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Articles by S Li
Total Records ( 23 ) for S Li
  H Liu , S Li , Y Zhang , Y Yan and Y. Li
 

Glutamate decarboxylase 65 (GAD65) produces -aminobutyric acid, the main inhibitory neurotransmitter in adult mammalian brain. Previous experiments, performed in brain, showed that GAD65 gene possesses two TATA-less promoters, although the significance is unknown. Here, by rapid amplification of cDNA ends method, two distinct GAD65 mRNA isoforms transcribed from two independent clusters of transcription start sites were identified in post-natal rat testis. RT–PCR results revealed that the two mRNA isoforms had distinct expression patterns during post-natal testis maturation, suggesting that GAD65 gene expression was regulated by alternative promoters at the transcription level. By using GAD65-specific antibodies, western blotting analysis showed that the 58-kDa GAD65, N-terminal 69 amino acids truncated form of full-length GAD65 protein, was developmentally expressed during post-natal testis maturation, suggesting that GAD65 gene expression in testis may also be regulated by post-translational processing. Confocal immunofluorescence microscopy revealed that GAD65 protein was presented in Leydig cells of Day 1 testis, primary spermatocytes and spermatids of post-natal of Day 90 testis. The above results suggested that GAD65 gene expression is dynamically regulated at multiple levels during post-natal testis maturation.

  H Zhou , Y Xiao , R Li , S Hong , S Li , L Wang , R Zeng and K. Liao
 

Adipocyte is not only a central player involved in storage and release of energy, but also in regulation of energy metabolism in other organs via secretion of peptides and proteins. During the pathogenesis of insulin resistance and type 2 diabetes, adipocytes are subjected to the increased levels of insulin, which may have a major impact on the secretion of adipokines. We have undertaken cleavable isotope-coded affinity tag (cICAT) and label-free quantitation approaches to identify and quantify secretory factors that are differentially secreted by 3T3-L1 adipocytes with or without insulin treatment. Combination of cICAT and label-free results, there are 317 proteins predicted or annotated as secretory proteins. Among these secretory proteins, 179 proteins and 53 proteins were significantly up-regulated and down-regulated, respectively. A total of 77 reported adipokines were quantified in our study, such as adiponectin, cathepsin D, cystatin C, resistin, and transferrin. Western blot analysis of these adipokines confirmed the quantitative results from mass spectrometry, and revealed individualized secreting patterns of these proteins by increasing insulin dose. In addition, 240 proteins were newly identified and quantified as secreted proteins from 3T3-L1 adipocytes in our study, most of which were up-regulated upon insulin treatment. Further comprehensive bioinformatics analysis revealed that the secretory proteins in extracellular matrix-receptor interaction pathway and glycan structure degradation pathway were significantly up-regulated by insulin stimulation.

  H Zemkova , M Kucka , S Li , A. E Gonzalez Iglesias , M Tomic and S. S. Stojilkovic
 

Anterior pituitary cells express cation-conducting P2X receptor channels (P2XRs), but their molecular identity, electrophysiological properties, cell-specific expression pattern, and physiological roles have been only partially characterized. In this study, we show by quantitative RT-PCR that mRNA transcripts for the P2X4 subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X4R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltage-insensitive Ca2+ influx. In the presence of ivermectin, a specific allosteric modulator of P2X4Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X4Rs, which facilitate Ca2+ influx and hormone secretion.

  L. R Meacham , C. A Sklar , S Li , Q Liu , N Gimpel , Y Yasui , J. A Whitton , M Stovall , L. L Robison and K. C. Oeffinger
 

Background  Childhood cancer survivors are at increased risk of morbidity and mortality. To further characterize this risk, this study aimed to compare the prevalence of diabetes mellitus (DM) in childhood cancer survivors and their siblings.

Methods  Participants included 8599 survivors in the Childhood Cancer Survivor Study (CCSS), a retrospectively ascertained North American cohort of long-term survivors who were diagnosed between 1970 and 1986 as well as 2936 randomly selected siblings of the survivors. The main outcome was self-reported DM.

