CSPG4, a potential therapeutic target, facilitates malignant progression of melanoma

Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified as a highly immunogenic tumor antigen on the surface of melanoma cells, is associated with melanoma tumor formation and poor prognosis in certain melanomas and several other tumor types. The complex mechanisms by which CSPG4 affects melanoma progression have started to be defined, in particular the association with other cell surface proteins and receptor tyrosine kinases (RTKs) and its central role in modulating the function of these proteins. CSPG4 is essential to the growth of melanoma tumors through its modulation of integrin function and enhanced growth factor receptor-regulated pathways including sustained activation of ERK 1,2. This activation of integrin, RTK, and ERK1,2 function by CSPG4 modulates numerous aspects of tumor progression. CSPG4 expression has further been correlated to resistance of melanoma to conventional chemotherapeutics. This review outlines recent advances in our understanding of CSPG4-associated cell signaling, describing the central role it plays in melanoma tumor cell growth, motility, and survival, and explores how modifying CSPG4 function and protein-protein interactions may provide us with novel combinatorial therapies for the treatment of advanced melanoma.     

Bacterial Protein N-Glycosylation: New Perspectives and Applications

Protein glycosylation is widespread throughout all three domains of life. Bacterial protein N-glycosylation and its application to engineering recombinant glycoproteins continue to be actively studied. Here, we focus on advances made in the last 2 years, including the characterization of novel bacterial N-glycosylation pathways, examination of pathway enzymes and evolution, biological roles of protein modification in the native host, and exploitation of the N-glycosylation pathways to create novel vaccines and diagnostics.     

Structure,variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> − 700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.     

ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides

High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n = 4x = 28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs.     

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.     

Redox regulation of glutenin subunit assembly in the plant endoplasmic reticulum

The glutenin fraction of wheat storage proteins consists of large polymers in which high‐ and low‐molecular‐weight subunits are connected by inter‐chain disulfide bonds. We found that assembly of a low‐molecular‐weight glutenin subunit in the endoplasmic reticulum is a rapid process that leads to accumulation of various oligomeric forms, and that this assembly is sensitive to perturbation of the cellular redox environment. In endoplasmic reticulum‐derived microsomes, low‐molecular‐weight glutenin subunits are subjected to hyper‐polymerization, indicating that cytosolic factors play an important role in limiting polymer size. Addition of physiological concentrations of reduced glutathione is sufficient to maintain the original polymerization pattern of the glutenin subunits upon cytosol dilution. Furthermore, we show that a low‐molecular‐weight glutenin subunit can be glutathionylated in endoplasmic reticulum‐derived microsomes, and that it can be directly reduced by glutathione in vitro. These results indicate that glutenin polymerization is sensitive to changes in the redox state of the cell, and suggest that the presence of a reducing cytosolic environment plays an important role in regulating disulfide bond formation in the endoplasmic reticulum of plant cells.     

Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Anti-inflammatory, hypolipidemic, and insulin-sensitizing effects of DHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated down-regulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R. Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex down-regulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.     

小麦低分子量麦谷蛋白亚基组成研究

利用改良的两步一向SDS—PAGE(two—step one—dimensional SDS—PAGE)分析了几种小麦低分子量麦谷蛋白亚基(LMW-GS)组成。70％热乙醇提取总谷蛋白,11％分离胶进行第一步SDS—PAGE分离．电泳1h后切取顶端1cm胶条并置于巯基乙醇溶液进行还原,还原后的胶条于11%～16．5％的梯度胶进行第二步SDS—PAGE分离。结果显示。两步一向SDS—PAGE可以彻底除去清蛋白、球蛋白和醇溶蛋白对LMW—GS分离的背景干扰。提高LMW—GS的分辨率。对几种小麦低分子量麦谷蛋白亚基分析表明：LMW-GS组合比HMW—GS更为丰富,每种小麦含有2～5种B亚基,2～4种C亚基．B、C亚基的总数量为4～8种。     

小麦高分子量麦谷蛋白亚基1Dx5启动子驱动外源基因的表达

将小麦高分子量麦谷蛋白亚基(HMW-GS)基因的胚乳组织特异性表达启动子驱动的外源突变型1Dx5基因和gus基因导入小麦中.对其转基因植株连续3代的跟踪研究表明,突变型1Dx5基因的重复序列导致其表达蛋白分子量增大,并影响其它1Bx17 1By18亚基基因的表达.组织化学分析观察到gus基因在1Dx5基因启动子驱动下的表达表现出胚乳组织特异性,在开花2周后开始表达,表达量呈持续上升,至腊熟期达到最高,其次为籽粒成熟期.     

高分子量麦谷蛋白１４和１５亚基的纯化、Ｎ—末端序列及部分生化特性研究

用ＳＤＳ－ＰＡＧＥ制备电泳技术结合一种新的凝胶中蛋白质显色方法,对普通小麦（Triticum aestivum)小偃六号的高分子量麦谷蛋白１４和１５亚基进行了有效的分离纯化,将其转印于ＰＶＤＦ膜上测定了Ｎ－端的氨基酸顺序,通过比较了发现它们与已知序列的其他的高分子是麦谷蛋白亚基高度同源。用两种双向电泳技术确定了它们的等电点（ＰＩ）属于碱性范围。