Molecular and phylogenetic characterization of a novel putative membrane transporter (SLC10A7), conserved in vertebrates and bacteria

The 'Solute Carrier Family SLC10' consists of six annotated members in humans, comprising two bile acid carriers (SLC10A1 and SLC10A2), one steroid sulfate transporter (SLC10A6), and three orphan carriers (SLC10A3 to SLC10A5). In this study we report molecular characterization and expression analysis of a novel member of the SLC10 family, SLC10A7, previously known as C4orf13. SLC10A7 proteins consist of 340-343 amino acids in humans, mice, rats, and frogs and show an overall amino acid sequence identity of >85%. SLC10A7 genes comprise 12 coding exons and show broad tissue expression pattern. When expressed in Xenopus laevis oocytes and HEK293 cells, SLC10A7 was detected in the plasma membrane but revealed no transport activity for bile acids and steroid sulfates. By immunofluorescence analysis of dual hemagglutinin (HA)- and FLAG-labeled SLC10A7 proteins in HEK293 cells, we established a topology of 10 transmembrane domains with an intracellular cis orientation of the N-terminal and C-terminal ends. This topology pattern is clearly different from the seven-transmembrane domain topology of the other SLC10 members but similar to hitherto uncharacterized non-vertebrate SLC10A7-related proteins. In contrast to the established SLC10 members, which are restricted to the taxonomic branch of vertebrates, SLC10A7-related proteins exist also in yeasts, plants, and bacteria, making SLC10A7 taxonomically the most widespread member of this carrier family. Vertebrate and bacterial SLC10A7 proteins exhibit >20% sequence identity, which is higher than the sequence identity of SLC10A7 to any other member of the SLC10 carrier family.     

Overexpression of cardiac actin with baculovirus is promoter dependent

The influence of the promoter and an N-terminal hexahistidine tag on human cardiac actin (ACTC) expression and function was investigated using four baculovirus constructs. It was found that both non-tagged ACTC and hisACTC expression from the p10 promoter was higher than from the polh promoter. Characterization showed that an N-terminal hexahistidine tag has a negative effect on ACTC. Recombinant ACTC inhibits DNase-I and binds myosin S1, indicative of proper folding. Our data support the hypothesis that the actin protein down-regulates the polh promoter.     

Leishmania major: Reactive oxygen species and interferon gamma induction by soluble lipophosphoglycan of stationary phase promastigotes

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is membrane glycoconjugate lipophosphoglycan (mLPG). In addition, it has been shown that mLPG could be used as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we attempted to compare the immunological properties of culture supernatant and membrane LPG prepared from stationary phase promastigotes of Leishmania major. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by thin layer chromatography. The effect of sLPG and mLPG on the production of reactive oxygen species (ROS) was studied using PBMCs isolated from healthy individuals. In addition, induction of IL-12, IFN-gamma and IL-10 secretion in the presence of sLPG and mLPG was investigated. Reactive oxygen species in addition to IL-10 and IL-12 were induced by both sLPG and mLPG. However, IFN-gamma production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design.     

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.     

Temperature-respiration relationships differ in mycorrhizal and non-mycorrhizal root systems of Picea abies (L.) Karst

Root respiration has been shown to increase with temperature, but less is known about how this relationship is affected by the fungal partner in mycorrhizal root systems. In order to test respiratory temperature dependence, in particular Q (10) of mycorrhizal and non-mycorrhizal root systems, seedlings of PICEA ABIES (L.) Karst. (Norway spruce) were inoculated with the ectomycorrhizal fungus PILODERMA CROCEUM (Eriksson and Hjortstam, SR430; synonym: PILODERMA FALLAX: [Libert] Stalpers) and planted in soil respiration cuvettes (mycocosms). Temperature dependence of hyphal respiration in sterile cultures was determined and compared with respiration of mycorrhizal roots. Respiration rates of mycorrhizal and non-mycorrhizal root systems as well as sterile cultures were sensitive to temperature. Q (10) of mycorrhizal root systems of 3.0 +/- 0.1 was significantly higher than that of non-mycorrhizal systems (2.5 +/- 0.2). Q (10) of P. CROCEUM in sterile cultures (older than 2 months) was similar to that of mycorrhizal root systems, suggesting that mycorrhizae may have a large influence on the temperature sensitivity of roots in spite of their small biomass. Our results stress the importance of considering mycorrhization when modeling the temperature sensitivity of spruce roots.     

