Structure of the receptor for insulin-like growth factor II: the puzzle amplified

The insulin-like growth factor II (IGF-II) is a polypeptide hormone with structural homologies to insulin and insulin-like growth factor I (IGF-I). In contrast to these other hormones, the in vivo function of IGF-II is not known. Although IGF-II can stimulate a broad range of biological responses in isolated cells, these responses have usually been found to be mediated by the insulin and IGF-I receptors. Recently, the receptor for IGF-II was found to also be the receptor for mannose-6-phosphate. Since this latter receptor has been implicated in targeting of lysosomal enzymes, the question is now raised of whether the same protein can also mediate metabolic responses to IGF-II.     

不同磷源对海洋卡盾藻生长和碱性磷酸酶活性的影响

[目的]研究不同磷源对海洋卡盾藻（Chattonella marina）生长和藻细胞碱性磷酸酶活性（APA）的影响。[方法]磷源分别为无机磷（NaH2PO4.2H2O）及有机磷源葡萄糖-6-磷酸（G-6-P）、甘油磷酸钠（G-P）、三磷酸腺苷（ATP）和卵磷脂（LEC）。[结果]海洋卡盾藻可以有效利用NaH2PO4.2H2O,对G-6-P、G-P、ATP也有一定的利用能力,但不能有效利用LEC。以NaH2PO4.2H2O、G-6-P、G-P和ATP为磷源培养的海洋卡盾藻的APA分别从起始的5.86 pmol/（cell.h）下降至第6天的1.25、1.12、1.58和1.72 pmol/（cell.h）,而后分别迅速上升至7.35、4.14、3.55和6.25 pmol/（cell.h）。[结论]该研究可为深入研究海洋卡盾的藻营养生理提供指导。     

Effect of galactose-1-phosphate on glucose oxidation by normal and galactosemic leukocytes

During incubation with galactose, galactosemic leukocytes accumulated more galactose-l-phosphate than did normal leukocytes. Concomitant determination of glucose oxidation, with C(14) glucose, revealed no inhibition of the hexosemonophosphate pathway. These results are at variance with recent studies in rat lens tissue, which suggests that intracellular galactose-1-phosphate depressed glucose-6-phosphate dehydrogenase activity and the oxidative pathway.     

IGF—II生理功能及其编码基因的结构研究

IGF-II属于胰岛素样生长因子家族中的重要成员,在哺乳动物的生长发育过程中发挥着重要的作用。本文从IGF-II生理功能,IGFs受体和结合蛋白,IGF-II蛋白及IGF-II基因结构,IGF-II基因表达和IGF-II基因印迹等方面进行了综述。     

Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro

Mannose 6-phosphate receptors carry newly synthesized lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes and then return to the TGN to carry out another round of lysosomal enzyme delivery. Although clathrin-coated vesicles mediate the export of mannose 6-phosphate receptors from the TGN, nothing is known about the transport vesicles used to carry these receptors back to the TGN. Two different in vitro assays used in this study show that an antibody that interferes with clathrin assembly blocks receptor-mediated endocytosis of transferrin, but has no effect on the recycling of the 300-kilodalton mannose 6-phosphate receptor from prelysosomes to the TGN. These results suggest that the transport of mannose 6-phosphate receptors from prelysosomes to the TGN does not involve clathrin.     

The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate.     

Crystal structure of a lipid G protein-coupled receptor

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P(1), resulting in the modulation of immune and stromal cell responses.     

水稻矮缩病毒非结构蛋白Pns6和外壳蛋白P8多克隆抗体的制备及其应用

利用RT-PCR的方法从感染水稻矮缩病毒(Rice dwarf virus,RDV)的水稻中克隆该病毒的运动蛋白基因S6和外壳蛋白基因S8,通过Gateway系统进行原核表达,获得表达载体p DEST17-Pns6和p DEST17-P8,将表达载体转化E.coli Rosetta,经IPTG诱导表达后获得分子质量约为59和46 ku含HIS标签的融合蛋白.以诱导表达的目的蛋白为抗原,免疫注射新西兰大白兔制备多克隆抗体.结果表明,Western blot检测制备的Pns6和P8抗体可特异性检测RDV,间接ELISA测定Pns6和P8抗体的效价均约为6 400倍,并建立了可靠、灵敏、特异的Dot-blot ELISA方法检测RDV.以上研究表明,RDV运动蛋白和外壳蛋白抗体均可应用于该病毒在田间的大规模调查和检测.     

