Degradation of Cell Wall of Mucor mucedo by Chitosanase from Bacillus R–4

To broaden our understanding of extracellular proteins of Aspergillus oryzae at the conidial germination stage, analyses of the secreted proteins during germination were carried out. Taka-amylase A (TAA), glucoamylase (GLAA), and aspergillopepsin A (PEPA) were identified as the main products by peptide mass fingerprinting. TAA and PEPA were detected simultaneously with the formation of germ tubes. With the development of germination, the pH of the medium fell from 5.5 to 3.5. The secreted PEPA had a pro-sequence and likely shifted from 42 kDa to 41 kDa below pH 4.6, indicating that the precursor of PEPA was secreted and underwent pH-dependent processing. Furthermore, the 41 kDa protein was trapped by the addition of pepstatin A, the specific inhibitor of PEPA, suggesting that the maturation of pro-PEPA was a stepwise autoprocessing upon acidification of the medium and itself was an intermediate of the processing. It was implied that PEPA plays an important role at the early germination stage.     

IL-4/IL-4R与肿瘤关系的研究进展

白细胞介素-4(interleukin-4,IL-4)是一种主要由2型T辅助细胞(Th2)分泌的多功能细胞因子,可特异性地与靶细胞表面的IL-4受体(interleukin-4 receptor,IL-4R)结合,发挥相应的生物学作用。在机体免疫反应中,IL-4/IL-4R可通过调节T细胞分化,抑制B淋巴细胞凋亡,诱发Ig E表型转换等方面引起哮喘、过敏等疾病。除此之外,IL-4/IL-4R还可以在肿瘤的发展中发挥不同的作用。因此,本文就IL-4/IL-4R对肿瘤发展影响的研究进展作一综述。     

Modulation of recombinant, α2*, α3* or α4*-nicotinic acetylcholine receptor (nAChR) function by nAChR β3 subunits

The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β3(V9'S) subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.     

The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein–Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.     

Metabolism of (−)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene to diol epoxides by liver microsomes

Metabolism of biosynthetic (?)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene by liver microsomes from control, phenobarbital-treated and 3-methylcholanthrene-treated rats was investigated. Although previous studies of the metabolism of related benzo[a]pyrene and benzo[e]pyrene dihydrodiols which also prefer the diaxial conformation had indicated that diol epoxides were minor metabolites, the diastereomeric chrysene 3,4-diol-1,2-epoxides-$\text{1}$ and $\text{?2}$ were major metabolites (66–90%). All three types of microsomes metabolized the chrysene 3,4-dihydrodiol at low but essentially similar rates (0.5–0.7 nmol substrate/nmol cytochrome P-450/min).     

Associations of the IL2Rα, IL4Rα, IL10Rα, and IFN γ R1 cytokine receptor genes with AIDS progression in a French AIDS cohort

We have performed an extensive analysis of Th1/Th2 cytokine receptors IL2Rα, IL4Rα, IL10Rα, and IFNγR1 gene polymorphisms to evaluate their impact on AIDS progression. The coding regions and promoters of these genes were sequenced in the genetics of resistance to immunodeficiency virus cohort, composed of 327 HIV-1-positive patients with extreme progression phenotypes, slow and rapid progressors, and of 446 healthy control subjects, all of them of Caucasian descent. Overall, 104 single nucleotide polymorphisms and four insertions/deletions with a minor allelic frequency higher than 1% were identified, 21 of them being newly characterized. We observed weak associations for 13 polymorphisms of IL2Rα, IL4Rα, IL10Rα, and IFNγR1, and 11 haplotypes of IL2Rα, IL4Rα, and IFNγR1. However, we could not relate these positive signals to any relevant biological information on the gene function. To affirm these putative associations in AIDS, further confirmation on other AIDS cohorts will be needed. This complete catalog of polymorphisms in IL2Rα, IL4Rα, IL10Rα, and IFNγR1 cytokine receptor genes should also be useful for investigating associations in other immune-related diseases.Electronic Supplementary Material Supplementary material is available for this article at     

Silencing of H4R inhibits the production of IL-1β through SAPK/JNK signaling in human mast cells

Context: Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.

