Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.     

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.     

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.     

A family with severe insulin resistance and diabetes due to a mutation in AKT2

Inherited defects in signaling pathways downstream of the insulin receptor have long been suggested to contribute to human type 2 diabetes mellitus. Here we describe a mutation in the gene encoding the protein kinase AKT2/PKBbeta in a family that shows autosomal dominant inheritance of severe insulin resistance and diabetes mellitus. Expression of the mutant kinase in cultured cells disrupted insulin signaling to metabolic end points and inhibited the function of coexpressed, wild-type AKT. These findings demonstrate the central importance of AKT signaling to insulin sensitivity in humans.     

The insulin receptor contains a calmodulin-binding domain

Substantial evidence suggests that calcium has a pivotal role in regulating the initial events through which insulin alters plasma membrane metabolism. Because binding of insulin to its receptor represents the initial site of insulin action in the plasma membrane, studies were undertaken to determine whether the insulin receptor is a calmodulin-binding protein. Preparations enriched in the insulin receptor and calmodulin-binding proteins were isolated from detergent-solubilized rat adipocyte membranes by chromatography with wheat germ agglutinin agarose and calmodulin-conjugated Sepharose, respectively. Substantial purification of a manganese-dependent, insulin-sensitive phosphoprotein of 95K identified as the beta subunit of the insulin receptor was accomplished. Binding and photocovalent cross-linking of iodine-125-labeled calmodulin to these affinity-purified preparations and to isolated plasma membranes, followed by immunoadsorption with insulin receptor antibodies bound to protein A Sepharose, resulted in significant purification of a binding complex of 110K to 140K. These results indicate that the adipocyte insulin receptor or a polypeptide closely associated with the receptor is a calmodulin-binding protein.     

中国荷斯坦牛TLR2基因SNPs的快速筛查及等位基因频率的估算

为了快速筛查Toll样受体2基因的SNPs,采用DNA池和直接测序法确定了荷斯坦牛TLR2基因的多个SNP突变方式,估算了各SNP的等位基因频率,并利用生物信息学方法预测突变对TLR2 mRNA和蛋白的影响。结果显示,在荷斯坦牛TLR2 基因内共发现6个核苷酸突变位点,分别为T602A、G10900C、G11047C、A11076T、C12077T、T10634G,其中T10634G为错义突变,导致第64位氨基酸由原来的谷氨酸(Gly)替换为天冬氨酸(Asp)。研究结果可为中国荷斯坦牛的抗病育种提供实验依据。     

Insulin receptor phosphorylation may not be a prerequisite for acute insulin action

An antiserum to the insulin receptor mimicked insulin's acute actions on glucose transport, phosphorylation of integral membrane proteins, and internalization of the insulin receptor in isolated rat adipose cells. These insulinomimetic actions of the antiserum occurred without the equivalent increase in phosphorylation of the beta subunit of the insulin receptor observed with insulin. Thus, a role of receptor phosphorylation in acute insulin action is now questioned.     

Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein

The Drosophila melanogaster gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode Caenorhabditis elegans, insulin/IGF signaling regulates adult longevity. We found that mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span was not a result of impaired oogenesis in chico females, nor was it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved.     

Production of alpha 1,3-galactosyltransferase-deficient pigs

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.     

团头鲂MHCⅡ α基因的SNP位点开发、鉴定及与抗病性状关联分析

用嗜水气单胞菌(Aeromonas hydrophila)感染团头鲂(Megalobrama amblycephala)区分抗病和易感组,通过PCR扩增及测序在MHCⅡα基因上筛选单核苷酸多态位点(single nucleotide polymorphisms,SNP),利用高分辨率熔解曲线法和限制性内切酶酶切法对SNP进行分型,分析其多态性及与抗病性状之间的关系。在MHCⅡa基因上共筛选出35个候选SNP位点,占MHCⅡa基因总碱基的1.45%,包含30个转换位点、5个颠换位点。其中外显子部分有16个,内含子部分7个,5′非编码区有1个,3′非编码区有11个。有13个SNP位点位于编码区,占总氨基酸位点的5.56%,位于839bp的T/A颠换和1 663bp的A/G转换是无义突变,其余11个SNP位点为有义突变。α1结构域的SNP位点分别占总碱基和总氨基酸位点的4.88%和10.98%,明显高于α2结构域的1.08%和3.22%。MHCⅡα基因的21个抗原结合位点(peptide binding region,PBR)中,有4个位点变异,变异率为19.05%;而non-PBR的变异率仅为8.20%(5/61)。用SPSS软件对分型成功的5个SNP位点在易感和抗病组各100尾中的基因型频率和等位基因频率进行了统计。通过卡方检验分析发现位于1 395bp(T/A)位点的基因型频率和等位基因频率在易感和抗病组中差异极显著,位于221bp(G/T)和1 859bp(G/T)位点的基因型频率和等位基因频率在易感组和抗病组中差异显著。本研究结果表明团头鲂MHCⅡα基因的多态性和抗细菌性败血症性状显著关联。     