Results  The mean ages of the survivors and the siblings were 31.5 years (age range, 17.0-54.1 years) and 33.4 years (age range, 9.6-58.4 years), respectively. Diabetes mellitus was reported in 2.5% of the survivors and 1.7% of the siblings. After adjustment for body mass index, age, sex, race/ethnicity, household income, and insurance, the survivors were 1.8 times more likely than the siblings to report DM (95% confidence interval [CI], 1.3-2.5; P < .001), with survivors who received total body irradiation (odds ratio [OR], 12.6; 95% CI, 6.2-25.3; P < .001), abdominal irradiation (OR, 3.4; 95% CI, 2.3-5.0; P < .001), and cranial irradiation (OR, 1.6; 95% CI 1.0-2.3; P = .03) at increased risk. In adjusted models, an increased risk of DM was associated with total body irradiation (OR, 7.2; 95% CI, 3.4-15.0; P < .001), abdominal irradiation (OR, 2.7; 95% CI, 1.9-3.8; P < .001), use of alkylating agents (OR, 1.7; 95% CI, 1.2-2.3; P < .01), and younger age at diagnosis (0-4 years; OR, 2.4; 95% CI, 1.3-4.6; P < .01).

Conclusion  Childhood cancer survivors treated with total body or abdominal irradiation have an increased risk of diabetes that appears unrelated to body mass index or physical inactivity.

  X Guo , X Xiao , S Li , P Wang , X Jia and Q. Zhang
 

Objective  To identify the genetic locus for X-linked nonsyndromic high myopia in a large Chinese family.

Methods  Phenotypic information and DNA samples were collected from 19 individuals in a Chinese family; 7 had high myopia and 12 were unaffected. We performed a linkage scan on the X chromosome and sequenced several candidate genes.

Results  High myopia in this family, presenting since early childhood and ranging from –6.00 to –15.00 diopters of sphere, is consistent with an X-linked recessive trait. The presence of a normal optic disc and the absence of color visual defects and other systemic abnormalities indicated that high myopia in this family is nonsyndromic. Our linkage analysis mapped the disease locus to Xq28, a 6.1-cM region between DXS8069 and Xqter, with 2-point logarithm of odds scores greater than 2.0 for 5 markers and a maximum logarithm of odds score of 3.59 at  = 0 for 2 markers. Sequence analysis of coding and adjacent intronic regions of GPR50, PRRG3, CNGA2, and BGN did not identify any potential causative mutation.

Conclusions  Nonsyndromic high myopia in a Chinese family was mapped to the MYP1 region, which confirmed and refined this region for high myopia. In addition, our results suggest that color visual defects and optic disc hypoplasia are not necessary signs of high myopia attributed to the MYP1 region.

Clinical Relevance  MYP1 is a common and the best locus for positional cloning of the gene responsible for high myopia. Our results suggest that MYP1 is also responsible for nonsyndromic high myopia.

  D. L Price , S. F Lin , Z Han , G Simpson , R. S Coffin , J Wong , S Li , Y Fong and R. J. Wong
 

Objective  To determine if prodrug conversion of fluorocytosine to fluorouracil by an engineered herpes virus, OncoVEXGALV/CD, enhances oncolytic therapy of head and neck squamous cell carcinoma.

Design  We assessed the ability of OncoVEXGALV/CD and OncoVEXGFP to infect, replicate within, and lyse 4 head and neck squamous cell carcinoma lines in vitro. The effects of adding fluorocytosine with OncoVEXGALV/CD were evaluated.

Results  Head and neck squamous cell carcinoma was permissive to green fluorescent protein expression in100% of cells by OncoVEXGFP at a multiplicity of infection of 1 after 48 hours and supported logarithmic viral replication. Virus caused more than 60% cell death 6 days after exposure to virus at a multiplicity of infection of 0.1 in 3 of the 4 cell lines. Fluorocytosine did not enhance cytotoxicity induced by OncoVEXGALV/CD at a multiplicity of infection of 0.1. However, for the least-sensitive SCC25 cell line, virus at a multiplicity of infection of 0.01 was cytotoxic to only 4% of cells after 6 days but was cytotoxic to 35% of cells with fluorocytosine.

Conclusions  OncoVEXGALV/CD efficiently infects, replicates within, and lyses head and neck squamous cell carcinoma at relatively low viral doses. Prodrug conversion by cytosine deaminase did not enhance therapy at viral doses that cause efficient cytotoxicity but may have beneficial effects in less-sensitive cell lines at low viral doses.

