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Articles by H Li
Total Records ( 41 ) for H Li
  Y Peng , H Li , M Wu , X Wang , S Fan , F Liu , B Xiang , Q Guo , X Tang and S. Shen
 

Colorectal cancer (CRC) is a common malignant tumor that is associated with an increased incidence of morbidity and mortality. Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate suppressor gene of tumor metastasis, which is down-regulated in CRC. In the present study, we constructed a colorectal tissue microarray to examine the expression profiles of NGX6, phospho-c-Jun N-terminal kinase (p-JNK), and phospho-extracellular signal-regulated kinase (p-ERK ) in CRC tissues. We found that the NGX6 expression was lower in CRC tissues and metastatic lymph nodes, whereas the expressions of p-JNK and p-ERK were higher in CRC tissues, than in normal intestinal mucosa. The expressions of NGX6, p-JNK, and p-ERK were associated with the clinical pathological features of colorectal tissues. NGX6 overexpression inhibited the activation and nuclear translocation of JNK1, which led to an accumulation of p-JNK in the cytoplasm, but did not inhibit the activation and nuclear translocation of ERK1/2. NGX6 also inhibited the expression of the transcription factors AP-1 (c-jun and c-fos) and Ets-1. In addition, NGX6 overexpression decreased the expression of cyclin D1 and dramatically suppressed the transcriptional efficiency of the cyclin D1 promoter. We propose that NGX6 expression is lost in the multi-step process of human colorectal carcinogenesis. Its overexpression can inhibit the expression of transcription factors AP-1 and Ets-1, and down-regulate the transcriptional activity of the cyclin D1 promoter in human CRC.

  H Li , Q Liu , X Hu , D Feng , S Xiang , Z He , J Zhou , X Ding , C Zhou and J. Zhang
 

Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) signaling, and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR). However, the details of how human ZCCHC12 involve in the NS-XLMR still remain unclear. In this study, we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization. Multiple-tissue northern blot analysis indicated that ZCCHC12 is highly expressed in human brain. Furthermore, in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain, midbrain, and diencephalon regions of mouse E10.5 embryos. Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcriptional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a co-activator. In conclusion, we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.

  S Dong , D Liang , N An , L Jia , Y Shan , C Chen , K Sun , F Niu , H Li and S. Fu
 

The aim of the present study is to investigate gene expression involved in the signal pathway of MAPK and death signal receptor pathway of FAS in lead-induced apoptosis of testicular germ cells. First, cell viabilities were determined by MTT assay. Second, using single cell gel-electrophoresis test (comet assay) and TUNEL staining technique, apoptotic rate and cell apoptosis localization of testicular germ cells were measured in mice treated with 0.15%, 0.3%, and 0.6% lead, respectively. Third, the immunolocalization of K-ras, c-fos, Fas, and active caspase-3 proteins was determined by immunohistochemistry. Finally, changes in the translational levels of K-ras, c-fos, Fas, and active caspase-3 were further detected by western blot analysis. Our results showed that lead could significantly induce testicular germ cell apoptosis in a dose-dependent manner (P < 0.01). The mechanisms were closely related to the increased expressions of K-ras, c-fos, Fas, and active caspase-3 in apoptotic germ cells. In conclusion, K-ras/c-fos and Fas/caspase-3 death signaling receptor pathways were involved in the lead-induced apoptosis of the testicular germ cells in mice.

  H Li and Y. Lu
 

Phenotypic inheritance induced by RNA has been documented in mouse and Caenorhabditis elegans. Here we report a similar inheritance in Drosophila. Mutant phenotypes of eye defects and antenna duplication generated from the crossing of one RNA interference (RNAi) transgenic line harboring one hairpin RNA transgene with a GAL4 driver line were inherited independently of the GAL4 driver. Hairpin RNA injection experiments demonstrated that the hairpin RNA could induce heritable mutant-like phenotypes on the eye and antenna. The penetrance of mutant phenotypes was reduced when the mutants were crossed to ago1 and piwi mutants. Our data suggest that hairpin RNA can induce phenotypic inheritance in Drosophila.

  S. A Lee , X. O Shu , H Li , G Yang , H Cai , W Wen , B. T Ji , J Gao , Y. T Gao and W. Zheng
 

Background: Soy food is a rich source of isoflavones—a class of phytoestrogens that has both antiestrogenic and anticarcinogenic properties.

Objective: The objective was to evaluate the association of adolescent and adult soy food intake with breast cancer risk in a cohort of 73,223 Chinese women who participated in the Shanghai Women's Health Study.

Design: A validated food-frequency questionnaire was used to assess usual dietary intake during adulthood and adolescence. After a mean follow-up of 7.4 y, 592 incident cases of breast cancer were identified for longitudinal analyses by using Cox regressions.

Results: Adult soy food consumption, measured either by soy protein or isoflavone intake, was inversely associated with the risk of premenopausal breast cancer, and the association was highly statistically significant (P for trend < 0.001). The multivariate-adjusted relative risks (RRs) for the upper intake quintile compared with the lowest quintile were 0.41 (95% CI: 0.25, 0.70) for soy protein intake and 0.44 (95% CI: 0.26, 0.73) for isoflavone intake. High intake of soy foods during adolescence was also associated with a reduced risk of premenopausal breast cancer (RR: 0.57; 95% CI: 0.34, 0.97). Women who consumed a high amount of soy foods consistently during adolescence and adulthood had a substantially reduced risk of breast cancer. No significant association with soy food consumption was found for postmenopausal breast cancer.

