Localization of low density lipoprotein receptor-related protein 1 to caveolae in 3T3-L1 adipocytes in response to insulin treatment

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitope-tagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.     

GGNBP1抗体制备及小鼠组织表达谱分析

体外实验研究表明配子生成素结合蛋白1(GGNBP1)可能与GGN1相互作用形成睾丸特异性复合物,在精子生成过程中发挥作用.从小鼠睾丸总RNA中反转录扩增Ggnbpl全长cDNA,构建表达质粒,在大肠杆菌中表达GGNBP1,经聚丙烯酰胺凝胶纯化后免疫新西兰白兔,制备兔多抗血清.镍离子金属螯合柱纯化表达的GGNBP1蛋白,与NHS活化基团交联,制备GGNBP1抗体亲和层析柱,纯化GGNBP1多抗.在293FT细胞中瞬时表达Myc-GGNBP1融合蛋白,用于Mvc单抗验证GGNBP1抗体特异性,结果证明获得了特异性的GGNBP1抗体.分别制备小鼠脑、心、肺、肝、脾、肾、肌肉、卵巢、睾丸和子宫组织匀浆,用GGNBP1抗体进行Western印迹分析,结果仅在睾丸组织匀浆中检测到GGNBP1特异性条带,证明GGNBP1是睾丸特异性表达蛋白.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin.

Pyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Man alpha 1-6(Man alpha 1-3)Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxy-succinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.     

Identification of high affinity receptors for human monocyte chemoattractant protein-1 on human monocytes

The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.     

Demonstration of the interaction of native C1 with monomeric immunoglobulins and C1 inhibitor

The association of native C1 with physiologically relevant proteins was studied by ultracentrifugation. 125I-C1 was centrifuged through numerous sucrose density gradients, each of which contained a different concentration of monomeric (19S) IgM throughout the gradient. The s-rate of C1 (16S) increased with increasing IgM input to a maximum of 32S. In the absence of C1q, the C1r2s2 subunit did not bind to the Ig. In gradients containing physiologic concentrations of IgM (1.3 mg/ml) at 0.14 M ionic strength, the observed s-rate of C1 was 21S. In the presence of 13 mg/ml IgG, C1 sedimented with an s-rate of 19S. Thus, under physiologic conditions, a significant fraction of native C1 is reversibly bound to monomeric Ig. SDS-PAGE analyses show that this interaction does not lead to C1 activation. The interaction of native C1 with C1 inhibitor (C1-In) was studied by ultracentrifugation at physiologic ionic strength. Purified 125I-C1-In alone sedimented with an s-rate of 4S. However in the presence of excess native C1, one-third of the C1-In co-sedimented with C1 at a 16S position. For these studies, 100 microM nitrophenylguanidinobenzoate (NPGB) was present throughout the sucrose density gradient to prevent C1 activation during centrifugation. As the concentration of NPGB was increased, the percent of 125I-C1-In at 16S decreased, indicating that C1-In was binding (reversibly) to the C1 active site region(s), which is at least partially accessible in uncleaved C1. In controls, when NPGB was omitted or activated C1 was used, the s-rate of 125I-C1-In was only 12S due to the release of C1rC1s(C1-In)2 from activated C1. Thus, under physiologic conditions native C1 is reversibly bound to C1-In.     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

Dimerize RACK1 upon transformation with oncogenic ras

From our previous studies, we learned that syndecan-2/p120-GAP complex provided docking site for Src to prosecute tyrosine kinase activity upon transformation with oncogenic ras. And, RACK1 protein was reactive with syndecan-2 to keep Src inactivated, but not when Ras was overexpressed. In the present study, we characterized the reaction between RACK1 protein and Ras. RACK1 was isolated from BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q61K)] of shrimp Penaeus japonicus and RACK1 was revealed to react with GTP-K(B)-Ras(Q61K), not GDP-K(B)-Ras(Q61K). This selective interaction between RACK1 and GTP-K(B)-Ras(Q61K) was further confirmed with RACK1 of human placenta and mouse RACK1-encoded fusion protein. We found that RACK1 was dimerized upon reaction with GTP-K(B)-Ras(Q61K), as well as with 14-3-3beta and geranylgeranyl pyrophosphate, as revealed by phosphorylation with Src tyrosine kinase. We reported the complex of RACK1/GTP-K(B)-Ras(Q61K) reacted selectively with p120-GAP. This interaction was sufficient to dissemble RACK1 into monomers, a preferred form to compete for the binding of syndecan-2. These data indicate that the reaction of GTP-K(B)-Ras(Q61K) with RACK1 in dimers may operate a mechanism to deplete RACK1 from reaction with syndecan-2 upon transformation by oncogenic ras and the RACK1/GTP-Ras complex may provide a route to react with p120-GAP and recycle monomeric RACK1 to syndecan-2.     