Specific block of calcium channel expression by a fragment of dihydropyridine receptor cDNA

Although the structure of rabbit skeletal muscle dihydropyridine (DHP) receptor, deduced from cDNA sequence, indicates that this protein is the channel-forming subunit of voltage-dependent calcium channel (VDCC), no functional proof for this prediction has been presented. Two DNA oligonucleotides complementary to DHP-receptor RNA sequences coding for putative membrane-spanning regions of the DHP receptor specifically suppress the expression of the DHP-sensitive VDCC from rabbit and rat heart in Xenopus oocytes. However, these oligonucleotides do not suppress the expression of the DHP-insensitive VDCC and of voltage-dependent sodium and potassium channels. Thus, the gene for DHP receptor of rabbit skeletal muscle is closely related, or identical to, a gene expressed in heart that encodes a component of the DHP-sensitive VDCC. The DHP-sensitive and DHP-insensitive VDCCs are distinct molecular entities.     

Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation

The canonical Wnt-beta-catenin signaling pathway is initiated by inducing phosphorylation of one of the Wnt receptors, low-density lipoprotein receptor-related protein 6 (LRP6), at threonine residue 1479 (Thr1479) and serine residue 1490 (Ser1490). By screening a human kinase small interfering RNA library, we identified phosphatidylinositol 4-kinase type II alpha and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled and dishevelled, the latter of which directly interacted with and activated PIP5KI. In turn, PtdIns (4,5)P2 regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a signaling mechanism for Wnt to regulate LRP6 phosphorylation.     

Binding of GGA2 to the lysosomal enzyme sorting motif of the mannose 6-phosphate receptor

The GGAs are a multidomain protein family implicated in protein trafficking between the Golgi and endosomes. Here, the VHS domain of GGA2 was shown to bind to the acidic cluster-dileucine motif in the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor (CI-MPR). Receptors with mutations in this motif were defective in lysosomal enzyme sorting. The hinge domain of GGA2 bound clathrin, suggesting that GGA2 could be a link between cargo molecules and clathrin-coated vesicle assembly. Thus, GGA2 binding to the CI-MPR is important for lysosomal enzyme targeting.     

Reexamination of glucose-6-phosphatase activity in the brain in vivo: no evidence for a futile cycle

Glucose-6-phosphatase activity in the rat brain in vivo was estimated by measuring the differential loss of tritium and carbon-14 from the glucose pool labeled by a mixture of [2-3H]glucose and [U-14C]glucose. The results provide no evidence of significant dephosphorylation of glucose-6-phosphate and do not support the hypothesis of a futile cycle involving glucose-6-phosphatase activity in the brain.     

鸡干扰素α受体Ⅱ胞外域的克隆及GST融合蛋白原核表达

应用RT-PCR技术扩增鸡干扰素α受体Ⅱ胞外域（CHIFNAR2 EC）基因片段,将扩增产物克隆至pMD-18T,转化J M109感受态。重组质粒与表达载体pGEX-6P-1经双酶切后构建重组表达质粒pGEX-6P-CHIF-NAR2EC,转入BL21,提取质粒酶切和测序鉴定。测序结果与参考序列比较,核苷酸同源性为99%。用IPTG诱导表达,对诱导条件进行初步优化,表达蛋白主要以包涵体的形式存在。通过切胶的方法进行纯化,获取具有较高纯度的融合蛋白。表达产物经SDS-PAGE和Western Blot检测显示融合蛋白分子量大小约为50 KD,采用抗GST抗体进行Western Blot,成功检测到特异性目的条带。     

PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity

The quantitatively minor phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] fulfills many cellular functions in the plasma membrane (PM), whereas its synthetic precursor, phosphatidylinositol 4-phosphate (PI4P), has no assigned PM roles apart from PI(4,5)P(2) synthesis. We used a combination of pharmacological and chemical genetic approaches to probe the function of PM PI4P, most of which was not required for the synthesis or functions of PI(4,5)P(2). However, depletion of both lipids was required to prevent PM targeting of proteins that interact with acidic lipids or activation of the transient receptor potential vanilloid 1 cation channel. Therefore, PI4P contributes to the pool of polyanionic lipids that define plasma membrane identity and to some functions previously attributed specifically to PI(4,5)P(2), which may be fulfilled by a more general polyanionic lipid requirement.     