Conclusion: This large, population-based, prospective cohort study provides strong evidence of a protective effect of soy food intake against premenopausal breast cancer.

  Q. P Wang , J. W Gu , X. H Zhan , H Li and X. H. Luo
  Background

Assessment of renal function in patients undergoing coronary artery bypass grafting (CABG) is important. Cystatin C has been proposed as an improved indicator of renal function. The aim of this study was to assess cystatin C as an early marker of changes in glomerular filtration rate (GFR) after CABG.

Methods

Blood samples were collected from 61 CABG patients at different time points. Using 51Cr-ethylenediaminetetraacetic acid (51Cr-EDTA) clearance as a ‘gold standard’, we compared the correlations and non-parametric receiver operator characteristic curves of serum cystatin C, serum creatinine and 24 h creatinine clearance (Ccr).

Results

The inverse of cystatin C correlated better with 51Cr-EDTA than those of serum creatinine and Ccr (r = 0.8578, 0.6771 and 0.6929, respectively). Cystatin C exhibited significantly superior diagnostic accuracy for detecting GFR <80 mL/min/1.73 m2 compared with serum creatinine (P = 0.013) and Ccr (P = 0.025); for detecting GFR <60 mL/min/1.73 m2, cystatin C had similar diagnostic accuracy to Ccr (P = 0.812) but was superior to creatinine (P = 0.033). At the best cut-off value, cystatin C had sensitivity 89% and specificity 93% for detecting GFR <80 mL/min/1.73 m2, sensitivity 86% and specificity 96% for detecting GFR <60 mL/min/1.73 m2.

Conclusions

Cystatin C is a better marker for detecting small temporary changes of GFR in CABG patients. This may allow better identification of patients with renal impairment.

  C Xia , Q Tong , Q Wang , Z Tang , L Qi , S Chi , M Zhang , X Wang , H Li and G. Xu
  Background

The in vitro directive of the European Union requires traceability to the international recommended reference procedures. The application of the reference procedures is necessary in order to evaluate the accuracy of -glutamyltransferase (GGT) assays of routine measurement systems in China.

Methods

Five frozen patient-pooled serum samples were assigned values by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure in order to evaluate the traceability of the results of GGT catalytic activity from six homogeneous systems. One of the serum samples was used to calibrate seven non-homogeneous systems.

Results

All of the homogeneous systems, except the Dade system (Dade Bering Inc, IL, USA), achieved traceability within the measurement range. The Roche and Hitachi systems were better than the other systems. After calibration, the variance of the non-homogeneous systems decreased dramatically from between 14.50% and 25.23% to between 1.25% and 3.09% and the bias decreased from between –11.4% and –4.1% to between 0.5% and 3.5%.

Conclusion

Manufacturers in China should ensure that their calibration systems correspond to the IFCC reference procedures. Fresh frozen pooled patient serum assigned by reference laboratories can be used to calibrate non-homogeneous systems in order to achieve traceability.

  H Li and R. Durbin
 

Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.

Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ~10–20x faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.

  K. E Holt , Y. Y Teo , H Li , S Nair , G Dougan , J Wain and J. Parkhill
 

Summary: Here, we present a method for estimating the frequencies of SNP alleles present within pooled samples of DNA using high-throughput short-read sequencing. The method was tested on real data from six strains of the highly monomorphic pathogen Salmonella Paratyphi A, sequenced individually and in a pool. A variety of read mapping and quality-weighting procedures were tested to determine the optimal parameters, which afforded ≥80% sensitivity of SNP detection and strong correlation with true SNP frequency at poolwide read depth of 40x, declining only slightly at read depths 20–40x.

  H Li , B Handsaker , A Wysoker , T Fennell , J Ruan , N Homer , G Marth , G Abecasis , R Durbin and 1000 Genome Project Data Processing Subgroup
 

Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.

  H Li and G. Yin
 

We propose a generalized method of moments approach to the accelerated failure time model with correlated survival data. We study the semiparametric rank estimator using martingale-based moments. We circumvent direct estimation of correlation parameters by concatenating the moments and minimizing a quadratic objective function. We establish the consistency and asymptotic normality of the parameter estimators, and derive the limiting distribution of the objective function. We carry out simulation studies to examine the finite-sample properties of the method, and demonstrate its substantial efficiency gain over the conventional method. Finally, we illustrate the new proposal with an example from a diabetic retinopathy study.