  S Li , Y Sun , C. P Liang , E. B Thorp , S Han , A. W Jehle , V Saraswathi , B Pridgen , J. E Kanter , R Li , C. L Welch , A. H Hasty , K. E Bornfeldt , J. L Breslow , I Tabas and A. R. Tall
 

Rationale: The complications of atherosclerosis are a major cause of death and disability in type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions.

Objective: To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis.

Methods and Results: We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr–/– mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr–/– mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr–/– macrophages showed an increased content of saturated fatty acids (FAs) and decreased -3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in -3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr–/– mice and this was reversed by a fish oil–rich diet.

Conclusions: The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of -3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

  S Li , B. J Scherlag , L Yu , X Sheng , Y Zhang , R Ali , Y Dong , M Ghias and S. S. Po
 

Background— We used high-frequency stimulation delivered during the refractory period of the atrium and pulmonary veins (PVs) to induce focal firing and atrial fibrillation (AF). This study was designed to demonstrate that bilateral low-level vagosympathetic nerve stimulation (LL-VNS) could suppress high-frequency stimulation-induced focal AF at atrial and PV sites.

Methods and Results— In 23 dogs anesthetized with Na-pentobarbital, electrodes in the vagosympathetic trunks allowed LL-VNS at 1 V below that which slowed the sinus rate or atrioventricular conduction. Multielectrode catheters were fixed at the right and left superior and inferior PVs and both atrial appendages. LL-VNS continued for 3 hours. At the end of each hour, the high-frequency stimulation algorithm consisting of a 40-ms train of stimuli (200 Hz; stimulus duration, 0.1 to 1.0 ms) was delivered 2 ms after the atrial pacing stimulus during the refractory period at each PV and atrial appendages site. The lowest voltage of high-frequency stimulation that induced AF was defined as the AF threshold. Five dogs without LL-VNS served as sham controls. Six dogs underwent LL-VNS after transection of bilateral vagosympathetic trunks. LL-VNS induced a progressive increase in AF threshold at all PV and atrial appendages sites, particularly significant (P<0.05) at the right superior PV, right inferior PV, left superior PV, and right atrial appendage. Bilateral vagosympathetic transection did not significantly alter the previous findings, and the 5 sham control dogs did not show changes in AF threshold at any site over a period of 3 hours.

Conclusions— LL-VNS may prevent episodic AF caused by rapid PV and non-PV firing.

  M. L Ji , J Wang , S Li and Z. C. Qi
 

A program mode is a regular trajectory of the execution of a program that is determined by the values of its input variables. By exploiting program modes, we may make worst-case execution time (WCET) analysis more precise. This paper presents a novel method to automatically find program modes and calculate the WCET estimates of programs. First, the modes of a program will be identified automatically by mode-relevant program slicing, and the precondition will be calculated for each mode using a path-wise test data generation method. Then, for each feasible mode, we show how to calculate its WCET estimate for modern reduced instruction set computer (RISC) processors with caches and pipelines and for traditional complex instruction set computer (CISC) processors. We also present a method to obtain the symbolic expression for each mode for CISC processors. The experimental results show the effectiveness of the method.

  A Banito , S. T Rashid , J. C Acosta , S Li , C. F Pereira , I Geti , S Pinho , J. C Silva , V Azuara , M Walsh , L Vallier and J. Gil
 

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16INK4a, and p21CIP1. Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.

  B Zheng , Z Wang , S Li , B Yu , J. Y Liu and X. Chen
 

Intergenic transcription by RNA Polymerase II (Pol II) is widespread in plant and animal genomes, but the functions of intergenic transcription or the resulting noncoding transcripts are poorly understood. Here, we show that Arabidopsis Pol II is indispensable for endogenous siRNA-mediated transcriptional gene silencing (TGS) at intergenic low-copy-number loci, despite the presence of two other polymerases—Pol IV and Pol V—that specialize in TGS through siRNAs. We show that Pol II produces noncoding scaffold transcripts that originate outside of heterochromatic, siRNA-generating loci. Through these transcripts and physical interactions with the siRNA effector protein ARGONAUTE4 (AGO4), Pol II recruits AGO4/siRNAs to homologous loci to result in TGS. Meanwhile, Pol II transcription also recruits Pol IV and Pol V to different locations at heterochromatic loci to promote siRNA biogenesis and siRNA-mediated TGS, respectively. This study establishes that intergenic transcription by Pol II is required for siRNA-mediated TGS, and reveals an intricate collaboration and division of labor among the three polymerases in gene silencing.

