Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils.

S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was greater than 60 min. The addition of resting PMN did not affect the T1/2 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10(-7) M) reduced measurable SNO-GSH (15 microM) at 5 min (48 +/- 5.0% control, P less than 0.05). Incubation (5 min, 37 degrees C) of PMN with 10 microM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 +/- 3 vs 78 +/- 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.     

The Mutation Bronze-Mutable 4 Derivative 6856 in Maize Is Caused by the Insertion of a Novel 6.7-Kilobase Pair Transposon in the Untranslated Leader Region of the Bronze-1 Gene

The Ds-controlled allele, bz-m4 Derivative 6856 [bz-m4 D6856], is reported to have an altered temporal- and tissue-specific pattern of gene expression. We have cloned this allele and have characterized it at the molecular level. The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site. The insert is 6.7-kbp long. Ds elements, each approximately 2 kbp in length, are at both ends of the insert. The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene. These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.     

微塑料对湖泊生态系统初级生产者的影响

塑料的广泛应用导致大量微塑料进入环境,尤其是水环境,从而产生环境风险。浮游植物等自养生物是湖泊系统的主要初级生产者,是湖泊食物链的关键组成部分,为食物链的上游提供能量和物质基础。同时,浮游植物也是对微塑料响应最敏感的类群。了解湖泊浮游植物等初级生产者对微塑料的响应是探究微塑料对湖泊生态系统功能影响的重要基础。总结了全球湖泊生态系统微塑料的丰度、类型、尺寸、来源等分布特征,系统分析了微塑料暴露对浮游植物等初级生产者细胞结构、基因表达和生长,以及对浮游植物叶绿素a含量、光合活性的影响,并总结了其中的影响机制。总体而言,微塑料会降低初级生产者的叶绿素a含量,剂量越高、尺寸越小,这种抑制作用越强烈;同时,微塑料也会作用于初级生产者,造成细胞膜损伤、DNA损伤,调控其相关功能基因表达,抑制其生长和光合活性等。然而,湖泊生态系统微塑料的实际检出浓度远低于室内暴露实验中的添加剂量,微塑料结构和组分也更为复杂,野外观测结果与室内培养实验之间还不能建立直接的对应关系。因此,未来的相关研究应集中在如何有效联系野外观测结果与室内培养实验结果,进一步聚焦建立可靠的、可应用推广的微塑料浓度与初级生产者之间的剂量-效应关系模型,探究微塑料对湖泊初级生产者的作用机制,为刻画微塑料对湖泊生态系统初级生产者及其功能的作用机制提供科学支撑。     

FH535 Inhibited Migration and Growth of Breast Cancer Cells

There is substantial evidence indicating that the WNT signaling pathway is activated in various cancer cell types including breast cancer. Previous studies reported that FH535, a small molecule inhibitor of the WNT signaling pathway, decreased growth of cancer cells but not normal fibroblasts, suggesting this pathway plays a role in tumor progression and metastasis. In this study, we tested FH535 as a potential inhibitor for malignant phenotypes of breast cancer cells including migration, invasion, and growth. FH535 significantly inhibited growth, migration, and invasion of triple negative (TN) breast cancer cell lines (MDA-MB231 and HCC38) in vitro. We demonstrate that FH535 was a potent growth inhibitor for TN breast cancer cell lines (HCC38 and MDA-MB-231) but not for other, non-TN breast cancer cell lines (MCF-7, T47D or SK-Br3) when cultured in three dimensional (3D) type I collagen gels. Western blotting analyses suggest that treatment of MDA-MB-231 cells with FH535 markedly inhibited the expression of NEDD9 but not activations of FAK, Src, or downstream targets such as p38 and Erk1/2. We demonstrated that NEDD9 was specifically associated with CSPG4 but not with β1 integrin or CD44 in MDA-MB-231 cells. Analyses of gene expression profiles in breast cancer tissues suggest that CSPG4 expression is higher in Basal-type breast cancers, many of which are TN, than any other subtypes. These results suggest not only a mechanism for migration and invasion involving the canonical WNT-signaling pathways but also novel strategies for treating patients who develop TN breast cancer.     

Population Genetics of the Washington National Primate Research Center's (WaNPRC) Captive Pigtailed Macaque (Macaca nemestrina) Population

Pigtailed macaques (Macaca nemestrina) provide an important model for biomedical research on human disease and for studying the evolution of primate behavior. The genetic structure of captive populations of pigtailed macaques is not as well described as that of captive rhesus (M. mulatta) or cynomolgus (M. fascicularis) macaques. The Washington National Primate Research Center houses the largest captive colony of pigtailed macaques located in several different housing facilities. Based on genotypes of 18 microsatellite (short tandem repeat [STR]) loci, these pigtailed macaques are more genetically diverse than captive rhesus macaques and exhibit relatively low levels of inbreeding. Colony genetic management facilitates the maintenance of genetic variability without compromising production goals of a breeding facility. The periodic introduction of new founders from specific sources to separate housing facilities at different times influenced the colony's genetic structure over time and space markedly but did not alter its genetic diversity significantly. Changes in genetic structure over time were predominantly due to the inclusion of animals from the Yerkes National Primate Research Center in the original colony and after 2005. Strategies to equalize founder representation in the colony have maximized the representation of the founders’ genomes in the extant population. Were exchange of animals among the facilities increased, further differentiation could be avoided. The use of highly differentiated animals may confound interpretations of phenotypic differences due to the inflation of the genetic contribution to phenotypic variance of heritable traits. Am. J. Primatol. 74:1017‐1027, 2012. © 2012 Wiley Periodicals, Inc.     

Glia Modulate NMDA-Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N-methyl-d -aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca²⁺ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells. The partially purified enzyme was active against glycogen, starch, maltotriose and maltose. It exhibited maximum catalytic activity against glycogen at pH 5.5. The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose. SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques. SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.