XMam1, Xenopus Mastermind1, induces neural gene expression in a Notch-independent manner

Mastermind, which is a Notch signal component, is a nuclear protein and is thought to contribute to the transactivation of target genes. Previously we showed that XMam1, Xenopus Mastermind1, was essential in the transactivation of a Notch target gene, XESR-1, and was involved in primary neurogenesis. To examine the function of XMam1 during Xenopus early development in detail, XMam1-overexpressed embryos were analyzed. Overexpression of XMam1 ectopically caused the formation of a cell mass with pigmentation on the surface of embryos and expressed nrp-1. The nrp-1-positive cell mass was produced by XMam1 without expression of the Notch target gene, XESR-1, and not by the activation form of Notch, NICD. The ectopic expression of nrp-1 was not inhibited by co-injection of XMam1 with a molecule known to inhibit Notch signaling. The nrp-1 expression was also recognized in the animal cap injected with XMam1DeltaN, which lacks the basic domain necessary for interacting with NICD and Su(H). These results show that XMam1 has the ability to induce the cell fate into the neurogenic lineage in a Notch-independent manner.     

High levels of RAE-1 isoforms on mouse tumor cell lines assessed by anti-"pan" RAE-1 antibody confer tumor susceptibility to NK cells.

Two sublines of the benzpyrene-induced mouse hepatoma cell line, G-1 and G-5, showed low and high metastatic ability, respectively, to the lung. We produced a polyclonal antibody (pAb) against RAE-1alpha. Five isoforms of RAE-1 have been identified to date, and this pAb recognized all isoforms and was named anti-"pan" RAE-1 pAb. The level of RAE-1 was approximately 5-fold higher in G-5 than in G-1, which was almost RAE-1-negative, as determined using anti-pan RAE-1 pAb. Expression levels of other markers including MHC class I (MHC-I) and Qa-1b were very low and indistinguishable in these sublines. NK-mediated cytotoxicity was determined with these sublines; G-5 was highly susceptible to NK-mediated cytolysis, while G-1 was relatively resistant. The NK-mediated G-5 > G-1 killing profile was diminished if the G-5 cells were pretreated with F(ab)(2)(') of anti-pan RAE-1 pAb. G-1, when transfected with Rae-1alpha cDNA, acquired NK-responsiveness similar to that of G-5. These and additional data using mouse cell lines with low MHC-I levels and various RAE-1 levels also demonstrated that RAE-1 level is critically associated with NK-susceptibility in tumor cells.     

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Alternol inhibits proliferation and induces apoptosis in mouse lymphocyte leukemia (L1210) cells

Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz–Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27^Kip1. Furthermore, the stimulatory effect of LKB1 on p27^Kip1 promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27^Kip1.     

^125I标记单抗LC—I与肺腺癌细胞体内外结合特性的研究

McAb LC-1 was derived from fusion of myeloma cells and murine spleen cells immunized with human lung adenocarcinoma SPC-A-1 cells. The immunoglobulin isotype of LC-1 belonged to IgM. LC-1 was direct against the common epitope of lung cancer. It not only reacted with small cell lung cancer but also with non small cell lung cancer. LC-1 was purified from ascitic fluid by euglobulin precipitation and Sephadex G-200 filtration chromatography, and was iodinated with Iodogen, the specific reactivity of 125I-labeled LC-1 was determined by comparing standard curve with self-displacement curve. The immunoreactive fraction of 125I-LC-1 was determined by its binding to excess of antigen. The RIA data were plotted in Scatchard-form as binding of SPC-A-1 cells to LC-1. The binding constant of LC-1 binding to SPC-A-1 was 4.8 x 10(8) M-1. The LC-1 binding sites on SPC-A-1 were 7.2 x 10(4) per cell. The RIA inhibition test showed that LC-1 and LAC-122 (another IgM isotype McAb reacted only with non small cell lung cancer) had no cross-reactivity. The treatment of SPC-A-1 cells by proteinase and sodium periodate inhibited LC-1 binding to these treated target cells by 39% and 66% respectively. These results suggested that the biochemical nature of antigen recognized by LC-1 was glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat alpha 1-microglobulin. Purification from urine and synthesis by hepatocyte monolayers