Objectives: To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.

Materials and methods: H4R expression was analyzed by RT-PCR and western blotting in human mast cell line-1 (HMC-1) cells and H4RsiRNA transfected cells. The effect of H4RsiRNA and H4R-antagonist on H4R mediated MC functions such as intracellular Ca²⁺ release, degranulation, IL-6 and IL-1β release, and the activation SAPK/JNK signaling pathways were studied. HMC-1 cells were stimulated with 10?μM of histamine (His) and 4-methylhistamine (4-MH) and pretreated individually with H4R-antagonist JNJ7777120 (JNJ), histamine H1 receptor (H1R)-antagonist mepyramine, and signaling molecule inhibitors SP600125 (SP) and Bay117082.

Results: We found that the HMC-1 cells expressed H4R and H4RsiRNA treatment down regulated the H4R expression in HMC-1 cells. Both His and 4-MH induced the intracellular Ca²⁺ release and degranulation whereas; H4R siRNA and JNJ inhibited the effect. Furthermore, the activation of H4R caused the phosphorylation of SAPK/JNK pathways. H4R gene silencing and pretreatment with SP and JNJ decreased His and 4-MH induced phosphorylation of SAPK/JNK. We found that the activation of H4R caused the release of IL-1β (124.22?pg/ml) and IL-6 (122.50?pg/ml) on HMC-1 cells. Whereas, SAPK/JNK inhibitor (68.36?pg/ml) inhibited the H4R mediated IL-1β release.

Conclusions: Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.     

Synthesis of novel (R)-4-fluorophenyl-1H-1,2,3-triazoles: A new class of α-glucosidase inhibitors

Diabetes is a non-communicable disease, which occurs either due to the lack of insulin or the inability of the human body to recognize it. The recent data indicates an increase in the trend of people diagnosed with Type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors are known to reduce the impact of carbohydrates on blood glucose level and prevent the digestion of carbohydrates. α-glucosidase inhibitors hold great potential for the treatment of T2DM. In search of better α-glucosidase inhibitors, a series of novel (R)-4-fluorophenyl-1H-1,2,3-triazole derivatives were synthesized (6 and 8a-n) and evaluated for their α-glucosidase inhibitory activity in vitro. All new compounds were characterized by ¹H NMR, ¹³C NMR, ¹⁹F NMR, ESI-MS, and where applicable by single crystal X-ray diffraction (8 m). A preliminary structure-activity relationship suggested that the presence of 1H-1,2,3-triazole ring in (R)-4-fluorophenyl-1H-1,2,3-triazole derivatives has remarkable contribution in the overall activity. Molecular docking studies were carried out to investigate the binding mode of compounds within the active site of the α-glucosidase enzyme. Docking results are in complete agreement with the experimental finding. This study unravelled a new class of triazole derivatives with α-glucosidase inhibitory activity.     

Lack of association or interactions between the IL-4, IL-4Rα and IL-13 genes, and rheumatoid arthritis

Introduction  

A feature of rheumatoid arthritis (RA) is an imbalance between proinflammatory and anti-inflammatory cytokines. Several recent studies have implicated polymorphism in the IL-4 signalling pathway in the development of erosive RA. The aim of the present study was to investigate the role of polymorphism in the IL-4, IL-4Rα and IL-13 genes in RA, including an examination of epistasis.     

The structure–function role of C-terminus in human bitter taste receptor T2R4 signaling

