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101.
Abstract: A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide‐bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human β‐defensins 1 and 2 (hBD‐1, hBD‐2) as well as of the novel murine β‐defensins 7 and 8 (mBD‐7 and mBD‐8) is reported. The peptides were synthesized by solid‐phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP‐1/hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver‐expressed anti microbial peptide (LEAP)‐1/hepcidin and hBD‐1, the identity of native and synthetic peptides was demonstrated by high‐pressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs. 相似文献
102.
二硫化碳对大鼠F_2代后遗影响的研究 总被引:3,自引:1,他引:2
本文报道大鼠妊娠7~14d以Cs_2染毒对F_2代的后遗影响。结果表明,CS_2对F_2代生长发育指标及F_1代脏器系数无明显影响,但对胎鼠有致畸作用,并可延续到第二代,以骨骼畸形为主,主要为胸骨缺失,其次为枕骨骨化延缓、肋骨延长和囱门增大等,且发生率几乎同胎鼠的F_1代。由此认为CS_2对后代的影响,主要是对F_2代的致畸作用较明显。 相似文献
103.
H.P.J. BENNETT N.G. SEIDAH S. BENJANNET S. SOLOMON M. CHRETIEN 《Chemical biology & drug design》1986,27(3):306-313
The cystine bridge structure of the amino-terminal fragment of human proopiomelanocortin has been reinvestigated. Highly purified amino-terminal fragment 1–76 was rapidly isolated from human pituitaries using only reverse-phase liquid chromatography (RP-HPLC). This peptide was then subjected to trypsin and V8-protease digestion and the products separated by RP-HPLC> and subjected to amino acid and microsequence analysis. The results show that disulfide bridges link Cys-2 to Cys-24 and Cys-8 to Cys-20. Amino acid analysis and amino sugar determination confirm (i) the previously proposed sequence and (ii) the suggestion of the presence of two glycosylation sites in this molecule. These are most probably located at Thr-45 (O-glycosylation) and at Asn-65 (N-glycosylation). 相似文献
104.
We attempted to generate a physicochemically stable cholera toxin B subunit (CTB) by de novo-introduction of intersubunit disulfide bonds between adjacent subunits. Genes encoding double mutant CTB (dmCTB) encompassing a pair of amino acids to be replaced with cysteine residues either at the N-terminal (T1C/T92C, Q3C/T47C), C-terminal (F25C/N103C, Y76C/N103C), or at the internal α-helix region (L77C/T78C), were engineered. One mutant with the N-terminal constraint [dmCTB(T1C/T92C)], expressed as pentamer retained monosialoganglioside G(M1) (GM1) binding affinity, and exhibited robust thermostability. However, when the mutant CTB was heat-treated in the presence of a reducing agent, the thermostable phenotype was abolished, indicating the observed phenotype is due to the introduction of intersubunit disulfide bonds. The mutant CTB also exhibited a strong acid stability at a pH as low as 1.2, as well as stability against incubation with sodium dodecyl sulfate at concentrations as high as 10%. Furthermore, intranasal administration of the mutant CTB to mice induced CTB-specific serum IgG even after heat treatment, while the wildtype CTB failed to show such heat-resistant mucosal immunogenicity. This study demonstrated that an enterotoxin B subunit could be transformed into a physicochemically stable pentamer by the de novo-introduction of peripherally arranged intersubunit disulfide crosslinks, which may prove to be a useful strategy for the development of molecularly stable enterotoxin B subunit-based vaccines and delivery molecules. 相似文献
105.
