127, 2010)

  D. M Janicke , J. S Harman , E. W Jamoom , S. L Simon , J Zhang and M. Dumont Driscoll
 

Objectives To examine the association between weight status and health service use, while considering the influence of psychosocial functioning and demographic variables. Methods Two hundred child–parent dyads were recruited from pediatric primary care clinics and completed measures of height, weight, and questionnaires assessing psychosocial functioning. Claims and expenditure data over a 12-month retrospective period were extracted from the Medicaid claims database. Results Children who were obese incurred greater health service use and expenditures than children who were of a healthy weight, even after controlling for psychosocial functioning and other demographic variables. Children who were overweight (but not obese) did not have differing levels of claims or expenditures than their healthy weight peers. Conclusions Understanding the impact of pediatric obesity on long-term expenditures is critical. These results provide some indication of the financial savings that might be achieved if obese children were supported to achieve a healthier weight status.

  A. A Richards , M. L Colgrave , J Zhang , J Webster , F Simpson , E Preston , D Wilks , K. L Hoehn , M Stephenson , G. A Macdonald , J. B Prins , G. J Cooney , A Xu and J. P. Whitehead
 

Adiponectin is an adipocyte-secreted, insulin-sensitizing hormone the circulating levels of which are reduced in conditions of insulin resistance and diabetes. Previous work has demonstrated the importance of posttranslational modifications, such as proline hydroxylation and lysine hydroxylation/glycosylation, in adiponectin oligomerization, secretion, and function. Here we describe the first functional characterization of adiponectin sialylation. Using a variety of biochemical approaches we demonstrated that sialylation occurs on previously unidentified O-linked glycans on Thr residues of the variable domain in human adiponectin. Enzymatic removal of sialic acid or its underlying O-linked sugars did not affect adiponectin multimer composition. Expression of mutant forms of adiponectin (lacking the modified Thr residues) or of wild-type adiponectin in cells defective in sialylation did not compromise multimer formation or secretion, arguing against a structural role for this modification. Activity of desialylated adiponectin was comparable to control adiponectin in L6 myotubes and acute assays in adiponectin–/– mice. In contrast, plasma clearance of desialylated adiponectin was accelerated compared with that of control adiponectin, implicating a role for this modification in determining the half-life of circulating adiponectin. Uptake of desialylated adiponectin by isolated primary rat hepatocytes was also accelerated, suggesting a role for the hepatic asialoglycoprotein receptor. Finally, after chronic administration in adiponectin–/– mice steady-state levels of desialylated adiponectin were lower than control adiponectin and failed to recapitulate the improvements in glucose and insulin tolerance tests observed with control adiponectin. These data suggest an important role for sialic acid content in the regulation of circulating adiponectin levels and highlight the importance of understanding mechanisms regulating adiponectin sialylation/desialylation.

  V Gordon , S Bhadel , W Wunderlich , J Zhang , S. B Ficarro , S. A Mollah , J Shabanowitz , D. F Hunt , I Xenarios , W. C Hahn , M Conaway , M. F Carey and D. Gioeli
 

Previously we determined that S81 is the highest stoichiometric phosphorylation on the androgen receptor (AR) in response to hormone. To explore the role of this phosphorylation on growth, we stably expressed wild-type and S81A mutant AR in LHS and LAPC4 cells. The cells with increased wild-type AR expression grow faster compared with parental cells and S81A mutant-expressing cells, indicating that loss of S81 phosphorylation limits cell growth. To explore how S81 regulates cell growth, we tested whether S81 phosphorylation regulates AR transcriptional activity. LHS cells stably expressing wild-type and S81A mutant AR showed differences in the regulation of endogenous AR target genes, suggesting that S81 phosphorylation regulates promoter selectivity. We next sought to identify the S81 kinase using ion trap mass spectrometry to analyze AR-associated proteins in immunoprecipitates from cells. We observed cyclin-dependent kinase (CDK)9 association with the AR. CDK9 phosphorylates the AR on S81 in vitro. Phosphorylation is specific to S81 because CDK9 did not phosphorylate the AR on other serine phosphorylation sites. Overexpression of CDK9 with its cognate cyclin, Cyclin T, increased S81 phosphorylation levels in cells. Small interfering RNA knockdown of CDK9 protein levels decreased hormone-induced S81 phosphorylation. Additionally, treatment of LNCaP cells with the CDK9 inhibitors, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole and Flavopiridol, reduced S81 phosphorylation further, suggesting that CDK9 regulates S81 phosphorylation. Pharmacological inhibition of CDK9 also resulted in decreased AR transcription in LNCaP cells. Collectively these results suggest that CDK9 phosphorylation of AR S81 is an important step in regulating AR transcriptional activity and prostate cancer cell growth.

  J Zhang and J. M. Zhou
 

Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) are recognized by host cell surface-localized pattern-recognition receptors (PRRs) to activate plant immunity. PAMP-triggered immunity (PTI) constitutes the first layer of plant immunity that restricts pathogen proliferation. PTI signaling components often are targeted by various Pseudomonas syringae virulence effector proteins, resulting in diminished plant defenses and increased bacterial virulence. Some of the proteins targeted by pathogen effectors have evolved to sense the effector activity by associating with cytoplasmic immune receptors classically known as resistance proteins. This allows plants to activate a second layer of immunity termed effector-triggered immunity (ETI). Recent studies on PTI regulation and P. syringae effector targets have uncovered new components in PTI signaling. Although MAP kinase (MAPK) cascades have been considered crucial for PTI, emerging evidence indicates that a MAPK-independent pathway also plays an important role in PTI signaling.

