Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis

Benzotriazole‐based coupling reagents have dominated the last two decades of solid phase peptide synthesis. However, a growing interest in synthesizing complex peptides has stimulated the search for more efficient and low‐cost coupling reagents, such as COMU which has been introduced as a nonexplosive alternative to the classic benzotriazole coupling reagents. Here, we present a comparative study of the coupling efficiency of COMU with the benzotriazole‐based HBTU and HCTU for use in in situ neutralization Boc‐SPPS. Difficult sequences, such as ACP(65–74), Jung–Redeman 10‐mer, and HIV‐1 PR(81–99), were used as model target peptides on polystyrene‐based resins, as well as polyethylene glycol‐based resins. Coupling yields obtained using fast in situ Boc‐SPPS cycles were determined with the quantitative ninhydrin test as well as via LC‐MS analysis of the crude cleavage products. Our results demonstrate that COMU coupling efficiency was less effective compared to HBTU and HCTU with HCTU ≥ HBTU > COMU, when polystyrene‐based resins were employed. However, when the PEG resin was employed in combination with a safety catch amide (SCAL) linker, more comparable yields were observed for the three coupling reagents with the same ranking HCTU ≥ HBTU > COMU. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Rumen and post-abomasal disappearance in lactating cows of amino acids and other components of barley grain treated with sodium hydroxide, formaldehyde or urea

Four rumen and duodenum cannulated, Holstein lactating cows were used in a change-over design to determine the effects of NaOH, formaldehyde (HCHO) or urea treated barley on disappearance of dry matter (DM), crude protein (CP), amino acids (AA), NDF, ADF and starch of barley in the rumen, post-abomasal (PAT) and total tract by the mobile nylon bag technique. Experimental treatments were coarse milled barley, barley treated with 35 g NaOH/kg, barley treated with 4 g formaldehyde/kg and barley treated with 35 g urea/kg, in which all chemical treated barley was milled coarse before feeding.

NaOH treatment reduced concentrations of lysine and cystine in the barley grain. All chemical treatments decreased rumen disappearances of barley CP but only NaOH and formaldehyde treatments also decreased total AA and some of the AA disappearances in the rumen. All chemical treatments increased DM, OM, CP, starch, NDF and ADF disappearance of barley in the PAT, but only NaOH and formaldehyde treatments increased total AA and most individual AA disappearances in the PAT. Chemical treatments increased disappearance of starch, methionine and glycine in the total tract (P<0.05).

Rumen disappearance of TAA was lower than for CP but PAT disappearance of TAA was more than for CP and finally total tract disappearance of TAA was more than for CP. Individual AA in barley disappeared at different rates in the rumen and PAT. Consequently, the proportion of digesta CP and AAs of barley, entering the intestine were changed by the chemical treatments. We concluded that, appropriate treatment of barley with NaOH or HCHO were provided substantial protection of CP and individual AA from rumen digestion and increased disappearance of most of barley nutrients in PAT, but, NaOH treatment reduced the AA quality of barley. Consequently, formaldehyde can therefore be considered better than NaOH and urea for treatment of barley grain.     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

抗A型产气荚膜梭菌α毒素单链抗体基因的克隆和表达

应用RT PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌α毒素单克隆抗体的杂交瘤细胞中 ,扩增出抗体V_H 和V_L 基因 ,连接成ScFv基因 ,并将其克隆至pGEM T载体中构建了重组质粒pXScFv 2E3。经序列分析证实 ,V_H 和V_L 基因及linker基因拼接正确 ,基因全长为 726bp ,编码 242个氨基酸。随后将其定向克隆于表达载体pHOG21,转化至大肠杆菌XL1 BLUE筛选出表达菌株XL1 BLUE(pHOG 2E3)。经ELISA和SDS PAGE分析表明 ,在20℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 25 %。并且ScFv基因表达产物能够中和α毒素的磷酯酶C活性     

泥炭藓: 一类具有重要生态、经济和科学价值的碳封存植物

全球气候变暖是人类面临最严峻的环境挑战。有效控制碳排放, 充分发挥生态系统的固碳能力是实现碳中和目标的重要手段。作为碳封存能力最强的一种湿地类型, 泥炭地是加快实现碳中和目标的关键陆地生态系统。作为泥炭地“有效的生态系统工程师”, 泥炭藓(Sphagnum)在泥炭地的碳汇功能、过滤淡水及保护土地免受洪水侵袭等方面具有极其重要的作用。100多年来, 泥炭藓广泛应用于医药保健、污染监测和废水处理等领域, 尤其是作为一类最值得信赖的土壤介质和保湿材料一直被广泛用于园艺产业。在全球气候变暖和“双碳”目标的大背景下, 泥炭藓已经成为生命科学和生态学研究的热点。该文主要从泥炭藓的形态、物种多样性和起源、生境与分布、繁殖和保护、培养与种植、环境指示和监测、用途和应用, 以及碳封存、储水和酸化能力等方面进行综述, 旨在为泥炭藓研究、泥炭地的保护和恢复以及泥炭藓开发利用和产业发展提供借鉴与参考。     