Tao Liu Yan Wang Xiaozhou Luo Jack Li Sean A. Reed Han Xiao Travis S. Young Peter G. Schultz 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(21):5910-5915
Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.We are developing strategies that begin to address the question of whether an expanded genetic code provides an evolutionary advantage to an organism. For example, it has been recently shown that a unique noncanonical amino acid mutation in TEM-1 β-lactamase significantly increases the enzyme’s catalytic activity for the substrate cephalexin, a result that cannot be recapitulated by substitution of canonical amino acids at this site (1). This same enzyme has been reengineered to be dependent on a noncanonical active site residue for activity, a dependency that was maintained for hundreds of generations without escape (2). Furthermore, addition of noncanonical amino acid building blocks to an unbiased library of ribosomally synthesized cyclic peptides provided a selective advantage in the evolution of inhibitors of cytotoxic intracellular proteases (3). Here we begin to explore whether noncanonical amino acids can provide Escherichia coli a selective growth advantage by increasing the thermal stability of essential proteins.Cysteine is unique among the 20 canonical amino acids in that it can form reversible covalent cross-links in proteins. Disulfide bonds can stabilize monomeric and multisubunit proteins (4), play a role in catalysis (5, 6), and regulate protein activity (7); because of these unique properties, disulfide bonds are highly conserved in protein evolution (8, 9). There has been considerable success in the use of structure-based design to engineer disulfide bonds into proteins for both biopharmaceutical and industrial applications (10, 11). However, the sites in proteins that can be cross-linked by a cystine disulfide are typically constrained to a distance between the two β-carbons of ∼5.5 Å (10) and a near 90° dihedral angle for the disulfide bond (12). Thus, the relatively long distances that might be required to bridge distinct protein domains or subunits, or steric constraints at specific sites may preclude the introduction of a natural disulfide bond. These challenges led us to explore whether we could overcome the geometrical constraints of the cysteine disulfide by genetically encoding noncanonical amino acids (NCAAs) with longer thiol-containing side chains.To this end, we designed a series of tyrosine derivatives (SetY, SprY, and SbuY) with para-substituted aliphatic thiols of various lengths (Fig. 1A). The calculated length between the two β-carbons of the SbuY-Cys disulfide bond is 14 Å when fully extended, which is significantly longer than a natural cystine cross-link. We genetically encoded these NCAAs in bacterial and mammalian cells by suppressing the nonsense codon TAG with an orthogonal, amber suppressor aminoacyl-tRNA synthetase (RS)/tRNA pair (13). Because statistical analyses of proteins have suggested that stabilizing disulfide mutations are most often found in regions of higher mobility near the protein surface and associated with longer loop lengths (>25 residues) (10, 14), we reasoned that these more flexible, extended disulfides might facilitate the introduction of stabilizing disulfide bonds into proteins. Using a N-terminal truncated β-lactamase as a model system (15), we carried out a growth-based selection under nonpermissive temperatures with a library of mutants in which the thiol-containing NCAAs were randomly incorporated, and identified a mutant enzyme cross-linked by an extended disulfide bond that is stabilized by ∼9 °C.Open in a separate windowFig. 1.Genetic incorporation of NCAAs containing long-side-chain thiols. (A) Structure of O-(2-mercaptoethyl)-l-tyrosine (SetY), O-(3-mercaptopropyl)-l-tyrosine (SprY), and O-(4-mercaptobutyl)-l-tyrosine (SbuY). (B) SDS/PAGE analysis of purified GFP (134TAG) expressed in E. coli DH10B using the MbXYRS/tRNApyl pair in the presence or absence of 1 mM NCAAs. GFP mutants were expressed in LB medium and purified by standard Ni-NTA affinity chromatography. (C) Mass spectral analysis of GFP mutants containing the corresponding NCAAs. The calculated masses for the mutant GFPs containing SetY, SprY, and SbuY are 28,005 Da, 28,019 Da, and 28,033 Da, respectively. (D) Fluorescence microscopy (10×) of 293T cells expressing an EGFP mutant (Tyr39TAG) in the presence or absence of NCAAs. 293T cells were transiently cotransfected with pCMV-XYRS and pEGFP-Tyr39TAG and were grown in DMEM supplemented with 10 FBS in the presence or absence of 250 μM NCAAs. 相似文献
106.
