Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus)

Three novel glutathione S-transferase (GSTs) cDNAs were cloned from a disk abalone (Haliotis dicus discus) cDNA library. Multiple alignment and phylogenetic analysis of three GSTs revealed that their closest relationship is with insect sigma GSTs. Recombinant GSTs were over-expressed in Escherichia coli as soluble fusion proteins. HdGSTS1 and HdGSTS2 were active towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid, whereas HdGSTS3 appeared to be a non-enzymatic GST. Two active GSTs had similar optimum conditions for enzymatic reaction at pH 8.0 and temperature of approximately 30 degrees C. Molecular modeling analysis of three GSTs implicates their diverse active sites as being responsible for their different enzymatic features. Three sigma GSTs had significantly different expression patterns and levels of expression in abalone tissues, indicating their different functions. After 48 h-exposure to three model marine pollutants, only HdGSTS1 exhibited a proper inducibility, exhibiting its good biomarker potential for organic contaminants in marine environment. In contrast, the other two sigma GSTs revealed a minor role in the response of pollutants exposure.     

Inhibition of casein kinase 1-epsilon induces cancer-cell-selective,PERIOD2-dependent growth arrest

Background  

Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.     

Trans-synaptic plasticity: presynaptic initiation, postsynaptic memory

A novel mechanism of persistent facilitation induced by serotonin at Aplysia synapses depends upon rapid postsynaptic protein synthesis and increased responsiveness to glutamate; whereas the memory for this synaptic change is postsynaptic, the initiating signal may be an increase in spontaneous release of glutamate from the presynaptic terminals.     

Stabilization of Metals in Subsurface by Biopolymers: Laboratory Drainage Flow Studies

Environmental contamination with heavy metals and radionuclides remains a major problem worldwide. The current clean-up methodologies are based on energy-intensive engineering processes, which are disruptive and costly. A new universal technology targeted for the permanent enclosure and fixation of nuclear and other extreme hazardous metallic wastes in subsurface sites is needed. Such technology will be useful in treating contamination at many sites in the U.S., with specific applications to Department of Energy (DOE) sites. Biopolymers are potential tools for such an innovative technology. Biopolymers have repeated sequences, and therefore provide ample opportunity for chemical reactions with metals, soil particles, and other biopolymers. They also have the additional ability of creating cross-linking interpenetrating networks that can encapsulate the contaminants. Based on this concept, in the present work five biopolymers (xanthan, chitosan, polyhydroxy butyrate, guar gum, polyglutamic acid) were investigated for potential use in the stabilization of metals in the subsurface. The effects of these biopolymers (used alone and in combinations) on soil characteristics (permeability, shear strength) and their metal uptake ability have been studied using laboratory drainage flow systems. Biopolymer solutions were run through the experimental sandpack columns, followed by copper solution and leaching agents (distilled water and hydrochloric acid). The permeability and shear strength of sand were evaluated. Copper uptake capacity of each biopolymer and combination of biopolymers was also studied along with subsequent leaching. All biopolymers tested improved sand characteristics (by decreasing permeability and increasing shear strength) and had good metal uptake ability (60–90%) with relatively low leachability (10–22%). While biopolymers used alone were more efficient in metal uptake, the combination of two biopolymers (xanthan and chitosan) had an increasing plugging effect. These results show the potential of using biopolymers in subsurface metal stabilization.     

Evidence that TGFA influences risk to cleft lip with/without cleft palate through unconventional genetic mechanisms

This study examined the association between markers in transforming growth factor alpha (TGFA) and isolated, non-syndromic cleft lip with/without palate (CL/P) using a case–parent trio design, considering parent-of-origin effects. We also tested for gene–environmental interaction with common maternal exposures, and for gene–gene interaction using markers in TGFA and another recognized causal gene, IRF6. CL/P case–parent trios from four populations (76 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 17 single nucleotide polymorphisms (SNPs) in TGFA. The transmission disequilibrium test was used to test individual SNPs, and the parent-of-origin likelihood ratio test (PO-LRT) was used to assess parent-of-origin effects. We also screened for possible gene–environment interaction using PBAT, and tested for gene–gene interaction using conditional logistic regression models. When all trios were combined, four SNPs showed significant excess maternal transmission, two of which gave significant PO-LRT values [rs3821261: P = 0.004 and OR(imprinting) = 4.17; and rs3771475: P = 0.027 and OR(imprinting) = 2.44]. Haplotype analysis of these two SNPS also supported excess maternal transmission. We saw intriguing but suggestive evidence of G × E interaction for several SNPs in TGFA when either individual SNPs or haplotypes of adjacent SNPs were considered. Thus, TGFA appears to influence risk of CL/P through unconventional means with an apparent parent-of-origin effect (excess maternal transmission) and possible interaction with maternal exposures.     

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

Effect of monoclonal antibody on expression of lipid metabolism related genes in porcine adipocytes

In order to study the mechanism of monoclonal antibody (McAb) against a porcine 40-kDa adipocyte-specific plasma membrane protein in reducing fat deposition, porcine primary adipocytes were treated with the McAb during the process of adipocyte differentiation; its effect on expression of lipid metabolism related genes was investigated. Adipocytes were treated with 1-methyl-3-isobutylmethylxanthine (IDX) plus 10 μg/mL of the McAb or without McAb. The mRNA levels of adipocyte differentiation related genes (PPARγ and C/EBPα), lipid metabolism related genes (FAS, HSL, CPT-1B, DGAT and A-FABP) and adiponectin gene (AdipoQ) were determined using real-time quantitative PCR. The results showed that the differentiated adipocyte number and triglyceride (TG) content in adipocytes treated with the McAb were lower than that in cells without McAb during the whole process of adipocyte differentiation. The McAb significantly reduced mRNA expression of PPARγ, C/EBPα, FAS, DGAT, A-FABP and adiponectin genes, but increased mRNA expression of HSL and CPT-1B genes during the medium and latter stage of adipocyte differentiation. This suggested that the McAb decreased triglycerol accumulation in adipocyte by both inhibiting adipocyte differentiation and regulating lipid metabolism, especially at the medium and latter stage of porcine adipocyte differentiation.     

Hitchhiking of Cu/Zn Superoxide Dismutase to Peroxisomes – Evidence for a Natural Piggyback Import Mechanism in Mammals

Most newly synthesized peroxisomal proteins are imported in a receptor-mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so-called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner 'copper chaperone of SOD1' (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D-liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1-dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS-mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co-import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.