  Y Zhang , S Li , L Yuan , Y Tian , J Weidenfeld , J Yang , F Liu , A. L Chokas and E. E. Morrisey
 

Cardiomyocyte proliferation is high in early development and decreases progressively with gestation, resulting in the lack of a robust cardiomyocyte proliferative response in the adult heart after injury. Little is understood about how both cell-autonomous and nonautonomous signals are integrated to regulate the balance of cardiomyocyte proliferation during development. In this study, we show that a single transcription factor, Foxp1, can control the balance of cardiomyocyte proliferation during development by targeting different pathways in the endocardium and myocardium. Endocardial loss of Foxp1 results in decreased Fgf3/Fgf16/Fgf17/Fgf20 expression in the heart, leading to reduced cardiomyocyte proliferation. This loss of myocardial proliferation can be rescued by exogenous Fgf20, and is mediated, in part, by Foxp1 repression of Sox17. In contrast, myocardial-specific loss of Foxp1 results in increased cardiomyocyte proliferation and decreased differentiation, leading to increased myocardial mass and neonatal demise. We show that Nkx2.5 is a direct target of Foxp1 repression, and Nkx2.5 expression is increased in Foxp1-deficient myocardium. Moreover, transgenic overexpression of Nkx2.5 leads to increased cardiomyocyte proliferation and increased ventricular mass, similar to the myocardial-specific loss of Foxp1. These data show that Foxp1 coordinates the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation of Fgf ligand and Nkx2.5 expression.

  G Zhang , G Guo , X Hu , Y Zhang , Q Li , R Li , R Zhuang , Z Lu , Z He , X Fang , L Chen , W Tian , Y Tao , K Kristiansen , X Zhang , S Li , H Yang , J Wang and J. Wang
 

Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in ~33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.

  S Li , T Hu , Y Chen , X Wang , T Liu , G Ma and Z. Su
 

Carboxylmethylated konjac glucomannan (CKGM) is a carboxylmethylated polymer of mannose and glucose that is derived from the plant Amorphophallus konjac cultivated in East Asia. The CKGM solution had a high volume-expanding efficacy and was evaluated as a plasma substitute in the present study. Ameliorative hemorrhagic shock rabbits were used as the model animals. The in vivo hemodynamic and hemorheologic properties, including blood pressure, blood viscosity, hematocrit, erythrocyte deformation index and erythrocyte aggregation index, were measured in animals treated in the CKGM solution. The in vitro colloid osmotic pressure (COP) of the CKGM solution was measured to estimate its plasma-expanding efficacy. These parameters of the CKGM-treated group were compared with groups exposed to four other treatments: human serum albumin (HSA), hydroxyethyl starch (HES), polygeline and normal saline. The CKGM solution showed an exceptionally higher COP than other therapy solutions. For example, the COP of 1% (weight in volume [w/v]) CKGM solution is comparable to those of 6% (w/v) HES solution and 5% (w/v) HSA solution. Accordingly, the CKGM solution can be transfused in a much lower dosage while maintaining its plasma-expanding efficacy. The CKGM-treated group showed an improved intravascular persistence and good hemodynamic and hemorheological properties. Biopsy analysis suggested no organ dysfunction in the group treated in CKGM solution. Moreover, the high plasma-expanding efficacy and inexpensive availability of the CKGM solution may facilitate its clinical application as a potential plasma substitute.

  J. F Brodeur , S Li , O Damlaj and V. P. Dave
 

TCR expression on double-positive (DP) thymocytes is a prerequisite for thymic selection that results in the generation of mature CD4+ and CD8+ single-positive T cells. TCR is expressed at very low level on preselection DP thymocytes and is dramatically up-regulated on positively selected thymocytes. However, mechanism governing TCR expression on developing thymocytes is not understood. In the present report, we demonstrate that the intra-cytoplasmic (IC) domain of CD3 plays a critical role in regulating TCR expression on DP thymocytes. We provide genetic and biochemical evidence to show that the CD3 IC domain mutations result in elevated expression of fully assembled TCR on DP thymocytes. We also demonstrate that TCR up-regulation on DP thymocytes in these transgenic mice occurs in a ligand-independent manner. Further, we show that the proline-rich sequence and endoplasmic reticulum (ER) retention motifs in the IC domain of CD3 play synergistic role in regulating TCR surface expression on DP thymocytes.