  K. L Penney , F. R Schumacher , H Li , P Kraft , J. S Morris , T Kurth , L. A Mucci , D. J Hunter , P. W Kantoff , M. J Stampfer and J. Ma
 

The role of selenium in prostate cancer (PCa) risk remains controversial, but many epidemiologic studies suggest an inverse association with more aggressive disease. A recently discovered selenoprotein, SEP15, which is highly expressed in the prostate, may play a role either independently or by modifying the effects of selenium. We genotyped four common single-nucleotide polymorphisms capturing common variation (frequency >5%; R2 > 0.8) within SEP15, as well as rs5859 in the 3' untranslated region, previously reported to reduce the efficiency of selenium incorporation into SEP15. We examined the association of these single-nucleotide polymorphisms with PCa risk and PCa-specific mortality, as well as their interactions with plasma selenium levels, in the Physicians' Health Study. In this nested case-control study (1,286 cases and 1,267 controls), SEP15 polymorphisms were not significantly associated with PCa risk. However, among the cases, three variants were significantly associated with PCa-specific mortality [rs479341 hazard ratio (HR), 1.94; 95% confidence interval (95% CI), 1.15-3.25; rs1407131 HR, 2.85; 95% CI, 1.45-5.59; rs561104 HR, 1.54; 95% CI, 1.12-2.11] with a recessive model. Additionally, rs561104 significantly modified the association of plasma selenium with PCa survival (Pinteraction = 0.02); an inverse relationship of high levels of selenium with PCa mortality was apparent only among those without the increased risk genotype. This study provides evidence that SEP15 genetic variation may influence PCa mortality. Additionally, the association of selenium with PCa mortality was modified by a variant, suggesting the possibility that some men with PCa may benefit more from selenium than others, depending on their genotype. Cancer Prev Res; 3(5); 604–10. ©2010 AACR.

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

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

  Y Adachi , R Li , H Yamamoto , Y Min , W Piao , Y Wang , A Imsumran , H Li , Y Arimura , C. T Lee , K Imai , D. P Carbone and Y. Shinomura
 

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal (GI) cancers. We have previously shown significant therapeutic activity for recombinant adenoviruses expressing dominant-negative insulin-like growth factor-I receptor (IGF-IR/dn), including suppression of tumor invasion. In this study, we sought to evaluate the mechanism of inhibition of invasion and the relationship between IGF-IR and matrix metalloproteinase (MMP) activity in GI carcinomas. We analyzed the role of IGF-IR on invasion in three GI cancer cell lines, colorectal adenocarcinoma, HT29; pancreatic adenocarcinoma, BxPC3 and gastric adenocarcinoma, MKN45, using a modified Boyden chamber method and subcutaneous xenografts in nude mice. The impact of IGF-IR signaling on the expression of MMPs and the effects of blockade of matrilysin or IGF-IR on invasiveness were assessed using recombinant adenoviruses, a tyrosine kinase inhibitor NVP-AEW541 and antisense matrilysin. Invasive subcutaneous tumors expressed several MMPs. IGF-IR/dn reduced the expression of these MMPs but especially matrilysin (MMP-7). Insulin-like growth factor (IGF) stimulated secretion of matrilysin and IGF-IR/dn blocked IGF-mediated matrilysin induction in three GI cancers. Both IGF-IR/dn and inhibition of matrilysin reduced in vitro invasion to the same degree. NVP-AEW541 also reduced cancer cell invasion both in vitro and in murine xenograft tumors via suppression of matrilysin. Thus, blockade of IGF-IR is involved in the suppression of cancer cell invasion through downregulation of matrilysin. Strategies of targeting IGF-IR may have significant therapeutic utility to prevent invasion and progression of human GI carcinomas.

  S. T Chiu , K. J Chang , C. H Ting , H. C Shen , H Li and F. J. Hsieh
 

Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in ApcMin/+ mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4–ephrin-A and EphB4–ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3–ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes’ stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca2+-dependent cell–cell adhesion), decreased mesenchymal markers (fibronectin and nuclear β-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL–Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3–ephrin-B interaction promotes MET by re-establishing epithelial cell–cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.

  Y Yao , H Li , Y Gu , N. E Davidson and Q. Zhou
 

Estrogen receptor (ER) mediates estrogen-dependent gene transcription, which plays a critical role in mammary gland development, reproduction and homeostasis. Histone acetyltransferases and class I and class II histone deacetylases (HDACs) cause posttranscriptional modification of histone proteins that participate in ER signaling. Here, we report that human SIRT1, a class III HDAC, regulates ER expression. Inhibition of SIRT1 activity by sirtinol suppresses ER expression through disruption of basal transcriptional complexes at the ER promoter. This effect leads to inhibition of estrogen-responsive gene expression. Our in vitro observations were further extended that SIRT1 knockout reduces ER protein in mouse mammary gland. Finally, ER-mediated estrogen response genes are also decreased in mouse embryonic fibroblasts derived from SIRT1-knockout mice. These results suggest that inhibition of SIRT1 deacetylase activity by either pharmacological inhibitors or genetic depletion impairs ER-mediated signaling pathways.

  A Zampetaki , L Zeng , A Margariti , Q Xiao , H Li , Z Zhang , A. E Pepe , G Wang , O Habi , E deFalco , G Cockerill , J. C Mason , Y Hu and Q. Xu
 

Background— Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development.

Methods and Results— En face staining of aortas from apolipoprotein E-knockout mice revealed increased expression of HDAC3, specifically in these branching areas in vivo, whereas rapid upregulation of HDAC3 protein was observed in endothelial cells exposed to disturbed flow in vitro. Interestingly, phosphorylation of HDAC3 at serine/threonine was observed in these cells, suggesting that disturbed flow leads to posttranscriptional modification and stabilization of the HDAC3 protein. Coimmunoprecipitation experiments showed that HDAC3 and Akt form a complex. Using a series of constructs harboring deletions, we found residues 136 to 206 of HDAC3 to be crucial in this interaction. Enforced expression of HDAC3 resulted in increased phosphorylation of Akt and upregulation of its kinase activity. In line with these findings, knockdown of HDAC3 with lentiviral vectors (shHDAC3) led to a dramatic decrease in cell survival accompanied by apoptosis in endothelial cells. In aortic isografts of apolipoprotein E-knockout mice treated with shHDAC3, a robust atherosclerotic lesion was formed. Surprisingly, 3 of the 8 mice that received shHDAC3-infected grafts died within 2 days after the operation. Miller staining of the isografts revealed disruption of the basement membrane and rupture of the vessel.

