G protein signaling from activated rat frizzled-1 to the beta-catenin-Lef-Tcf pathway

The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.     

Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I

To investigate the mechanism by which immune activation augments replication of the human immunodeficiency virus type 1 (HIV-1) in infected T cells, four different classes of T cell mitogens were evaluated for their effects on the HIV-1 long terminal repeat (LTR). Phytohemagglutinin (PHA), a mitogenic lectin; phorbol 12-myristic 13-acetate, a tumor promoter; ionomycin, a calcium ionophore; and tat-1, the trans-activator protein from the human T cell leukemia/lymphoma virus type I (HTLV-I) each stimulated the HIV-1 LTR. Studies of deleted forms of the LTR supported a central role in these responses for the HIV-1 enhancer, which alone was sufficient for mitogen inducibility, but also suggested that other 5' positive and negative regulatory elements contribute to the overall magnitude of the response. Synergistic activation of the HIV-1 LTR (up to several thousandfold) was observed with combinations of these mitogens and the HIV-1--derived tat-III protein. Cyclosporin A, an immunosuppressive agent, inhibited PHA-mediated activation of the HIV-1 LTR but was without effect in the presence of other mitogens. Thus, HIV-1 gene expression and replication appear to be regulated, via the HIV-1 LTR, by the same mitogenic signals that induce T cell activation.     

A distinct signaling pathway used by the IgG-containing B cell antigen receptor

The immunoglobulin G (IgG)-containing B lymphocyte antigen receptor (IgG-BCR) transmits a signal distinct from that of IgM-BCR or IgD-BCR, although all three use the same signal-transducing component, Igalpha/Igbeta. Here we demonstrate that the inhibitory coreceptor CD22 down-modulates signaling through IgM-BCR and IgD-BCR, but not that through IgG-BCR, because of the IgG cytoplasmic tail, which prevents CD22 phosphorylation. These results suggest that the cytoplasmic tail of IgG specifically enhances IgG-BCR signaling by preventing CD22-mediated signal inhibition. Enhanced signaling through IgG-BCR may be involved in efficient IgG production, which is crucial for immunity to pathogens.     

A role for the protease falcipain 1 in host cell invasion by the human malaria parasite

Cysteine proteases of Plasmodium falciparum are required for survival of the malaria parasite, yet their specific cellular functions remain unclear. We used a chemical proteomic screen with a small-molecule probe to characterize the predominant cysteine proteases throughout the parasite life cycle. Only one protease, falcipain 1, was active during the invasive merozoite stage. Falcipain 1-specific inhibitors, identified by screening of chemical libraries, blocked parasite invasion of host erythrocytes, yet had no effect on normal parasite processes such as hemoglobin degradation. These results demonstrate a specific role for falcipain 1 in host cell invasion and establish a potential new target for antimalarial therapeutics.     

Activation of Drosophila Toll during fungal infection by a blood serine protease

Drosophila host defense to fungal and Gram-positive bacterial infection is mediated by the Spaetzle/Toll/cactus gene cassette. It has been proposed that Toll does not function as a pattern recognition receptor per se but is activated through a cleaved form of the cytokine Spaetzle. The upstream events linking infection to the cleavage of Spaetzle have long remained elusive. Here we report the identification of a central component of the fungal activation of Toll. We show that ethylmethane sulfonate-induced mutations in the persephone gene, which encodes a previously unknown serine protease, block induction of the Toll pathway by fungi and resistance to this type of infection.     

中性蛋白酶水解蛋清蛋白的工艺

通过单因子试验及正交试验,确定中性蛋白酶水解制备蛋清蛋白肽的最佳工艺条件为底物溶液pH值为7.0、加酶量5 000 U.g-1、水解温度为55℃,水解6 h,氮回收率为49.22%。     

胰蛋白酶对核桃蛋白水解条件的优化研究

【目的】确定蛋白酶水解核桃蛋白的最佳工艺条件。【方法】在单因素分析的基础上,采用响应面分析方法对核桃蛋白的水解条件进行了优化。【结果】胰蛋白酶水解核桃蛋白的最佳反应条件为:反应时间4.0 h,底物质量浓度27 g/L,温度53.4℃,pH8.0,加酶量(E/S)8.3 g/kg,各因素对核桃蛋白水解的影响顺序为温度>加酶量>底物质量浓度。【结论】实际验证表明,利用优化的水解条件可取得较好的水解效果,水解度可达14.497%。     

赖氨酸对原代奶牛乳腺上皮细胞中酪蛋白及mTOR信号通路相关基因表达的影响

为了在探究单一添加不同浓度的赖氨酸对原代奶牛乳腺上皮细胞的增殖,以及mTOR信号通路介导的酪蛋白合成相关基因的影响.试验用厄尔平衡溶液(EBSS)代替正常培养基来处理原代奶牛乳腺上皮细胞,在此基础上依次添加不同浓度的赖氨酸,采用MTT法检测细胞12h和24h的增殖和利用qRT-PCR技术检测4个编码酪蛋白基因和8个mTOR信号通路中与乳蛋白合成翻译相关基因的相对表达量.结果表明:添加赖氨酸12h,浓度为0.5~24mmol/L时,原代奶牛乳腺上皮细胞增殖数量增加(P=0.05);添加赖氨酸24h,浓度为0.5~8mmol/L时,细胞增殖数量增加(P0.01).当赖氨酸的添加浓度为0.5~16 mmol/L时,CSN1S1、CSN1S2、CSN2和CSN3基因表达均显著上调(P0.01).当赖氨酸添加量为0.5~16mmol/L时,mTOR和raptor基因的表达量显著上调(P0.05).在添加不同浓度的赖氨酸时,下游通路信号因子S6K1、4EBP1和eEF2基因表达均显著上调(P0.01);rps6基因表达上调,eIF4E基因表达下调,但差异均不显著.以上结果表明,补充赖氨酸能显著促进乳腺上皮细胞中酪蛋白的合成,这一过程与mTOR信号通路介导蛋白质合成相关.     