  S Li , B Han , G Liu , J Ouellet , F Labrie and G. Pelletier
 

The sex steroids, estrogens, progesterone, and androgens, all play a role in mammary development and function. To precisely identify the sites of action of these steroids, we studied the localization of the estrogen receptor (ER) and ERβ, the progesterone receptor A (PRA) and PRB, and androgen receptors (AR) in the normal human mammary gland. Immunocytochemical localization of ER, ERβ, PRA, PRB, and AR was performed with reduction mammoplasty specimens from premenopausal women. ER, PRA, PRB, and AR were localized mostly to the inner layer of epithelial cells lining acini and intralobular ducts, as well as to myoepithelial cells scattered in the external layer of interlobular ducts. AR was also found in some stromal cells. ERβ staining was more widespread, resulting in epithelial and myoepithelial cells being labeled in acini and ducts as well as stromal cells. These results suggest that all sex steroids can directly act on epithelial cells to modulate development and function of the human mammary gland. Estrogens and androgens can also indirectly influence epithelial cell activity by an action on stromal cells. (J Histochem Cytochem 58:509–515, 2010)

  S Li , Q Wang , Y Wang , X Chen and Z. Wang
 

It is well established that epidermal growth factor (EGF) induces the cytoskeleton reorganization and cell migration through two major signaling cascades: phospholipase C-1 (PLC-1) and Rho GTPases. However, little is known about the cross talk between PLC-1 and Rho GTPases. Here we showed that PLC-1 forms a complex with Rac1 in response to EGF. This interaction is direct and mediated by PLC-1 Src homology 3 (SH3) domain and Rac1 106PNTP109 motif. This interaction is critical for EGF-induced Rac1 activation in vivo, and PLC-1 SH3 domain is actually a potent and specific Rac1 guanine nucleotide exchange factor in vitro. We have also demonstrated that the interaction between PLC-1 SH3 domain and Rac1 play a significant role in EGF-induced F-actin formation and cell migration. We conclude that PLC-1 and Rac1 coregulate EGF-induced cell cytoskeleton remodeling and cell migration by a direct functional interaction.

  S Li , D Zhang , L Yang , J. V Burnier , N Wang , R Lin , E. R Lee , R. I Glazer and P. Brodt
 

The IGF-I receptor (IGF-IR) was identified as a tumor progression factor, but its role in invasion and metastasis has been the subject of some controversy. Previously we reported that in murine lung carcinoma M-27 cells, overexpression of IGF-IR increased the synthesis and activation of matrix metalloproteinase (MMP)-2 via Akt/phosphatidylinositol 3-kinase signaling. In contrast, we show here that in these and other cells, IGF-IR overexpression reduced the constitutive and phorbol 12-myristate 13-acetate (PMA)-inducible expression of three protein kinase C (PKC)-regulated metalloproteinases, MMP-3, MMP-9, and MMP-13, in cultured cells as well as in vivo in sc tumors. To elucidate the underlying mechanism, we analyzed the effect of IGF-IR on PKC expression and activity using wild-type and IGF-IR-overexpressing (M-27IGFIR) tumor cells. Our results show that overexpression and activation of IGF-IR reduced PKC- expression, PKC activity, and downstream ERK1/2 signaling, and these effects were reversed in cells expressing kinase (Y1131,1135,1136F) or C-terminal (Y1250/51F) domain mutants of IGF-IR. This reduction was due to transcriptional down-regulation of PKC- as evidenced by reduced PKC- mRNA expression in a phosphatidylinositol 3-kinase-dependent manner and a blockade of PKC- promoter activation as revealed by a reporter gene assay. Finally, reconstitution of PKC- levels could restore MMP-9 expression levels in these cells. Collectively, these results show that IGF-IR can inhibit PKC- gene transcription and thereby block the synthesis of PMA-regulated MMPs, suggesting that within the same cells, IGF-IR can act as both a positive and negative regulator of MMP expression and function.