Stress relaxation of wood partially non-crystallized using aqueous NaOH solutions

Wood samples (Picea jezoensis Carr.) were treated with aqueous NaOH solutions (0-0.20 concentration fraction, 12 conditions), and bending tests were performed to measure stress relaxation. The relationship between mechanical properties and NaOH concentration is discussed. The relaxation modulus and relaxation rate were divided into three concentration ranges. Both decreased slightly for NaOH concentrations less than 0.10, decreased drastically for concentrations between 0.11 and 0.14, and decreased slightly for concentrations greater than 0.15. The change in relaxation behavior upon NaOH treatment was due to an increase in molecular chain mobility in non-crystallized regions along the microfibril longitudinal axis in wood as well as lignin swelling. Furthermore, the molecular chain response in this region required time; thus, the dependence of crystallinity on the relaxation rate was apparent in the long time region.     

West Nile Virus Exposure in Black Bears of Northeastern Wisconsin

ABSTRACT Knowledge of the distribution and pathology of West Nile virus (WNV) in black bears is a necessary tool that allows wildlife managers to implement a management plan, set harvest quotas, and relocate nuisance bears. We studied the presence and significance of WNV titers in free-roaming black bears (Ursus americanus) in northeastern Wisconsin between February 2003 and March 2005. Serum neutralizing antibodies to WNV, with confirmation by plaque-reduction neutralization test to both WNV and Saint Louis encephalitis, identified exposure in 13 of 74 (17.6%) bears. This compares with a 6% infection rate in black bears in Virginia and 22% in European brown bears (Ursus arctos). Pathologic effects from exposure to WNV were not seen in any of the black bears studied.     

GC32细胞对三种虫媒病毒的敏感性研究

用胃癌细胞株ＧＣ３２,对乙型脑炎、基孔肯雅、兰加特病毒进行敏感性研究。并用ＢＨＫ２１细胞作对照,发现ＧＣ３２细胞对两种病毒的敏感性与对照细胞ＢＨＫ２１极为接近。ＢＨＫ２１和ＧＣ３２细胞对基孔肯雅、乙型脑炎和兰加特的免疫荧光实验,于感染后４８ｈ或７２ｈ都出现＋＋＋～＋＋＋＋的阳性结果。两种细胞对基孔肯雅和乙型脑炎病毒都形成空斑,ＢＨＫ２１细胞对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为５．６５和５．３６;ＧＣ３２对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为６．４８和５．６１。实验表明,ＧＣ３２细胞可以作为有关病毒实验的理想细胞株。     

HCV Antibody Response and Genotype Distribution in Different Areas and Races of China

Hepatitis C virus (HCV) heterogeneity accounts for the failure of effective vaccine development and the lack of successful anti-viral therapy in some patients. Little is known about the immune response to HCV peptides and the region or race specific genotypes in China. The objective of this study was to characterize HCV antibody immune response to HCV peptides and HCV genotypes in different regions and races of China. A total of 363 serum samples were collected from HCV carriers in 6 regions in China. The immune response to HCV peptides was evaluated by ELISA. HCV genotypes were examined using nested RT-PCR. We found that the anti-HCV antibody neutralization rates were significantly different among the serum samples from different areas or from different races in the same area. For samples from Tibet and Sinkiang, the rates of neutralization by HCV peptides were only 3.2% and 30.8%, respectively. The genotypes of samples from Tibet and Sinkiang were apparently heterogeneic and included type I, II, III and multiple types (I/II/III, I/II, I/III, II/III). One specific sample with multiple-genotype (I/II/III) HCV infection was found to consist of type I, II, III, II/III and an unclassified genotype. These studies indicate that the anti-HCV antibody immune response to HCV peptides varied across regions and among races. The distribution of HCV genotypes among Tibetans in Tibet and Uighurs in Sinkiang was different from that in the inner areas of China. In addition, a “master” genotype, type II, was found to exist in HCV infection with multiple HCV genotypes.