Alkaline phosphatase conjugated protein A as a sensitive reagent to immunoscreen an expression cDNA plasmid library: isolation of cDNA to the calcium-binding protein of the chick embryonic chorioallantoic membrane

A highly efficient immunoscreening procedure has been developed to isolate cDNA clones to the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM). A library of total CAM cDNA was constructed using the expression plasmid vector, pUC 19. Bacterial clones containing plasmids with CaBP cDNA inserts were detected immunohistochemically based on their expression of hybrid CaBP protein sequences. For immunodetection, nitrocellulose bacterial colony replicas were treated with specific antibodies to the CaBP followed by incubation with Staphylococcus aureus Protein A conjugated with alkaline phosphatase (AP) which served as a secondary immunoreagent. Positive clones were then histochemically identified based on AP enzyme activity. The identity of the immunopositive clones was further verified by in vitro translation of mRNA selected by hybridization to the cloned cDNA. The AP-based immunoscreening procedure yields stable reaction products with relatively low background, and should find general application for isolating specific cDNA clones from expression cDNA libraries.     

地塞美松中间体的C1,4脱氢和11α-羟基化

地塞美松 (Ｄｅｘａｍｅｔｈａｓｏｎｅ)为高效肾上腺皮质激素药物 ,临床上广泛使用。开拓用梯可吉宁 (Ｔｉｇｏｇｅｎｉｎ)为起始原料生产从化学结构和合成技术上讲较为合理 ,而且适合于资源综合利用[1] 。在合成过程中 ,除了Ａ环需引入Ｃ1,2 和Ｃ4 ,5两个双键 (含Ｃ-3 羟基氧化 )外 ,还需用微生物法在Ｃ-11位引入羟基。国外常采用化学法脱氢 ,然后再用霉菌 11α 羟基化[2 ,3 ] 。本文报道用两类微生物菌种 ,节杆菌 (Ａｒｔｈｒｏｂａｃｔｅｒｓｐ .)ＡＸ86和绿僵菌 (Ｍｅｔａｒｈｉｚｉｕｍｓｐ .)Ｍ 88,混合转化一步完成脱氢和羟基化反应…     

甾体1，4-脱氢和11α-羟基化反应的两种不同微生物转化

Two kinds of micro-organism, Arthrobacter sp. AX86(1,4-dehdrogenator)and Absidia sp. A28(11α-hydroxylator) were used in this experiment. Two different fermentation techniques were performed to accomplish the multiple conversional reactions for producing 16β-methyl-11α, 17α, 21-trihydroxy-1,4-pregnadiene-3,20-dione (Ⅲ) from 16β-methyl-3β, 17α,21-trihydroxy-5α-pregnane-20-one-21-acetate(1) 1)To produce product(Ⅲ)by means of a two-step fermentation method which were independently performed first by Arthrobacter and next by Abslaia, and 2)the product was obtained by a sequential fermentation system of aforesaid two micro-organisms in a single fermentor without isolation of the intermediates from the mixture. Our results showed that in both fermentation systems high yield of product was obtained. However, according to the technical simplicity, shorter duration of fermentation cycle and efficient yield of product, the second method is better than the first one.     

Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses,but no correlation to viral load

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.     

中条山油松人工林下物种对群落物种丰富度分布格局的贡献

该研究以中条山油松人工林群落为研究对象,研究林下不同大小的子群落对群落物种丰富度分布格局的贡献,并确定影响该区域群落物种丰富度分布格局的关键种,为区域物种多样性保护提供理论依据.结果 表明:(1)该地区林下物种频度分布格局呈明显右偏,且不同样方物种丰富度存在明显差异.(2)常见种对群落丰富度分布格局的贡献大于稀有种.(...     

Acyl-CoA: sn-Glycerol-3-Phosphate O-Acyltransferase in Rat Brain Mitochondria and Microsomes

Abstract: The subcellular distribution of acyl-CoA: sn -glycerol-3-phosphate O-acyltransferase between brain mitochondria and microsomes was investigated. The activities associated with purified rat brain mitochondrial and microsomal preparations could be distinguished by differences in their acyl-CoA specificity, products of acylation, and sensitivity to N -ethylmaleimide, trypsin, acetone, and polymyxin B. It was concluded that both brain mitochondria and microsomes possess the acyltransferase.     

Does rearing an aphid parasitoid on one host affect its ability to parasitize another species?

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