Conclusions— Our findings demonstrated that HDAC3 serves as an essential prosurvival molecule with a critical role in maintaining the endothelial integrity via Akt activation and that severe atherosclerosis and vessel rupture in isografted vessels of apolipoprotein E-knockout mice occur when HDAC3 is knocked down.

  A Margariti , A Zampetaki , Q Xiao , B Zhou , E Karamariti , D Martin , X Yin , M Mayr , H Li , Z Zhang , E De Falco , Y Hu , G Cockerill , Q Xu and L. Zeng
 

Rationale: Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function.

Objective: This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism.

Methods and Results: Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of β-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other β-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased β-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced β-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to β-catenin and forms a complex with 14-3-3 , , and proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLC-IP3K (phospholipase C–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with β-catenin, disrupting the complex and releasing β-catenin to translocate into the nucleus.

Conclusions: These findings demonstrate that HDAC7 interacts with β-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth.

  T Ago , J Kuroda , J Pain , C Fu , H Li and J. Sadoshima
 

Rationale: NADPH oxidases are a major source of superoxide (O2) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood.

Objective: The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart.

Methods and Results: Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging. Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O2 production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone- and diphenyleneiodonium-sensitive O2 production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4.

Conclusions: Upregulation of Nox4 by hypertrophic stimuli and aging induces oxidative stress, apoptosis and LV dysfunction, in part because of mitochondrial insufficiency caused by increased O2 production and consequent cysteine oxidation in mitochondrial proteins.

  J Cheng , Y Wang , Y Ma , B. T. y Chan , M Yang , A Liang , L Zhang , H Li and J. Du
  Rationale:

Mechanical stress plays an important role in proliferation of venous smooth muscle cells (SMCs) in neointima, a process of formation that contributes to failure of vein grafts. However, it is unknown what intracellular growth signal leads to proliferation of venous SMCs.

Objective:

The objective of this study is to identify mechanisms of mechanical stretch on neointima formation.

Methods and Results:

By a microarray analysis, we found that mechanical cyclic stretch (15% elongation) stimulated the transcription of SGK-1 (serum-, glucocorticoid-regulated kinase-1). Mechanical stretch–induced SGK-1 mRNA expression was blocked by actinomycin D. The mechanism for the SGK-1 expression involved MEK1 but not p38 or JNK signaling pathway. SGK-1 activation in response to stretch is blocked by insulin-like growth factor (IGF)-1 receptor inhibitor and mammalian target of rapamycin complex (mTORC)2 inhibitor (Ku-0063794) but not mTORC1 inhibitor (rapamycin). Mechanical stretch–induced bromodeoxyuridine incorporation was reduced by 83.5% in venous SMCs isolated from SGK-1 knockout mice. In contrast, inhibition of Akt, another downstream signal of PI3K resulted in only partial inhibition of mechanical stretch–induced proliferation of venous SMCs. Mechanical stretch also induced phosphorylation and nuclear exportation of p27kip1, whereas knockout of SGK-1 attenuated this effect of mechanical stretch on p27kip1. In vivo, we found that placement of a vein graft into artery increased SGK-1 expression. Knockout of SGK-1 effectively prevented neointima formation in vein graft. There is significant lower level of p27kip1 located in the nucleus of neointima cells in SGK-1 knockout mice compared with that of wild-type vein graft. In addition, we also found that wire injury of artery or growth factors in vitro increased expression of SGK-1.

Conclusions:

These results suggest that SGK-1 is an injury-responsive kinase that could mediate mechanical stretch–induced proliferation of vascular cells in vein graft, leading to neointima formation.

  N Mdivani , H Li , M Akhalaia , M Gegia , L Goginashvili , D. S Kernodle , G Khechinashvili and Y. W. Tang
 

Background: Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify the response to anti-TB drugs are desirable.

Methods: We developed 2 real-time PCR assays that use hydrolysis probes to target DNA of the IS6110 insertion element and mRNA for antigen 85B. The nucleic acids are extracted directly from concentrated sputum samples decontaminated with sodium hydroxide and N-acetyl-l-cysteine. We prospectively compared these assays with results obtained by sputum mycobacterial culture for patients receiving anti-TB therapy.

Results: Sixty-five patients with newly diagnosed TB and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2 and at months 1, 2, and 4 after therapy initiation. Both the DNA PCR assay (98.5% positive) and the mRNA reverse-transcription PCR (RT-PCR) assay (95.4% positive) were better than standard Ziehl–Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum samples. The overall agreement between culture and mRNA RT-PCR results for all 286 sputum samples was 87.1%, and compared with culture, the mRNA RT-PCR assay’s diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled those of culture at the follow-up time points.

