Native N-glycopeptide thioester synthesis through N→S acyl transfer

Peptide thioesters are important tools for the total synthesis of proteins using native chemical ligation (NCL). Preparation of glycopeptide thioesters, that enable the assembly of homogeneously glycosylated proteins, is complicated by the perceived fragile nature of the sugar moiety. Herein, we demonstrate the compatibility of thioester formation via N→S acyl transfer with native N-glycopeptides and report observations that will aid in their preparation.     

An insertion mutant in DQA1*0501 restores susceptibility to HLA-DM: implications for disease associations

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2-DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of α-II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three α-chain mutations (α+53G, α+53R, or αY22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.     

Structural organization of essential iron-sulfur clusters in the evolutionarily highly conserved ATP-binding cassette protein ABCE1

The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular mechanism has, however, not been elucidated. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines, predicted to coordinate iron-sulfur clusters. Here we present detailed information on the type and on the structural organization of the Fe-S clusters in ABCE1. Based on biophysical, biochemical, and yeast genetic analyses, ABCE1 harbors two essential diamagnetic [4Fe-4S](2+) clusters with different electronic environments, one ferredoxin-like (CPX(n)CX(2)CX(2)C; Cys at positions 4-7) and one unique ABCE1-type cluster (CXPX(2)CX(3)CX(n)CP; Cys at positions 1, 2, 3, and 8). Strikingly, only seven of the eight conserved cysteines coordinating the Fe-S clusters are essential for cell viability. Mutagenesis of the cysteine at position 6 yielded a functional ABCE1 with the ferredoxin-like Fe-S cluster in a paramagnetic [3Fe-4S](+) state. Notably, a lethal mutation of the cysteine at position 4 can be rescued by ligand swapping with an adjacent, extra cysteine conserved among all eukaryotes.     

牛乳酪蛋白源抗菌肽的研究进展

抗菌肽是具有抗菌活性的肽类物质的总称。除了广泛存在于生物体内的内源性抗菌肽外,已从酪蛋白的酶解产物中获得了多种外源性抗菌肽。抗菌肽的抗菌机理独特,有望成为新一代抗菌剂。简要综述了牛乳酪蛋白源抗菌肽的种类、抗菌机理及其研究展望。     

Late mitotic failure in mice lacking Sak, a polo-like kinase

Polo-like kinases in yeast, flies, and mammals regulate key events in mitosis. Such events include spindle formation at G2/M, the anaphase-promoting complex (APC) at the exit from mitosis, the cleavage structure at cytokinesis, and DNA damage checkpoints in G2/M. Polo-like kinases are distinguished by two C-terminal polo box (pb) motifs, which localize the enzymes to mitotic structures. We previously identified Sak, a novel polo-like kinase found in Drosophila and mammals. Here, we demonstrate that the Sak kinase has a functional pb domain that localizes the enzyme to the nucleolus during G2, to the centrosomes in G2/M, and to the cleavage furrow during cytokinesis. To study the role of Sak in embryo development, we generated a Sak null allele, the first polo-like kinase to be mutated in mice. Sak(-/-) embryos arrested after gastrulation at E7.5, with a marked increase in mitotic and apoptotic cells. Sak(-/-) embryos displayed cells in late anaphase or telophase that continued to express cyclin B1 and phosphorylated histone H3. Our results suggest that Sak is required for the APC-dependent destruction of cyclin B1 and for exit from mitosis in the postgastrulation embryo.     

蒙古国喀尔喀部蒙古族4项群体指趾遗传学特征研究

在内蒙古民族大学调查了160例(男66例,女94例)来自蒙古国的喀尔喀蒙古族留学生的拇指类型、环食指长、指甲形状、足趾长4项群体指趾遗传学特征。研究结果:1)直型拇指率为44.38%,环指长率78.13%,指甲形状长型率58.75%、方型率6.88%、扁型率34.38%,足趾类型拇趾长率35.00%;2)指甲形状出现率性别间差异具统计学意义,两两特征间均未表现出明显相关关系;3)与我国10个蒙古族族群相比,多数差异具有统计学意义。     

Effects of Removal of Muscle Receptor Organ Input on the Temporal Structure of Non-Giant Swimming Cycles in the Crayfish, Cherax Destructor

Most studies of the muscle receptor organs (MROs) of decapod crustaceans have focused on their role in local reflex loops. This may not be their only function. We examine their involvement in the regulation of non-giant swimming cycles by removing stretch receptor (SR) input from the MROs in abdominal segments 2-5 of the crayfish Cherax destructor . SR input was left intact in two control groups, one of which had sham surgery and the other no surgery at all. We recorded electromyograms (EMGs) from selected uropod muscles during tailflipping in sequences of non-giant swimming in tethered animals. The removal of SR input had a significant effect. The opener muscle period was shorter in the experimental group than in either of the control groups. This suggests that by using SR afference, crayfish sacrifice speed for increased control of the swimming movement.     

红松人工林优势木竞争指数影响因子

在温带湿润气候区东段不同立地条件下的红松人工林内设置面积为600 m~2(20 m×30 m)的70块矩形固定样地。在每个样地内,选取5颗最高且长势较好的优势木作为研究的对象木,用Voronoi图确定优势木相应的竞争木,测定每一块样地内优势木与竞争木之间的距离。采用Hegyi单木竞争指数模型,分析优势木在不同样地水平上的种内竞争强度,探讨林分生长因子、地形因子、土壤养分因子对优势木竞争指数的影响,并对这3类因子与优势木竞争指数进行相应的拟合和相关性分析。结果表明：红松人工林优势木竞争强度随着优势木胸径的增大而变小,并且两者之间的关系服从幂函数;红松优势木的竞争指数与其树高、胸径、冠幅呈极显著的相关关系(P<0.01);坡向、坡位、海拔对竞争指数影响极显著(P<0.01);红松人工林优势木竞争指数的大小与土壤氮磷钾含量均呈极显著的相关关系(P<0.01);pH值对优势木竞争指数的影响不显著。当红松人工林优势木平均胸径达到45cm,优势木平均树高和冠幅大于周围竞争木时,其对周围资源的利用程度增大,林木会发生自然稀疏现象,其所受的竞争压力减小。红松喜光性强,对水分的要求高,...     

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H- heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.      

质粒超螺旋比例对其细胞转染效率及表达的影响

检测质粒开环、闭环比例不同时对其细胞转染效率及表达的影响。构建携带人肝细胞生长因子基因的质粒(pcDNA3-HGF)及携带LacZ报告基因的质粒(pcDNA3-LacZ),用核酸纯化试剂盒提取开环、闭环比例不同的质粒,然后用LipofectAMINE介导它们分别转染NIH3T3细胞,采用X-Gal染色和ELISA法分别观察转染效率和表达活性。结果表明,用超螺旋比例分别为85.45%和48.44%的质粒pcDNA3-LacZ转染生长旺盛的NIH3T3细胞,48h检测转染效率分别为23.4%±3.8%和9.3%±2.5%,前者约是后者的2.5倍。用超螺旋比例分别为93.28%和40.53%的质粒pcDNA3-HGF转染1×106NIH3T3细胞,ELISA法检测48h细胞培养上清中HGF的表达量分别为46.5±6.3ng和25.6±4.2ng,前者是后者的1.8倍。初步认为质粒开环、闭环比例不同对其细胞转染效率及表达有一定影响,超螺旋比例高时其转染效率及表达量较高。