Human phosphoglycerate kinase and inactivation of the X chromosome

The fibroblasts derived from the skin of a woman heterozygous for an X-linked deficiency of phosphoglycerate kinase represented a mosaic. Two of 22 clones with normal glucose-6-phosphate dehydrogenase activity and hypoxanthine(guanine) phosphoribosyltransferase activity had no phosphoglycerate kinase activity detected by electrophoresis. Because the loci for glucose-6-phosphate dehydrogeniase and hypoxanthine(guanine)phosphoribosyltransferase are already known to undergo inactivation and to be on the short arm of the X chromosome and the locus for phosphoglycerate kinase is on the long arm, these observations support the conclusion that the entire human X chromosome can be involved in X inactivation.     

A key enzyme in the biogenesis of lysosomes is a protease that regulates cholesterol metabolism

Mucolipidosis II is a severe lysosomal storage disorder caused by defects in the α and β subunits of the hexameric N-acetylglucosamine-1-phosphotransferase complex essential for the formation of the mannose 6-phosphate targeting signal on lysosomal enzymes. Cleavage of the membrane-bound α/β-subunit precursor by an unknown protease is required for catalytic activity. Here we found that the α/β-subunit precursor is cleaved by the site-1 protease (S1P) that activates sterol regulatory element-binding proteins in response to cholesterol deprivation. S1P-deficient cells failed to activate the α/β-subunit precursor and exhibited a mucolipidosis II-like phenotype. Thus, S1P functions in the biogenesis of lysosomes, and lipid-independent phenotypes of S1P deficiency may be caused by lysosomal dysfunction.     

Isozymes of aldolase

A new method of starch-gel electrophoresis using a technique of staining dependent on enzyme activity has been employed to demonstrate the isozymes of aldolase from a variety of human, rat, and frog tissues. Five of these isozymes were detected in man, seven in the rat, and at least four in the frog. The abilities of these isozymes to cleave fructose-1 ,6-diphosphate and fructose-1-phosphate were compared.     

Specificity of Drosophila cytonemes for distinct signaling pathways

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. We investigated the specificity of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. We conclude that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme.     

Ribose intervention in the cardiac pentose phosphate pathway is not species-specific

Ribose is cardioprotective in the rat in a variety of pathophysiological conditions. The metabolic basis for this effect is the low capacity of the oxidative pentose phosphate pathway in the myocardium. Ribose bypasses this pathway, elevates the available pool of 5-phosphoribosyl-l-pyrophosphate, and thus stimulates the biosynthesis of adenine nucleotides. In this study reported here the activity of glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the oxidative pentose phosphate shunt, was very low in the human heart and was of the same order of magnitude in the myocardium of various animal species. Furthermore, ribose had a similar stimulating effect on myocardial adenine nucleotide biosynthesis in the guinea pig, in which hemodynamic parameters are different from those in the rat. It is concluded that the metabolic basis for the effectiveness of ribose is similar in all species investigated.     

Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region

The function of the c-Myc oncoprotein and its role in cell growth control is unclear. A basic region of c-Myc is structurally related to the basic motifs of helix-loop-helix (HLH) and leucine zipper proteins, which provide sequence-specific DNA binding function. The c-Myc basic region was tested for its ability to bind DNA by attaching it to the HLH dimerization interface of the E12 enhancer binding factor. Dimers of the chimeric protein, termed E6, specifically bound an E box element (GGCCACGTGACC) recognized by other HLH proteins in a manner dependent on the integrity of the c-Myc basic motif. Methylation of the core CpG in the E box recognition site specifically inhibited binding by E6, but not by two other HLH proteins. Expression of E6 (but not an E6 DNA binding mutant) suppressed the ability of c-myc to cooperate with H-ras in a rat embryo fibroblast transformation assay, suggesting that the DNA recognition specificity of E6 is related to that of c-Myc in vivo.