Conclusions: The continued presence of viable M. tuberculosis according to culture and results obtained by RT-PCR analysis of antigen 85B mRNA correlated clinically with resistance to anti-TB drugs, whereas the DNA PCR assay showed a high false-positive rate. This mRNA RT-PCR assay may allow rapid monitoring of the response to anti-TB therapy.

  S Huang , H Li , X Ding and C. Xiong
 

Background: We recently detected cell-free seminal RNA (cfsRNA) and set out to study its concentration, integrity, stability in healthy individuals, and mechanisms for its protection from ribonucleases.

Methods: We quantified cfsRNA by reverse-transcription quantitative real-time PCR (RT-qPCR) targeting of the 5' region of the ACTB (actin, beta) transcript. cfsRNA integrity was analyzed by microcapillary electrophoresis and by amplification of full-length ACTB and DDX4 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 4] transcripts, including measurement of the relative amounts of different regions of ACTB and DDX4 transcripts. Stability of cfsRNA was measured by time-course analysis of different regions of ACTB and DDX4 transcripts. To investigate whether cfsRNA was protected in complexed forms, we processed seminal plasma in 2 ways: filtration through pores of different sizes and Triton X-100 treatment before RNA recovery.

Results: cfsRNA concentrations varied from 0.87–3.64 mg/L [mean (SD), 1.75 mg/L (0.92 mg/L)]. Most cfsRNA was present in partially degraded forms, with smaller amounts of middle and 3' amplicons compared with 5' amplicons. Although the 3' region of the DDX4 transcript was degraded completely by 90 min, the 5' regions of ACTB and DDX4 transcripts were stable up to 24 h. Filtration through 0.22-µm pores reduced ACTB and DDX4 mRNA concentrations by 72% and 61%, respectively. Nearly all seminal ACTB and DDX4 mRNA disappeared after Triton X-100 treatment.

Conclusions: Although cfsRNA was partially degraded, it represented diverse transcript species and was abundant, fairly stable, and associated with particles in healthy individuals. cfsRNA may represent a potential noninvasive biomarker of the male reproductive system and of germline epigenetics.

  H Li , M. J Stampfer , L Mucci , N Rifai , W Qiu , T Kurth and J. Ma
 

Background: Adipocytokines may mediate the association between adiposity and lethal prostate cancer outcomes.

Methods: In the Physicians’ Health Study, we prospectively examined the association of prediagnostic plasma concentrations of adiponectin and leptin with risk of developing incident prostate cancer (654 cases diagnosed 1982–2000 and 644 age-matched controls) and, among cases, risk of dying from prostate cancer by 2007.

Results: Adiponectin concentrations were not associated with risk of overall prostate cancer. However, men with higher adiponectin concentrations had lower risk of developing high-grade or lethal cancer (metastatic or fatal disease). The relative risk (95% CI) comparing the highest quintile to the lowest (Q5 vs Q1) was 0.25 (95% CI 0.07–0.87; Ptrend = 0.02) for lethal cancer. Among all the cases, higher adiponectin concentrations predicted lower prostate cancer–specific mortality [hazard ratio (HR)Q5 vs Q1= 0.39; 95% CI 0.17–0.85; Ptrend = 0.02], independent of body mass index (BMI), plasma C-peptide (a marker of insulin secretion), leptin, clinical stage, and tumor grade. This inverse association was apparent mainly among men with a BMI ≥25 kg/m2 (HRQ5 vs Q1= 0.10; 95% CI 0.01–0.78; Ptrend = 0.02), but not among men of normal weight (Ptrend = 0.51). Although the correlation of leptin concentrations with BMI (r = 0.58, P < 0.001) was stronger than that of adiponectin (r = –0.17, P < 0.001), leptin was unrelated to prostate cancer risk or mortality.

Conclusions: Higher prediagnostic adiponectin (but not leptin) concentrations predispose men to a lower risk of developing high-grade prostate cancer and a lower risk of subsequently dying from the cancer, suggesting a mechanistic link between obesity and poor prostate cancer outcome.

  J. J Zhang , L. D Wang , H Li and Y. C. Zhao
  Background

To assess the response time and the details of the operating procedure of the Beijing 120 Emergency Medical Service (EMS).

Methods

Between June and December 2005, 51 918 EMS cases recorded in the Dispatch Center of the Beijing Emergency Medical Center were analysed.

Results

The median response time of the 51 918 cases was 16.5 min; the cumulative proportions were 2.28%, 9.64% and 18.04% for less than 5 min, 8 min and 10 min of response time respectively, whereas the proportion was 19.20% for more than 30 min of response time.

Conclusions

On the basis of this analysis, the response time of the Beijing 120 EMS system was found to be longer than that indicated by the national standards.

  H Li , A Mittal , D. Y Makonchuk , S Bhatnagar and A. Kumar
 

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder of skeletal muscle caused by mutation in dystrophin gene. Although the degradation of skeletal muscle extracellular matrix, inflammation and fibrosis are the common pathological features in DMD, the underlying mechanisms remain poorly understood. In this study, we have investigated the role and the mechanisms by which increased levels of matrix metalloproteinase-9 (MMP-9) protein causes myopathy in dystrophin-deficient mdx mice. The levels of MMP-9 but not tissue inhibitor of MMPs were drastically increased in skeletal muscle of mdx mice. Besides skeletal muscle, infiltrating macrophages were found to contribute significantly to the elevated levels of MMP-9 in dystrophic muscle. In vivo administration of a nuclear factor-kappa B inhibitory peptide, NBD, blocked the expression of MMP-9 in dystrophic muscle of mdx mice. Deletion of Mmp9 gene in mdx mice improved skeletal muscle structure and functions and reduced muscle injury, inflammation and fiber necrosis. Inhibition of MMP-9 increased the levels of cytoskeletal protein β-dystroglycan and neural nitric oxide synthase and reduced the amounts of caveolin-3 and transforming growth factor-β in myofibers of mdx mice. Genetic ablation of MMP-9 significantly augmented the skeletal muscle regeneration in mdx mice. Finally, pharmacological inhibition of MMP-9 activity also ameliorated skeletal muscle pathogenesis and enhanced myofiber regeneration in mdx mice. Collectively, our study suggests that the increased production of MMP-9 exacerbates dystrophinopathy and MMP-9 represents as one of the most promising therapeutic targets for the prevention of disease progression in DMD.

  J Hong , H Li , M Chen , Y. C. Q Zang , S. M Skinner , J. M Killian and J. Z. Zhang
 

MBP-specific autoreactive T cells are considered pro-inflammatory T cells and thought to play an important role in the pathogenesis of multiple sclerosis (MS). Here, we report that MBP83–99-specific T cells generated from MS patients (n = 7) were comprised of pro-inflammatory and regulatory subsets of distinct phenotypes. The pro-inflammatory phenotype was characterized by high production of IFN-, IL-6, IL-21 and IL-17 and low expression of FOXP3, whereas the regulatory subset expressed high levels of FOXP3 and exhibited potent regulatory functions. The regulatory subset of MBP-specific T cells appeared to expand from the CD4+CD25 T-cell pool. Their FOXP3 expression was stable, independent of the activation state and it correlated with suppressive function and inversely with the production of IFN-, IL-6, IL-21 and IL-17. In contrast, the phenotype and function of FOXP3low MBP-specific T cells were adaptive and dependent on IL-6. The higher frequency of FOXP3high MBP-specific T cells was observed when IL-6 was neutralized in the culture of PBMC with MBP. The study provides new evidence that MBP-specific T cells are susceptible to pro-inflammatory cytokine milieu and act as either pro-inflammatory or regulatory T cells.

  W Cao , C Xu , G Lou , J Jiang , S Zhao , M Geng , W Xi , H Li and Y. Jin
  Objective

The aim of this study was to assess the efficacy and toxicity of the combination of paclitaxel and nedaplatin as a first-line chemotherapy for patients with advanced esophageal cancer.

Methods

Patients with advanced esophageal cancer received 175 mg/m2 of paclitaxel over a 3 h infusion, followed by nedaplatin 80 mg/m2 in a 1 h infusion on day 1 every 3 weeks until the documented disease progression, unacceptable toxicity or patient's refusal.

Results

Between March 2005 and December 2007, 48 patients entered in the study. Forty-six (95.8%) of the 48 patients were assessable for response. The overall response rate was 41.7% (95% CI, 27.8–55.7%) with 2 complete responses and 18 partial responses. The median follow-up period was 20.5 months (range, 12.5–27.2 months). The median overall time to progression and overall survival (OS) were 6.1 months (95% CI, 4.8–7.4 months) and 11.5 months (95% CI, 9.1–13.9 months), respectively. The estimate of OS at 12 and 24 months was 43.8% (95% CI, 29.7–77.8%) and 10.4% (95% CI, 1.8–19.1%), respectively. Most patients experienced anemia, during their course of therapy with 6 (13.0%) patients for grade 3/4 anemia, and grade 1 or 2 anemia was detected in 23 (50%) patients. Grade 3 leucopenia, neutropenia and thrombocytopenia were documented in 8 (17.4%), 9 (17.4%) and 2 (4.3%) patients, respectively. Grade 3 nausea and vomiting were detected in 3 (6.5%) and 2 (4.3%) patients, respectively. Two patients (4.3%) were hospitalized because of treatment-related complications. The treatment was well tolerated and no toxic death occurred.

Conclusions

Combination of paclitaxel and nedaplatin is a tolerated treatment modality with promising activity in previously untreated advanced esophageal cancer.

  H Li , Y Bao , A Xu , X Pan , J Lu , H Wu , H Lu , K Xiang and W. Jia
 

Objective: Fibroblast growth factor (FGF) 21, a hormone primarily secreted by liver, has recently been shown to have beneficial effects on glucose and lipid metabolism and insulin sensitivity in animal models. This study investigated the association of serum FGF21 levels with insulin secretion and sensitivity, as well as circulating parameters of lipid metabolism and hepatic enzymes in Chinese subjects.

Design: Serum FGF21 levels were determined by ELISA in 134 normal glucose tolerance (NGT), 101 isolated-impaired fasting glucose, and 118 isolated-impaired glucose tolerance (I-IGT) Chinese subjects, and their association with parameters of adiposity, glucose, and lipid profiles, and levels of liver injury markers was studied. In a subgroup of this study, the hyperglycemic clamp technique was performed in 31 NGT, 17 isolated-impaired fasting glucose, and 15 I-IGT subjects to measure insulin secretion and sensitivity to test the associations with serum FGF21.