Rat alpha 1-microglobulin was isolated from the urine of rats treated with sodium chromate, and was purified by the use of gel chromatography, affinity chromatography on concanavalin-A-Sepharose and ion-exchange chromatography. The protein was heterogeneous in charge, had a tendency to form dimers, and was associated with a brown-coloured chromophore. The size of the protein (25 kDa) was similar to guinea pig alpha 1-microglobulin but smaller than the human protein, when measured with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunological cross-reaction with human and guinea pig alpha 1-microglobulin was demonstrated. The concentration of alpha 1-microglobulin in rat serum was 16.4 mg/l (SD = 8.5 mg/l, n = 13) and rat serum alpha 1-microglobulin was eluted from a gel chromatography column at two different positions corresponding to monomeric alpha 1-microglobulin and IgA. The latter alpha 1-microglobulin activity could be absorbed by anti-IgA serum. Rat alpha 1-microglobulin and albumin were continuously released into the medium of rat hepatocyte monolayers, and alpha 1-microglobulin was isolated from the medium by the use of immunoprecipitation with anti-(alpha 1-microglobulin). Tritiated leucine, added to the medium, was incorporated into the protein, suggesting a de novo synthesis of alpha 1-microglobulin by the hepatocytes. The size of hepatic alpha 1-microglobulin was similar to that of purified urinary rat alpha 1-microglobulin, when determined with sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

Inflammation-specific T1 imaging using anti-intercellular adhesion molecule 1 antibody-conjugated gadolinium diethylenetriaminepentaacetic acid

To examine inflammatory tissue, an initial and common symptom of various types of pathogenesis, we designed inflammation-targeted T(1) contrast agents prepared by bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) with anti-intercellular adhesion molecule 1 (ICAM-1) antibody. The anti-ICAM-1 antibody was coupled with DTPA and was then conjugated with Gd. The specific binding of the Gd-DTPA-anti-ICAM-1 antibody complex to the ICAM-1-expressing cells was examined in the cultured endothelial cells where ICAM-1 expression was stimulated. Inflammation-specific T(1) imaging was then assessed using a mouse abscess model with the 1.5-Tesla module. The Gd-DTPA-anti-ICAM-1 antibody displayed increased r1, which was two times higher than that of Gd-DTPA and showed predominant binding to cultured endothelial cells, which expressed a high level of ICAM-1. Moreover, the inflammation-specific T(1) enhancement was imaged with the Gd-DTPA-anti-ICAM-1 antibody in the mouse acute inflammation model. The Gd-DTPA-anti-ICAM-1 antibody showed significantly increased vascular circulation time, which thereby offered a greater chance for its binding to the target cells. The Gd-DTPA-anti-ICAM-1 antibody displays a potential targeted T(1) contrast agent specific to the inflammatory tissue that expresses ICAM-1.     

Responses of banana fruit to treatment with 1-methylcyclopropene

Experiments were conducted to determine levels of 1-methylcyclopropene (1-MCP) exposure needed to prevent ethylene-stimulated banana fruit ripening, characterise responses of ethylene-treated fruit to subsequent treatment with 1-MCP, and to test effects of subsequent ethylene treatment on 1-MCP-treated fruit softening. Fruit softening was measured at 20°C and 90% relative humidity. One hour exposure at 20°C to 1000 nl 1-MCP/l essentially eliminated ethylene-stimulated ripening effects. Exposure for 12 h at 20°C to just 50 nl 1-MCP/l was similarly effective. Fruit ripening initiated by ethylene treatment could also be delayed with subsequent 1-MCP treatment. However, 1-MCP treatment only slowed down ripening of ethylene-treated fruit when applied at 1 day after ethylene and was ineffective when applied 3 or 5 days after ethylene treatment. The ripening response of fruit treated with 1-MCP and subsequently treated with ethylene varied with interval time between 1-MCP and ethylene treatments. As time increased, the response of 1-MCP-treated fruit to ethylene was enhanced. Responses to 0.1, 1, 10 or 100 µl ethylene/l concentrations were similar. Enzyme kinetic analysis applied to 1-MCP effects on ethylene-induced softening of banana fruit suggested that 1-MCP inhibition is by noncompetitive antagonism of ethylene binding.     

Covalent modification of human immunodeficiency virus type 1 p6 by SUMO-1

The p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX. Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6. To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system. HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9. Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached. SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease. Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment. As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type. Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content. Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis. HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication.