Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Enzymatic synthesis of 5-bromo-2’-deoxyuridine-2-14C and of 5-iodo-2’-deoxyuridine-2-14C and their incorporation into deoxyribonucleic acid (Allium cepa)

5-Bromo-2’-deoxyuridine-2-¹⁴C was prepared from 5-bromouracil-2-¹⁴C and 2’-de-oxyguanosine using trans-N-deoxyribosylase fromLactobacillus helveticus and incorporated into DNA ofAllium cepa roots. After isolating the DNA and hydrolyzing it enzymatically to deoxynucleoside-5’-phosphates a radioactive nucleotide was detected which yielded 5-bromo-2’-deoxyuridine-2-¹⁴C on enzymatic dephosphorylation. The incorporation of 5-iodo-2’-deoxy-uridine-2-¹⁴C was followed only by microautoradiography.     

L-乳酸脱氢酶热变性的热力学研究

用差示扫描量热法对L-乳酸脱氢酶的热变性进行了研究(温度扫描范围为290—390K,酶蛋白溶液浓度为0.28—0.72mg蛋白/mg溶液)。实验观察到当酶溶液浓度在0.62—0.72mg蛋白/mg溶液范围内有一个吸热转变,酶溶液浓度小于0.62mg蛋白/mg溶液时有两个未完全分开的吸热转变。 这个酶的量热焓与范德霍夫焓的比远大于1,而接近于2,这表明乳酸脱氢酶的变性过程不是一个简单的两态转变,从热力学和吸热峰的形状、大小分析,可以推断乳酸脱氢酶分子是由两个以弱相互作用相连结的合作结构区组成,而每一个结构区是由两个相互作用很强的亚基组成。也就是说乳酸脱氨酶的变性过程包括两个半独立的合作结构区的转变,每一个结构区的转变都近似一个两态转变,ΔHeal与ΔHvh的比值是随着两个半独立部分相互作用的增强,即蛋白浓度的增加而减小。随着蛋白浓度的减小,蛋白质周围水分子增多,酶分子中两个半独立部分的相对独立性增强,这可由热谱图上一个吸热转变变成两个半独立的转变得到证实。     

Production of monoclonal antibodies against avian LHRH-I

Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse. This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

伊贝母体细胞无性系的建立及其胚状体的发生

本文报道了伊贝母体细胞无性系的建立及其胚状体的发生。已继代培养三年零六个月共30多代的鳞芽愈伤组织,目前仍有分化能力。通过愈伤组织形态细胞学的观察,发现伊贝母体细胞无性系形成小鳞茎的途径有二:一是由特化了的愈伤组织表皮细胞。经多次分裂发育成不定芽而形成小鳞茎;二是由愈伤组织表层或内层特化了的胚性细胞,经多次分裂发育成胚状体而形成小鳞茎。不定芽和胚状体的形态发生是有区别的。     

糙叶败酱挥发油镇静作用的研究

本文观察了败酱科植物糙叶败酱(Patrinia scabra Bunge)根和根茎中制得的挥发油的镇静作用,井与黄花败酱挥发油做了比较。结果表明,此油灌胃给予数组小鼠,剂量0.45ml/kg,显示如下的作用:[1]能显著延长由于腹腔注射戊巴比妥钠(50mg/kg)引起的小鼠睡眠时间,但其作用强度弱于黄花败酱挥发油。(2)一次灌胃给予小鼠大剂量10.46g/kg的糙叶败酱挥发油,连续观察10天,动物外观正常,无一死亡,体重增加与对照组相似。