J. Cho 《Journal of thrombosis and haemostasis》2013,11(12):2084-2091
Protein disulfide isomerase (PDI) catalyzes disulfide bond oxidation, reduction and isomerization during protein synthesis in the endoplasmic reticulum (ER). In addition to its critical role in the ER, in vitro and in vivo studies with blocking antibodies and conditional knockout mice have demonstrated that cell surface PDI is required for thrombosis, hemostasis and vascular inflammation in a manner dependent on its isomerase activity. This review will focus on our current understanding of the pathophysiologic role of PDI in regulating integrin‐mediated platelet and neutrophil functions during vascular disease. 相似文献
107.
108.
探讨肝移植术后新发急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)的诊断和治疗方法.方法 2003年10月至2012年12月在中山大学附属第三医院肝移植中心定期随访的肝移植患者中发现1例APL患者,回顾性分析该例患者的临床表现、诊断、治疗和预后情况.检索Pubmed、万方医学数据库等国内外数据库,分析肝移植术后白血病患者,特别是APL患者的年龄、性别、原发病、免疫抑制方案、术后发病时间、白血病分类、染色体变异类型、治疗、预后及死因等特点.结果 本例患者于肝移植术后52个月门诊随访时发现外周血白细胞减少,无明显症状和体征,无感染与出血倾向,重组人粒细胞集落刺激因子治疗无效,经骨髓穿刺检查诊断为急性髓细胞白血病(acute myeloid leukemia,AML)-M3型,即APL.经维A酸和复方黄黛片(主要化学成份为二硫化二砷)联合治疗,患者获得完全缓解.检索文献共报道肝移植术后白血病34例(包括本例),其中22例诊断为AML(成人18例,儿童4例),术后发病时间中位数为26个月(38 d至8年).18例AML中,以APL最多见(8/18,44%)且伴有异常染色体核型t(15; 17),治疗采用维A酸、三氧化二砷及联合柔红霉素、阿糖胞苷等化学药物治疗(化疗)药物.22例AML死亡13例(59%),主要死于弥散性血管内凝血(disseminated intravascular coagulation,DIC)、感染、肝衰竭和心力衰竭.APL和非APL患者中分别有2例(2/8)和11例死亡(79%,11/14),两组差异有统计学意义(P<0.05).APL患者中6例化疗后完全缓解,另2例死于DIC和败血症.结论 肝移植术后新发AML总体预后不佳,其中APL的预后好于非APL.早期诊断、早期治疗是提高肝移植术后APL疗效的关键措施. 相似文献
109.
目的 研究不同生殖阶段暴露于二硫化碳(CS2)对小鼠子宫和胚胎组织粘附分子表达水平的影响。方法 分别给处于卵泡发育期、胚胎植入期(分为植入早期和晚期)的雌性小鼠腹腔注射CS2(631.4 mg/kg),每天1次,连续2 d,于受孕第9d收集样本,用western blotting测定胚胎和子宫组织中粘附分子纤维粘连蛋白(FN)及其受体蛋白整合素β1的表达水平。结果 与对照组比较,胚胎植入期染毒组胚胎和子宫组织中的FN与整合素β1表达水平各分别下降了54.8%、10.4%和52.5%、21.1%,差异均有统计学意义(P<0.05);在卵泡发育期染毒组,子宫组织中FN与整合素β1表达水平分别下降了69.4%和16.0%,差异有统计学意义(P<0.05),但胚胎组织中仅见FN表达水平明显降低(P<0.01);与对照组比较,植入期染毒组的胚泡植入数减少了37.5%,差异有统计学意义(P<0.01),但卵泡发育期染毒组仅表现为轻微下降(P>10.05)。结论 胚胎植入期暴露于CS2明显减少小鼠胚胎和子宫组织中粘附分子FN和整合素β的表达水平,粘附分子表达水平的降低与胚胎植入障碍相关联。 相似文献
110.