  J Ackerman , R Sharma , J Hitchcock , T Hayashi , Y Nagai , S Li , S Lu , J Miret , K Tang , F Spence and J. Aubrecht
 

We have developed the bioluminescent Salmonella reverse mutation assay as a tool for detecting mutagenicity applicable for high-throughput screening of new chemicals. In this study, we report the inter-laboratory evaluation of the assay using 10 model chemicals in five independent laboratories located in the USA (Groton, CT; Cambridge, MA and La Jolla, CA), Europe (Sandwich, Kent, UK) and Asia (Nagoya, Japan). The studies were performed in blinded fashion in all sites except for Groton and Cambridge laboratories. The chemicals were tested in at least three independent experiments using strains TA98-lux and TA100-lux in the presence and absence of metabolic activation. The results were statistically evaluated and compared to published results. Seven of the 10 compounds were positive in either TA98-lux and/or TA100-lux in the presence or absence of metabolic activation. The positive compound set included: nitrofurazone, 3-3'-dimethoxybenzidine, benzo[a]pyrene, 1,4-benzoquinone dioxime, 2-amino-5-nitrophenol, 2-bromo-4,6-dinitroaniline and busulfan. The remaining three compounds, namely, anthracene, crystal violet and benzyl chloride were negative in both Salmonella strains. Final results for individual compounds yielded 100% agreement among the laboratories and published data. Detailed comparison of all 40 individual test conditions yielded 93% (37 of 40) agreement among participating laboratories. We conclude that the bioluminescent Salmonella reverse mutation assay is a robust, accurate and economical higher throughput assay applicable for the mutagenicity screening of chemicals.

  J. X Yin , R. F Yang , S Li , A. O Renshaw , Y. L Li , H. D Schultz and M. C. Zimmerman
 

Reactive oxygen species (ROS), particularly superoxide (O2·–), have been identified as key signaling intermediates in ANG II-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O2·– in ANG II-stimulated neurons; however, additional sources of O2·–, including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O2·– in neurons and that increased scavenging of mitochondria-produced O2·– attenuates ANG II-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG II (100 nM) increased mitochondria-localized O2·– levels, as measured by MitoSOX Red fluorescence. This response was significantly attenuated in neurons overexpressing the mitochondria-targeted O2·–-scavenging enzyme Mn-SOD. To examine the biological significance of the ANG II-mediated increase in mitochondria-produced O2·–, we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II-induced inhibition of voltage-gated K+ current (IKv) in neurons. Adenovirus-mediated Mn-SOD overexpression or pretreatment with the cell-permeable antioxidant tempol (1 mM) significantly attenuated ANG II-induced inhibition of IKv. In contrast, pretreatment with extracellular SOD protein (400 U/ml) had no effect. Mn-SOD overexpression also inhibited ANG II-induced activation of Ca2+/calmodulin kinase II, a redox-sensitive protein known to modulate IKv. These data indicate that ANG II increases mitochondrial O2·–, which mediates, at least in part, ANG II-induced activation of Ca2+/calmodulin kinase II and inhibition of IKv in neurons.

  K Felio , H Nguyen , C. C Dascher , H. J Choi , S Li , M. I Zimmer , A Colmone , D. B Moody , M. B Brenner and C. R. Wang
 

Group 1 CD1 (CD1a, CD1b, and CD1c)–restricted T cells recognize mycobacterial lipid antigens and are found at higher frequencies in Mycobacterium tuberculosis (Mtb)–infected individuals. However, their role and dynamics during infection remain unknown because of the lack of a suitable small animal model. We have generated human group 1 CD1 transgenic (hCD1Tg) mice that express all three human group 1 CD1 isoforms and support the development of group 1 CD1–restricted T cells with diverse T cell receptor usage. Both mycobacterial infection and immunization with Mtb lipids elicit group 1 CD1–restricted Mtb lipid–specific T cell responses in hCD1Tg mice. In contrast to CD1d-restricted NKT cells, which rapidly respond to initial stimulation but exhibit anergy upon reexposure, group 1 CD1–restricted T cells exhibit delayed primary responses and more rapid secondary responses, similar to conventional T cells. Collectively, our data demonstrate that group 1 CD1–restricted T cells participate in adaptive immune responses upon mycobacterial infection and could serve as targets for the development of novel Mtb vaccines.

  J Kranich , N. J Krautler , J Falsig , B Ballmer , S Li , G Hutter , P Schwarz , R Moos , C Julius , G Miele and A. Aguzzi
 

Progressive accumulation of PrPSc, a hallmark of prion diseases, occurs when conversion of PrPC into PrPSc is faster than PrPSc clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrPSc accumulation and increased prion titers in prion-infected C57BL/6 x 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8–/– mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrPSc accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8.

 
 
 
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