Bitter taste, in humans, is sensed by 25 G protein-coupled receptors, referred to as bitter taste receptors (T2Rs). The diverse roles of T2Rs in various extraoral tissues have implicated them as a potential target for therapeutic intervention. Structure–function studies have provided insights into the role of transmembrane and loop regions in the activation mechanism of T2Rs. However, studies aimed at deciphering the role of their carboxyl-terminus (C-terminus) are limited. In this study, we identified a KLK/R motif in the C-terminus that is conserved in 19 of the 25 T2Rs. Using site-directed mutagenesis we studied the role of 16 residues in the C-terminus of T2R4. The C-terminus of T2R4 is polybasic with 6 of the 16 residues consisting of lysines, constituting two separate KK motifs. We analyzed the effect of the C-terminus mutations on plasma membrane trafficking, and characterized their function in response to the T2R4 agonist quinine. The majority of the mutants showed defective receptor trafficking with ≤ 50% expression on the cell surface. Interestingly, mutation of the distal Lys296 of the KLK motif in T2R4 resulted in constitutive activity. The K296A mutant displayed five-fold basal activity over wild type T2R4, while the conservative substitution K296R showed wild type characteristics. The Lys294, Leu295 and Lys296 of the KLK motif in T2R4 were found to perform crucial roles, both, in receptor trafficking and function. Results from this study provide unique mechanistic insights into the structure–function role of the C-terminus in T2R signaling.     

Oncostatin M (OSM) primes IL-13- and IL-4-induced eotaxin responses in fibroblasts: Regulation of the type-II IL-4 receptor chains IL-4Rα and IL-13Rα1

Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in regulation of various chronic inflammatory processes. Previous work has shown that OSM induces eosinophil accumulation in mouse lungs in vivo and stimulates the eosinophil-selective chemokine eotaxin-1 synergistically with IL-4 in vitro. To examine the role of receptor regulation by OSM in synergistic eotaxin-1 responses, we here examine the modulation of the type-II IL-4 receptor (IL-4Rα and IL-13Rα1) by OSM and other gp130/IL-6 cytokine family members using NIH3T3 fibroblasts and primary mouse lung fibroblasts. We first show that OSM with either IL-13 or IL-4 synergistically induces eotaxin-1 expression in a dose-dependent fashion. Analysis of IL-4Rα expression at the protein (Western blot and FACS) and RNA (TAQMAN) levels showed that OSM markedly elevates expression by 3 h. OSM enhanced IL-13Rα1 mRNA and induced a smaller but detectable increase in total IL-13Rα1 protein. Priming fibroblasts with OSM for 6 h markedly enhanced subsequent IL-13 and IL-4-induced eotaxin-1 responses and STAT6 tyrosine-641 phosphorylation. Regulation of IL-4Rα by OSM was sensitive to inhibition of the PI3′K pathway by LY294002. These studies provide novel mechanistic insights in OSM role in regulation of synergistic eotaxin-1 responses and IL-4Rα expression in fibroblasts.     

4D和4R对小麦三种同工酶合成的影响

利用聚丙烯酰胺凝胶电泳对小麦品种天选15号、天选15号—4D缺体,黑麦品种德国白粒及利用“缺体回交法”培育的小麦(4D)一黑麦(4R)异代换系幼苗的过氧化物酶同工酶、细胞色素氧化酶同工酶及酯酶同工酶进行了研究,结果表明4D染色体对小麦的一种过氧化物酶同工酶和一种细胞色素氧化酶同工酶量的合成具有控制作用,在小麦4D缺体的遗传背景下,黑麦4R染色体能够补偿由于4D缺失引起的这两种同工酶合成降低的效应。4D对小麦幼苗期酯酶同工酶的合成没有明显的作用,4R在小麦4D缺体遗传背景下对酯酶同工酶的合成也没有明显的影响     

Interaction of novel Dobzhansky–Muller type genes for the induction of hybrid lethality between Gossypium hirsutum and G. barbadense cv. Coastland R4-4

Hybrid lethality was identified in interspecific hybrids between two cotton species, Gossypium hirsutum and G. barbadense cv. Coastland R4-4 (R4-4). Genetic analysis indicated that the lethal symptom was controlled by two dominant complementary genes, one from G. hirsutum and another from R4-4. Microsatellite mapping identified the location of the causal gene in G. hirsutum as chromosome D8, while the R4-4 gene was placed on chromosome D11. Our data indicate that these genes conform to the Dobzhansky–Muller model, and are novel for the induction of hybrid lethality in Gossypium. Following the genetic nomenclature, we propose that the two novel Dobzhansky–Muller genes from G. hirsutum and from R4-4 be named Le ₃ and Le ₄ , respectively. Given what we know about their inheritance patterns, their genotypes should be Le ₃ Le ₃ le ₄ le ₄ in G. hirsutum, and le ₃ le ₃ Le ₄ Le ₄ in R4-4. Data from this study supported previous information in that expression of the lethal symptom might be affected by the dosage of causal alleles and the environment in which plants are growing.     