Type I and type II GABAA-benzodiazepine receptors produced in transfected cells

GABAA (gamma-aminobutyric acid A)-benzodiazepine receptors expressed in mammalian cells and assembled from one of three different alpha subunit variants (alpha 1, alpha 2, or alpha 3) in combination with a beta 1 and a gamma 2 subunit display the pharmacological properties of either type I or type II receptor subtypes. These receptors contain high-affinity binding sites for benzodiazepines. However, CL 218 872, 2-oxoquazepam, and methyl beta-carboline-3-carboxylate (beta-CCM) show a temperature-modulated selectivity for alpha 1 subunit-containing receptors. There were no significant differences in the binding of clonazepam, diazepam, Ro 15-1788, or dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to all three recombinant receptors. Receptors containing the alpha 3 subunit show greater GABA potentiation of benzodiazepine binding than receptors containing the alpha 1 or alpha 2 subunit, indicating that there are subtypes within the type II class. Thus, diversity in benzodiazepine pharmacology is generated by heterogeneity of the alpha subunit of the GABAA receptor.     

Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor

The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.     

苏钟猪TLR4多态性及编码区C1027A功能分析

【目的】研究苏钟猪TLR4的多态性及其编码区第1 027 bp处突变对TLR4蛋白功能的影响。【方法】采用PCR-SSCP的方法检测TLR4在苏钟猪中的多态性并利用real-time PCR方法检测不同基因型（CC型和AC型）的猪肺泡巨噬细胞经脂多糖（lipopolysaccharide,LPS）诱导后,TLR4及促炎症因子TNF-α、IL-1β表达量的变化情况,探讨该处突变对TLR4识别LPS的影响。【结果】①共检测到3个突变：G962A、C1027A、G960A,其中前两个为有义突变,且C1027A突变可引起编码氨基酸性质的改变;②LPS能快速诱导猪肺泡巨噬细胞中TLR4及促炎症因子TNF-α和IL-1β表达水平上调且TLR4编码区C1027A不同基因型（CC型和AC型）的猪肺泡巨噬细胞（pulmonary alveolar macrophages,PAMs）对LPS的敏感性及反应强度存在显著性差异。【结论】苏钟猪TLR4编码区的序列相对保守、多态含量较低,群体遗传变异程度较小。TLR4编码区C1027A突变能影响TLR4识别LPS的能力,等位基因C为苏钟猪抗革兰阴性菌感染的优势基因。     

A single amino acid mutation contributes to adaptive beach mouse color pattern

Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor (Mc1r) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Florida's Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.     

Fish oil prevents insulin resistance induced by high-fat feeding in rats

Non-insulin-dependent diabetes mellitus is an increasingly prevalent disease in Western and developing societies. A major metabolic abnormality of non-insulin-dependent diabetes is impaired insulin action (insulin resistance). Diets high in fat from vegetable and nonaquatic animal sources (rich in linoleic acid, an omega-6 fatty acid, and saturated fats) lead to insulin resistance. In rats fed high-fat diets, replacement of only 6 percent of the linoleic omega-6 fatty acids from safflower oil with long-chain polyunsaturated omega-3 fatty acids from fish oil prevented the development of insulin resistance. The effect was most pronounced in the liver and skeletal muscle, which have important roles in glucose supply and demand. The results may be important for therapy or prevention of non-insulin-dependent diabetes mellitus.     

Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex

Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces.     

A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function

The Drosophila melanogaster gene insulin-like receptor (InR) is homologous to mammalian insulin receptors as well as to Caenorhabditis elegans daf-2, a signal transducer regulating worm dauer formation and adult longevity. We describe a heteroallelic, hypomorphic genotype of mutant InR, which yields dwarf females with up to an 85% extension of adult longevity and dwarf males with reduced late age-specific mortality. Treatment of the long-lived InR dwarfs with a juvenile hormone analog restores life expectancy toward that of wild-type controls. We conclude that juvenile hormone deficiency, which results from InR signal pathway mutation, is sufficient to extend life-span, and that in flies, insulin-like ligands nonautonomously mediate aging through retardation of growth or activation of specific endocrine tissue.     

亚洲玉米螟的抗药性监测及双酰胺类药剂抗性靶标分子检测

[目的]研究监测和检测亚洲玉米螟田间种群对双酰胺类等药剂的抗性,为指导田间合理用药及抗性治理提供依据.[方法]于2018和2019年,采用人工饲料浸药法,测定新疆乌鲁木齐市、伊宁市、疏勒县、泽普县和昌吉市亚洲玉米螟田间种群对氯虫苯甲酰胺、溴氰虫酰胺、阿立卡(噻虫嗪·高效氯氟氰菊酯)和亮泰(阿维菌素·氯虫苯甲酰胺)的抗性...     

Biosynthesis of the Torpedo californica acetylcholine receptor alpha subunit in yeast

Yeast cells were transformed with a plasmid containing complementary DNA encoding the alpha subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the alpha subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.