大气颗粒物(PM2.5、PM10)对地表景观结构的响应研究进展

颗粒物PM_(2.5)、PM_(10)是近年来我国大气首要污染物,威胁环境和人类健康。地表景观结构直接或间接影响PM_(2.5)、PM_(10)浓度,了解其影响过程和机理对于改善生态环境具有重要意义。系统总结了国内外关于PM_(2.5)、PM_(10)对地表景观结构响应的研究成果,指出研究中出现不确定性的可能影响因素,并对今后的发展方向进行展望。得出基本结论:(1)地表景观类型的构成及其格局显著影响大气颗粒物浓度,对PM_(2.5)、PM_(10)起到"源"和"汇"的作用。(2)地表景观结构引起局地气候变化并影响颗粒物的迁移转化,但其影响过程和机理复杂,研究结论并不明确。(3)颗粒物浓度和地表景观数据主要通过实际监测或遥感处理方法获得,但因为获取方法、监测点微观环境及遥感影像等因素影响,导致数据具有不确定性,加上时空尺度相对应的复杂性,大大限制了地表景观结构与PM_(2.5)、PM_(10)响应关系的研究进展,是未来要突破的难点。(4)PM_(2.5)、PM_(10)对地表景观结构响应的区域时空差异及过程,局地小气候变化对颗粒物浓度的影响过程和强度,主要景观类型尤其是水体、湿地景观对大气颗粒物浓度的影响过程、机理与贡献程度等是未来需要关注的方向。     

Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N5‐formyltetrahydrofolate and UDP‐Ara4N

ArnA from Escherichia coli is a key enzyme involved in the formation of 4‐amino‐4‐deoxy‐l ‐arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram‐negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N‐ and C‐terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N¹⁰‐formyltetrahydrofolate. Here we describe the structure of the isolated N‐terminal domain of ArnA in complex with its UDP‐sugar substrate and N⁵‐formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.     

Estrogen induced synthesis of specific proteins in human breast cancer cells

Human breast cancer cells in tissue culture (MCF-7) were pretreated with the antiestrogen nafoxidine to arrest cellular proliferation and then were given estradiol to release this block and stimulate DNA synthesis and cell division. During this period of growth stimulation intracellular proteins, labeled by a double isotope method, were analyzed on SDS-polyacrylamide gel electrophoresis. Estradiol directly increases the rates of synthesis of specific proteins which migrate on SDS-gels at molecular weights of 24,000 and 36,000. Nafoxidine-pretreatment alone does not induce these same proteins, and no changes in the rates of specific protein synthesis occur in cells grown on control medium for the same length of time as on estradiol. Induced synthesis of these proteins is observed only during the period of estrogen stimulation of cell proliferation following pretreatment with nafoxidine. We do not detect induction when cells are incubated with estradiol without antiestrogen-pretreatment. Since rescue of antiestrogen growth inhibition is also the only condition under which MCF-7 cell division can be reproducibly stimulated by estrogen, these proteins may be related to estrogen effects on cellular proliferation.     

Development of chemiluminescence method for determination of 10‐hydroxycamptothecin based on luminol–[Ag(HIO6)2]5− reaction in alkaline solution

A novel chemiluminescence (CL) method was developed for the determination of 10‐hydroxycamptothecin(HCPT) based on the CL reaction between [Ag(HIO₆)₂]^5? and luminol in alkaline solution. CL emission of Ag(III) complex–luminol in alkaline medium was very different from that in acidic medium. A possible mechanism of enhanced CL emission was suggested. The enhanced effect of HCPT on CL emission of the [Ag(HIO₆)₂]^5?–luminol system was found. The enhanced degree of CL emission was proportional to HCPT concentration. The effect of the reaction conditions on CL emission was examined. Under optimal conditions, the limit of detection was 6.5 × 10^?9 g mL^?1. The proposed method was applied for the determination of HCPT in real samples with the recoveries of 93.2–109% with the RSD of 1.7–3.3%. Copyright © 2010 John Wiley & Sons, Ltd.