The application of phosphoramidate ProTide technology to the potent anti-HCV compound 4′-azidocytidine (R1479)

We report the design, synthesis and evaluation of a family of ca 50 phosphoramidate ProTides of the potent anti-HCV compound 4′-azidocytidine (R1479), with variation on the ester, amino acid and aryl moiety of the ProTide. Sub-μM inhibitors of HCV emerge. The compounds are all non-cytotoxic in the replicon assay. We herein report detailed SARs for each of the regions of the ProTide.     

Incomplete Deletion of IL-4Rα by LysMCre Reveals Distinct Subsets of M2 Macrophages Controlling Inflammation and Fibrosis in Chronic Schistosomiasis

Mice expressing a Cre recombinase from the lysozyme M-encoding locus (Lyz2) have been widely used to dissect gene function in macrophages and neutrophils. Here, we show that while naïve resident tissue macrophages from IL-4Rα^flox/deltaLysM^Cre mice almost completely lose IL-4Rα function, a large fraction of macrophages elicited by sterile inflammatory stimuli, Schistosoma mansoni eggs, or S. mansoni infection, fail to excise Il4rα. These F4/80^hiCD11b^hi macrophages, in contrast to resident tissue macrophages, express lower levels of Lyz2 explaining why this population resists LysM^Cre-mediated deletion. We show that in response to IL-4 and IL-13, Lyz2^loIL-4Rα⁺ macrophages differentiate into an arginase 1-expressing alternatively-activated macrophage (AAM) population, which slows the development of lethal fibrosis in schistosomiasis. In contrast, we identified Lyz2^hiIL-4Rα⁺ macrophages as the key subset of AAMs mediating the downmodulation of granulomatous inflammation in chronic schistosomiasis. Our observations reveal a limitation on using a LysM^Cre mouse model to study gene function in inflammatory settings, but we utilize this limitation as a means to demonstrate that distinct populations of alternatively activated macrophages control inflammation and fibrosis in chronic schistosomiasis.     

牦牛MC4R基因及其蛋白结构预测分析

根据普通牛MC4R基因序列设计同源引物,扩增克隆牦牛MC4R基因,对牦牛与普通牛MC4R基因及其蛋白结构进行生物信息学分析。与普通牛MC4R基因相比,牦牛在该基因存在5个多态位点,其中3个在密码子第3位,2个位点在密码子第1位;4个牦牛个体中有1个与其他个体存在2个变异位点且在密码子第3位;牦牛与普通牛在MC4R蛋白2个氨基酸的差异只引起两物种MC4R蛋白二级结构组分的细微差异。MC4R蛋白在牦牛和普通牛之间保守性较强,MC4R基因可能在哺乳类数百万年的进化历程中受到较强的进化抑制或净化进化。为研究MC4R基因在牦牛食欲控制、体重调节、脂肪沉积和能量平衡调节等方面提供基础资料。     

虹鳟MC4R基因的PCR扩增及其应用

黑素细胞皮质激素受体(MC4R)是跨膜G蛋白偶联受体。MC4R在人和鼠的体重、能量稳态和采食量的调控中具有重要作用,是第一个发现的与人类显性遗传疾病性肥胖相关的靶位点。虹鳟(Oncorhynchus mykiss)属于冷水性鱼类,具有很好的药用和食用价值,但生长缓慢。本研究根据斑马鱼的MC4R基因保守区的核苷酸序列设计引物,通过PCR扩增出虹鳟的MC4R基因,纯化后测序。本实验测出虹鳟MC4R基因968bp,并发现其与其它鱼类的MC4R进行了同源性分析,构建基因进化树。