Membrane topology of mouse stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.     

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)₄G-NH₂] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS₂ bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)₃ ^3? → M(II)(TRI LXC)₃ ^?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)₂(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)₃ ^?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)₂ ⁺ at pH < 5 followed by a two-proton dissociation step (pK _a2) yielding M(II)(TRI LXC)₃ ^?. Pb(II)(TRI LXC)(HTRI LXC)₂ ⁺ converts to Pb(II)(TRI LXC)₃ ^? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK _a of binding to the “d”-substituted peptide, (TRI L12C, pK _a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK _a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K _bind = 3.2 × 10⁷ M^?1) compared with that with TRI L16C (K _bind = 1.2 × 10⁷ M^?1) at pH > 8.     

Crumbs proteins stabilize the cone mosaics of photoreceptors and improve vision in zebrafish

Although the unique organization of vertebrate cone mosaics was first described long ago,both their underlying molecular basis and physiological significance are largely unknown.Here,we demonstrate that Crumbs proteins,the key regulators of epithelial apical polarity,establish the planar cellular polarity of photoreceptors in zebrafish.Via heterophilic Crb2a-Crb2b interactions,the apicobasal polarity protein Crb2b restricts the asymmetric planar distribution of Crb2a in photoreceptors.The planar polarized Crumbs proteins thus balance intercellular adhesions and tension between photoreceptors,thereby stabilizing the geometric organization of cone mosaics.Notably,loss of Crb2b in zebrafish induces a nearsightedness-like phenotype in zebrafish accompanied by an elongated eye axis and impairs zebrafish visual perception for predation.These data reveal a detailed mechanism for cone mosaic homeostasis via previously undiscovered apical-planar polarity coordination and propose a pathogenic mechanism for nearsightedness.     

QTL-seq identifies an early flowering QTL located near Flowering Locus T in cucumber

Key message

Next-generation sequencing enabled a fast discovery of a major QTL controlling early flowering in cucumber, corresponding to the FT gene conditioning flowering time in Arabidopsis.

Abstract

Next-generation sequencing technologies are making it faster and more efficient to establish the association of agronomic traits with molecular markers or candidate genes, which is the requirement for marker-assisted selection in molecular breeding. Early flowering is an important agronomic trait in cucumber (Cucumis sativus L.), but the underlying genetic mechanism is unknown. In this study, we identified a candidate gene for early flowering QTL, Ef1.1 through QTL-seq. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Muromskij” (early flowering) and “9930” (late flowering) suggested quantitative nature of flowering time in cucumber. Genome-wide comparison of SNP profiles between the early and late-flowering bulks constructed from F₂ plants identified a major QTL, designated Ef1.1 on cucumber chromosome 1 for early flowering in Muromskij, which was confirmed by microsatellite marker-based classical QTL mapping in the F₂ population. Joint QTL-seq and traditional QTL analysis delimited Ef1.1 to an 890 kb genomic region. A cucumber gene, Csa1G651710, was identified in this region, which is a homolog of the FLOWERING LOCUS T (FT), the main flowering switch gene in Arabidopsis. Quantitative RT-PCR study of the expression level of Csa1G651710 revealed significantly higher expression in early flowering genotypes. Data presented here provide support for Csa1G651710 as a possible candidate gene for early flowering in the cucumber line Muromskij.     

Screening and Mutagenesis of Aspergillus niger for the Improvement of Glucose-6-Phosphate Dehydrogenase Production

The Aspergillus niger strain ZBY-7 was selected as the original strain of glucose-6-phosphate dehydrogenase production. After mutagenesis of the strain by means of UV irradiation and nitrosoguanidine, mutants of Aspergillus niger resistant to a certain metabolic inhibitor were obtained. Five of the mutants showed increased glucose-6-phosphate dehydrogenase production. The mutant resistant to antimycin A (Aspergillus niger AM-23) produced the highest level of glucose-6-phosphate dehydrogenase (695.9% of that produced by the original strain).     

Gβ1γ2蛋白的纯化及其与腺苷酸环化酶的互作关系

对G蛋白β₁γ₂亚基及偶联组分在信号传导中的作用进行了初步研究. 以离体昆虫细胞Sf9(Spodoptera frugiperda, 秋粘虫卵巢细胞)或H5(Trichoplusia ni, 粉纹夜蛾细胞)为生物反应器, 高效表达了G蛋白β₁γ₂亚基. 用Ni-NTA亲和层析柱, 经快速蛋白纯化技术(FPLC)获得高纯度的Gβ₁γ₂. 活性测定表明, 纯化的GGβ₁γ₂能显著刺激腺苷酸环化酶(AC2)的活力. 应用基于表面胞质团共振现象的BIAcore技术, 采用生物传感芯片NTA(biosensor chip NTA)直接证明腺苷酸环化酶2 (AC₂)的胞质末端C₂区域为G蛋白β₁γ₂亚基的结合区, 从而明确了二者互作的活性位点和结合位点同位于该区域.     

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).     

Development of Fatal Intestinal Inflammation in MyD88 Deficient Mice Co-infected with Helminth and Bacterial Enteropathogens

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection.     

氨氧化古菌的生态学研究进展

上百年来细菌一直被认为是地球氨氧化过程的主要驱动者,2005年海洋中分离到迄今唯一的非极端环境泉古菌,发现其氧化氨态氮获得能源生长,是氨氧化古菌。氨氧化古菌和细菌对地球氨氧化过程的相对贡献率,是目前全球氮循环研究最重要的微生物生态学问题之一。已有的证据表明古菌在海洋氨氧化过程中发挥了重要作用,细菌则是土壤氨氧化过程的主要驱动者。本文重点探讨了原位自然环境下氨氧化古菌的生态学研究进展。     

头端延髓腹外侧区注射5—羟色胺对应激性高血粘度...

Experiments were carried out on 62 wistar rats. The hyperviscosity and elevation of blood pressure were induced by hanging and restraining the rats with their four limbs tied on a frame. It was found that microinjection of 5-HT (25 micrograms/10 microliters) into the 4th ventricle of the brain or bilateral microinjection of 5-HT (4 micrograms/0.5 microliters/site) into rostral ventrolateral medulla (rVLM) reduced stress-induced hyperviscosity (p < 0.01) and elevation of blood pressure (p < 0.01). The effect of 5-HT injected into the 4th ventricle or rVLM was blocked by bilateral microinjection of cinanserine (4 micrograms/0.5 microliter/site) into rVLM. These results suggest that microinjection of 5-HT into 4th ventricle and rVLM could reduce stress-induced hyperviscosity and elevation of blood pressure and these effects were probably mediated via 5-HT receptors in the rVLM.