Fish oils inhibit endothelial cell production of platelet-derived growth factor-like protein

Diets rich in fish and fish oils are associated with a reduced risk of cardiovascular disease and atherosclerosis. The interaction of a commercial fish oil extract (MaxEPA) with vascular endothelial cells (ECs) was studied as a possible mechanism for this protective effect. MaxEPA almost completely inhibited EC production of platelet-derived growth factor-like protein (PDGFc) while other lipids had a lesser effect or no effect. Overall protein synthesis was not reduced, nor was the inhibition due to defective secretion or increased degradation of the growth factor. Antioxidants suppressed the inhibitory activity of MaxEPA indicating that free radical oxidative processes were required for the inhibition. These results suggest that fish oils may suppress intimal smooth muscle cell proliferation by decreasing the production of EC-derived paracrine growth factors. This inhibitory process represents a possible molecular mechanism for the antiatherosclerotic action of marine lipids.     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease

Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.     

荞麦蛋白的酶水解工艺条件研究

为确定木瓜蛋白酶水解荞麦蛋白的最佳工艺条件,以水解度(DH)为指标,系统分析了pH、温度、酶用量、时间、底物浓度等5个因素对木瓜蛋白酶水解荞麦蛋白的影响,并在此基础上进行了二次回归正交旋转试验(4因素全面试验),建立了木瓜蛋白酶水解荞麦蛋白的数学模型。结果表明,木瓜蛋白酶在底物浓度80 g/L、温度45℃、酶用量20 000 U/g、pH值7.0、反应时间为3 h时,荞麦蛋白的水解度可达14.38%。各因素对荞麦蛋白水解的影响顺序依次为:温度>pH值>酶用量>底物浓度。     

Censoring of autoreactive B cell development by the pre-B cell receptor

Antibody diversity occurs randomly as B cells recombine their immunoglobulin (Ig) heavy- and light-chain genes during development. This process inevitably generates reactivity against self structures, and several mechanisms prevent the development of autoreactive B cells. We report here a role for the pre-B cell receptor, composed of Ig heavy and surrogate light chains, in the negative selection of cells expressing Ig heavy chains with the potential to generate autoantibodies. Surrogate light-chain-deficient (SLC-/-) mice harbored elevated levels of antinuclear antibodies (ANAs) in their serum and showed evidence of escape of pre-B cells expressing prototypic autoantibody heavy chains from negative selection, leading to mature autoantibody secreting CD21-CD23- B cells in the periphery. Thus, the pre-B cell receptor appears to censor the development of certain autoantibody-secreting cells and may represent an important factor in multifactorial autoimmune diseases.     

Interferon: production by chick erythrocytes activated by cell fusion

In the presence of Sendai virus inactivated with ultraviolet light, nucleated chick erythrocytes can be fused with several types of human cells to form heterokaryons. Although chick erythrocytes alone cannot be stimulated by Sendai virus to produce interferon, fusion with a human cell (AH-1) which itself may produce human interferon results in heterokaryons in which the erythrocyte genome is activated and chick interferon is produced. When nucleated chick erythrocytes are fused with another type of human cell (HeLa clone S-3) which does not produce human interferon when stimulated, no chick interferon is detectable, despite morphologic changes suggestive of activation of the erythrocyte nuclei.     

Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway

The Nedd8 ubiquitin-like protein modification pathway regulates cell-cycle progression. Our analysis of Nedd8 requirements during Caenorhabditis elegans embryogenesis indicates that the cytoskeleton is another target. Nedd8 conjugation negatively regulated contractility of the microfilament-rich cell cortex during pronuclear migration and again during cytokinesis. The Nedd8 pathway also was required after meiosis to negatively regulate katanin, a microtubule-severing complex, permitting the assembly of a large mitotic spindle. We propose that Nedd8-modified cullin, as part of an E3 ubiquitin ligase complex, targets katanin for degradation during the transition from meiosis to mitosis.     

Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein

The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.     

Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility

EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.     

Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1

Intraperitoneal administration of human recombinant interleukin-1 (IL-1) to rats can increase blood levels of corticosterone and adrenocorticotropic hormone (ACTH). The route by which IL-1 affects pituitary-adrenal activity is unknown. That the IL-1-induced pituitary-adrenal activation involves an increased secretion of corticotropin-releasing factor (CRF) is indicated by three lines of evidence. First, immunoneutralization of CRF markedly attenuated the IL-1-induced increase of ACTH blood levels. Second, after blockade of fast axonal transport in hypothalamic neurons by colchicine, IL-1 administration decreased the CRF immunostaining in the median eminence, indicating an enhanced release of CRF in response to IL-1. Third, IL-1 did not stimulate ACTH release from primary cultures of anterior pituitary cells. These data further support the notion of the existence of an immunoregulatory feedback circuit between the immune system and the brain.