Results: The serum FGF21 levels in I-IGT were significantly higher than NGT subjects [164.6 pg/ml (89.7, 261.0) vs. 111.8 pg/ml (58.0, 198.9); P < 0.05], and correlated positively with several parameters of adiposity. Multiple stepwise regression analysis showed an independent association of serum FGF21 with serum triglycerides, total cholesterol, and -glutamyltransferase (all P < 0.05). However, FGF21 did not correlate with insulin secretion and sensitivity, as measured by hyperglycemic clamp and a 75-g oral glucose tolerance test.

Conclusions: Serum levels of FGF21 are closely related to adiposity, lipid metabolism, and biomarkers of liver injury but not insulin secretion and sensitivity in humans.

  Y Wu , H Li , R. J. F Loos , Q Qi , F. B Hu , Y Liu and X. Lin
 

We previously found that plasma RBP4 levels were strongly associated with metabolic syndrome components. This study aimed to determine whether RBP4 variants are associated with the metabolic syndrome components and plasma RBP4 levels, and to investigate whether the associations between plasma RBP4 and the metabolic syndrome components are causal. Five tagSNPs were tested for their associations with plasma RBP4 levels and metabolic syndrome components in a population-based sample of 3,210 Chinese Hans. A possible causal relationship between plasma RBP4 levels and hypertriglyceridemia was explored by Mendelian randomization. Plasma RBP4 levels were significantly associated with rs10882273 (βz –0.10SD[–0.17, –0.03], P = 0.0050), rs3758538 (βz –0.13SD[–0.24, –0.02], P = 0.0249) in all participants, and with rs17108993 in Shanghai participants (βz –0.19SD[–0.32, –0.05], P = 0.0061). The single nucleotide polymorphism (SNP) rs3758538 was significantly associated with hypertriglyceridemia (OR 0.62[0.45–0.85], P = 0.0026) and triglycerides (βz –0.19SD[–0.30, –0.07], P = 0.001) in all participants. In Mendelian randomization analysis, the observed effect size of association between rs3758538 and hypertriglyceridemia was different from the expected effect size (P = 0.0213). This is the first study to show that the RBP4 variants are significantly associated with plasma RBP4 levels and hypertriglyceridemia risk in Chinese Hans. However, results of Mendelian randomization do not support the hypothesis that RBP4 levels are causally related to hypertriglyceridemia risk.

  M. J Doubell , H Yamazaki , H Li and Y. Kokubu
 

An advanced model of the Turbulence Microstructure Acquisition Profiler, TurboMAP-L, to investigate the coupling between phytoplankton and turbulence in aquatic systems is described. The profiler provides simultaneous measurement of turbulence, hydrographic and biological parameters at sampling rates between 64 and 512 Hz. Specifically, the addition of a new laser fluorescence-based sensor extends the measurement of in situ chlorophyll fluorescence distributions to millimeter scales. Complementary information on phytoplankton patch and particulate matter size and spatial structure are obtained through the attachment of a separate CMOS mini-camera system. Images of the TurboMAP-L LED fluorescence/turbidity sensor sample volume are obtained with 330 x 330 µm resolution. Results with respect to the performance of the laser sensor and camera system are presented from laboratory tests and field experiments conducted in coastal waters off Tokyo, Japan.

  S Liu , W Tang , J Fang , J Ren , H Li , Z Xiao and L. D. Quarles
 

We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts associated with abnormal Fgf23 production and mineralization in Hyp mice. We found evidence that elevation of Fgf23 expression in osteocytes is associated with increments in Fgf1, Fgf7, and Egr2 and decrements in Sost, an inhibitor in the Wnt-signaling pathway, were observed in Hyp bone. β-Catenin levels were increased in Hyp cortical bone, and TOPflash luciferase reporter assay showed increased transcriptional activity in Hyp-derived osteoblasts, consistent with Wnt activation. Moreover, activation of Fgf and Wnt-signaling stimulated Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the extracellular matrix protein Dmp1. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced posttranslational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regard to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH-altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone.

  H Li and F. Zhao
 

Based on a multivariate extension of the constrained locally polynomial estimator of Aït-Sahalia and Duarte (2003), we provide one of the first nonparametric estimates of probability densities of LIBOR rates under forward martingale measures and state-price densities (SPDs) implicit in interest rate cap prices. The forward densities and SPDs depend significantly on the slope and volatility of LIBOR rates, and mortgage markets activities have strong impacts on the shape of the forward densities. The SPDs exhibit a pronounced U-shape as a function of future LIBOR rates, suggesting that the state prices are high at both extremely low and high interest rates, which tend to be associated with recessions and periods of high inflation, respectively. Our results provide nonparametric evidence of unspanned stochastic volatility and suggest that the unspanned factors could be partly driven by activities in the mortgage markets.

  H Li , R Daculsi , M Grellier , R Bareille , C Bourget and J. Amedee
 

In our previous studies, roles of gap junction and vascular endothelial growth factor in the cross-talking of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) have been extensively studied. The present study focused on the investigation of the roles of neural (N)-cadherin in early differentiation of HBMSCs in direct-contact cocultures with HUVECs for 24 and 48 h. Quantitative real-time polymerase chain reaction, immunofluorescence, Western blot, as well as functional studies were applied to perform the studies at both protein and gene levels. Results showed that cocultured cells expressed much higher N-cadherin than monocultured cells after 24 and 48 h of culture. We observed that N-cadherin concentrated in the membrane of cocultured HBMSCs (co-HBMSCs) while distributed within the cytoplasm of monocultured HBMSCs, which indicated that the cell-cell adhesion was improved between cocultured cells. In addition, more β-catenin was found to translocate into the cocultured cells nuclei and more T cell factor-1 (TCF-1) were detected in cocultured cells than in the monocultured cells. Moreover, mRNA levels of early osteoblastic markers including alkaline phosphatase (ALP) and type I collagen (Col-I) of co-HBMSCs were significantly upregulated, whereas neutralization of N-cadherin led to a downregulation of ALP and Col-I in both of the HBMSCs and co-HBMSCs compared with untreated cells. Taking our findings together it can be concluded that cocultures of HBMSCs with HUVECs increased N-cadherin expression and improved cell-cell adhesion. Whether this applies only to osteoprogenitor cells or to all the cell types in the culture will need to be determined by further studies. Subsequently, signaling transduction might be induced with the participation of β-catenin and TCF-1. With the N-cadherin-mediated cell-cell adhesion and signaling transductions, the early osteoblastic differentiation of co-HBMSCs was significantly upregulated.

  E Zaks Makhina , H Li , A Grishin , V Salvador Recatala and E. S. Levitan
 

Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K+ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K+]. One of the identified compounds, gambogic acid (GA), potently (EC50 ≤ 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC50 = ~10 µm) non-selective K+ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K+ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels.

  H Li , G Liu , J Yu , W Cao , V. G Lobo and J. Xie
 

Alternative pre-mRNA splicing is often controlled by cell signals, for example, those activating the cAMP-dependent protein kinase (PKA) or the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). We have shown that CaMKIV regulates alternative splicing through short CA repeats and hnRNP L. Here we use a splicing reporter that shows PKA/CaMKIV promotion of exon inclusion to select from exons containing random 13-nt sequences for RNA elements responsive to the kinases in cultured cells. This selection not only identified both PKA- and CaMKIV-responsive elements that are similar to the CaMKIV-responsive RNA element 1 (CaRRE1) or CA repeats, but also A-rich elements not previously known to respond to these kinases. Consistently, hnRNP L is identified as a factor binding the CA-rich elements. Analyses of the motifs in the highly responsive elements indicate that they are indeed critical for the kinase effect and are enriched in alternative exons. Interestingly, a CAAAAAA motif is sufficient for the PKA/CaMKIV-regulated splicing of the exon 16 of the CaMK kinase β1 (CaMKK2) transcripts, implying a role of this motif in signaling cross-talk or feedback regulation between these kinases through alternative splicing. Therefore, these experiments identified a group of RNA elements responsive to PKA and CaMKIV from in vivo selection. This also provides an approach for selecting RNA elements similarly responsive to other cell signals controlling alternative splicing.

  D Grandy , J Shan , X Zhang , S Rao , S Akunuru , H Li , Y Zhang , I Alpatov , X. A Zhang , R. A Lang , D. L Shi and J. J. Zheng
 

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.

  J Hollien , J. H Lin , H Li , N Stevens , P Walter and J. S. Weissman
 

Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box–binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.

  J. F Salazar Gonzalez , M. G Salazar , B. F Keele , G. H Learn , E. E Giorgi , H Li , J. M Decker , S Wang , J Baalwa , M. H Kraus , N. F Parrish , K. S Shaw , M. B Guffey , K. J Bar , K. L Davis , C Ochsenbauer Jambor , J. C Kappes , M. S Saag , M. S Cohen , J Mulenga , C. A Derdeyn , S Allen , E Hunter , M Markowitz , P Hraber , A. S Perelson , T Bhattacharya , B. F Haynes , B. T Korber , B. H Hahn and G. M. Shaw
 

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4+ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

  B Liu , J Yao , Y Wang , H Li and F. Qin
 

Protons, which are released during inflammation and injury, regulate many receptors and ion channels involved in pain transduction, including capsaicin channels (transient receptor potential vanilloid receptors 1). Whereas extracellular acidification both sensitizes and directly activates the channel, it also causes concomitant reduction of the unitary current amplitudes. Here, we investigate the mechanisms and molecular basis of this inhibitory effect of protons on channel conductance. Single-channel recordings showed that the unitary current amplitudes decreased with extracellular pH in a dose-dependent manner, consistent with a model in which protons bind to a site within the channel with an apparent pKa of ~6. The inhibition was voltage dependent, ~65% at –60 mV and 37% at +60 mV when pH was reduced from 7.4 to 5.5. The unitary current amplitudes reached saturation at [K+] ≥ 1 M, and notably the maximum amplitudes did not converge with different pHs, inconsistent with a blockade model based on surface charge screening or competitive inhibition of permeating ions. Mutagenesis experiments uncovered two acidic residues critical for proton inhibition, one located at the pore entrance and the other on the pore helix. Based on homology to the KcsA structure, the two acidic residues, along with another basic residue also on the pore helix, could form a triad interacting with each other through extensive hydrogen bonds and electrostatic contacts, suggesting that protons may mediate the interactions between the selectivity filter and pore helix, thereby altering the local structure in the filter region and consequently the conductance of the channel.

 
 
 
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