  G. J Pons Estel , L. A Gonzalez , J Zhang , P. I Burgos , J. D Reveille , L. M Vila and G. S. Alarcon
 

Objective. To determine the features predictive of atherosclerotic cardiovascular damage in patients with SLE.

Methods. SLE LUMINA (LUpus in MInorities: NAture vs nurture) patients (n = 637), aged >=16 years, disease duration <=5 years at baseline (T0), of African–American, Hispanic and Caucasian ethnicity were studied. Atherosclerotic cardiovascular damage was defined by the following items of the SLICC Damage Index (SDI) cardiovascular domain: angina or coronary artery by pass surgery, myocardial infarction and/or congestive heart failure; factors associated with its occurrence were examined by univariable and multivariable regression analyses.

Results. Forty-three (6.8%) of 637 patients developed cardiovascular damage over a mean ± s.d. total disease duration of 6.6 ± 3.6 years. Nearly 90% of the patients were women with a mean ± s.d. age of 36.5 (12.6) years; all ethnic groups were represented. By multivariable analyses, after adjusting for the cardiovascular manifestations present, age [odds ratio (OR) = 1.06; 95% CI 1.03, 1.09], male gender (OR = 3.57; 95% CI 1.35, 9.09) SDI at baseline (OR = 1.28; 95% CI 1.09, 1.50) and CRP levels [highest tertile (OR = 2.63; 95% CI 1.17, 5.91)] were associated with the occurrence of cardiovascular damage, whereas the number of years of education was negatively associated with such outcome (OR = 0.85; 95% CI 0.74, 0.94).

Conclusions. Our data suggest that atherosclerotic cardiovascular damage in SLE is multifactorial; traditional (age, gender) and disease-related factors (CRP levels, SDI at baseline) appear to be important contributors to such an occurrence. Tight control of the inflammatory process must be achieved to prevent it.

  J Zhang and J. H. C. Wang
 

Background: Platelet-rich plasma (PRP) has been used to enhance tendon healing in clinical settings. However, the cellular mechanisms underlying PRP treatment of injured tendons remain unclear. The aim of this study was to determine the effects of PRP, in the form of PRP-clot releasate (PRCR), on tendon stem cells (TSCs), a newly discovered cell population in tendons.

Hypothesis: The PRCR treatment promotes differentiation of TSCs into tenocytes that are activated to proliferate quickly and increase collagen production.

Study Design: Controlled laboratory study.

Methods: After PRCR treatment, cell morphology, expression of stem/progenitor cell marker nucleostemin, and population doubling time were examined. In addition, gene and protein analyses were performed using reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blot to characterize the type of cells that had differentiated after PRCR treatment.

Results: The TSCs without PRCR treatment were small and exhibited an irregular shape, whereas with increasing PRCR dosage, TSCs became large, well spread, and highly elongated with downregulation of nucleostemin expression. The PRCR treatment also markedly enhanced TSC proliferation, tenocyte-related gene and protein expression, and total collagen production, all of which indicated that PRCR treatment induced differentiation of TSCs into activated tenocytes.

Conclusion: The PRCR treatment promotes differentiation of TSCs into active tenocytes exhibiting high proliferation rates and collagen production capability.

Clinical Relevance: The findings of this study suggest that PRP treatment of injured tendons is "safe" as it promotes TSC differentiation into tenocytes rather than nontenocytes, which would compromise the structure and function of healing tendons by formation of nontendinous tissues. Moreover, they suggest that PRP treatment can enhance tendon healing because tenocytes induced to differentiate by PRP are activated to proliferate quickly and produce abundant collagen to repair injured tendons that have lost cells and matrix.

  T Yamashiro , H Kuge , J Zhang and K. Honke
 

Aldosterone synthesis in the zona glomerulosa of the adrenal gland is catalysed by aldosterone synthase (CYP11B2). The CYP11B2 expression is induced by angiotensin II (Ang II), mediated by increase of intracellular Ca2+ level. Since calcineurin (CN) is an important mediator activated by Ca2+, we investigated the issue of whether CN is involved in the Ang II-induced CYP11B2 expression in human adrenocortical H295R cells. First, CN inhibitors, cyclosporine A (CysA) and tacrolimus (FK506) inhibited the Ang II-induced elevation of CYP11B2 mRNA level. Second, enforced expression of a constitutively active CN increased the CYP11B2 mRNA level. Third, depletion of CN by siRNA technique blocked the Ang II-induced elevation of CYP11B2 mRNA level. Fourth, in reporter assays using a luciferase gene connected to a 5'-flanking region (from –134 to +43 bp) of the hCYP11B2 gene, both CysA and FK506 inhibited the Ang II-mediated up-regulation of luciferase activity. Finally, activation of CN in living H295R cells following the Ang II treatment was confirmed using a fluorescence resonance energy transfer-based sensor. Taken together, we conclude that CN mediates the Ang II-induced aldosterone synthesis through up-regulation of the CYP11B2 transcription.

  Y. C Han , C. Y Park , G Bhagat , J Zhang , Y Wang , J. B Fan , M Liu , Y Zou , I. L Weissman and H. Gu
 

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.

 
 
 
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