An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe.

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.     

Pre‐copulation intervals,copulation frequencies,and initial progeny sex ratios in two biotypes of whitefly,Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) is a species complex, and its systematic classification requires controlled crossing experiments among its genetic groups. Accurate information on pre‐copulation intervals, copulation frequencies, and initial frequency of egg fertilization of newly emerged adults is critical for designing procedures for collecting the virgin adults necessary for these experiments. In the literature, considerable variation is reported between B. tabaci populations, with respect to the length of the pre‐copulation interval and the initial frequency of egg fertilization. Here, we used a video‐recording method to observe continuously the copulation behaviour of the Mediterranean/Asia Minor/Africa (B biotype) and the Asia II (ZHJ1 biotype) groups of B. tabaci. We also recorded the initial frequency of egg fertilization, as determined by the sex of the progeny. When adults were caged in female–male pairs on leaves of cotton plants, the earliest copulation events occurred 2–6 h after emergence; at 12 h after emergence 56–84% of the females had copulated at least once, and nearly all (92–100%) had copulated at least once by 36 h after emergence. Both females and males copulated repeatedly. Approximately 80 and 20% of copulation events occurred during the photophase and scotophase, respectively. By 72 h post‐emergence, the females of the B and ZHJ1 biotypes had copulated on average 6.1 and 3.9 times, respectively. When adults were caged in groups on plants 1–13 h after emergence, 30–35% of the eggs deposited during this period were fertilized, and approximately 90% of females were fertilized by the end of the 13 h. Although timing of copulation differed in detail between the two genetic groups, the results demonstrate that B. tabaci adults can start to copulate as early as 2–6 h post‐emergence and the majority of females can become fertilized on the day that they emerge.     

Influence of GATC sequences on Escherichia coli DNA mismatch repair in vitro.

The effect of the number and position of DNA adenine methylation (dam) sites, i.e., d(GATC) sequences, on mismatch repair in Escherichia coli was investigated. The efficiency of repair was measured in an in vitro assay which used an f1 heteroduplex containing a G/T mismatch within the single EcoRI site. Both an increase in the number of dam sites and a shortened distance between dam site and mismatched site increased the efficiency of mismatch repair. The sequences adjacent to d(GATC) also affected the efficiency of methylation-directed mismatch repair. Furthermore, heteroduplexes with one extra dam site located close to either the 5' or 3' end of the excised base increased the repair efficiency to about the same extent. The findings suggest that the mismatch repair pathway has no preferred polarity.     

白兰瓜果实输入~(14)C-葡萄糖的转化与蔗糖合成

~(14)C 追踪试验结果表明,白兰瓜幼果中输入的~(14)C-葡萄糖,50%以上转化为稀酸水解和稀酸不水解的结构物质;果实发育后期,输入后48小时,在果肉和种子中分别只有18%和32%的~(14)C 参入结构物质。根据醇溶性糖的纸层析鉴定,幼果薄片渗入的~(14)C-葡萄糖仅转化为果糖,而发育后期果实则更多转化为蔗糖。显然,幼果的代谢模式是使物质和能量导向结构物质的形成;而后期果实生长已基本停止,物质代谢的方向又转向蔗糖合成的轨道上来。蔗糖合成底物试验结果表明,供给幼果不同底物都只有很低的蔗糖合成活性;发育后期果实供给UDPG+F-6-P 底物时可测出较高的蔗糖合成活性,初步推测白兰瓜中蔗糖合成主要是通过蔗糖磷酸酯合成酶来实现的。     

异大茴香脑新资源——齿叶黄皮的研究

齿叶黄皮是生长于广东省北部石灰岩山地的一种芸香科常绿小乔木,其叶富含精油,枝叶经水蒸汽蒸馏出油率为0.7%。精油经毛细管气相色谱、气相色谱/质谱联用、红外吸收光谱、核磁共振波谱等方法分析,确证含异大茴香脑(isoanethole)93.10%。异大茴香脑对霉菌有较强抑菌活性,并可通过异构化一步转变为大茴香脑。     

Formation of lymphocyte colonies under serum-free culture conditions in normal individuals and patients with chronic lymphocytic leukemia

This paper describes a culture system which supports the formation of B cell and some T cell colonies under serum-free conditions in peripheral blood samples of normal individuals and patients with chronic lymphocytic leukemia (CLL) of B cell type. In this system, serum is replaced by bovine serum albumin, transferrin, cholesterol, insulin and catalase or horseradish peroxidase. In addition, it is necessary to add staphylococcus protein A, mitomycin-treated T cells as feeders and phytohemagglutinin leukocyte-conditioned medium as a source of growth factors. The plating efficiency is greatly enhanced when normal cells are incubated with galactose oxidase prior to plating and when CLL cells are exposed sequentially to neuraminidase and galactose oxidase.     

Genetic polymorphism of GPT and GLO-I in Galicia (northwest Spain): a comparative study

GPT and GLO-I phenotypes were determined by means of isoelectric focusing and starch gel electrophoresis, respectively, in a sample of the Galician population (Northwest Spain); GPT: n = 302, GLO-I: n = 500. The gene frequencies come to: GPT1 = 0.5099, GPT2 = 0.4901; GLO1 = 0.4930, GLO2 = 0.5070. No rare variants were found. The Galician gene frequencies are compared with those obtained on other populations from different parts of the world.     

Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.     

N-Deacetylation of 2-acetamido-2-deoxy-hexosecontaining oligosaccharides and polysaccharides by calcium in liquid ammonia

N-Deacetylation of 2-acetamido-2-deoxy-hexose residues is accomplished in liquid ammonia containing calcium. Oligosaccharides, lacto-N-fucopentaose II and lacto-N-difucohexaose I, containing 3,4-disubstitutedN-acetylhexosamine residues are quantitativelyN-deacetylated. When applied to polysaccharides, however, only partialN-deacetylation was achieved.Author for correspondence. AXRD     

水翁花蕾和水翁叶精油的化学成分研究

水翁的花营和鲜叶经水蒸汽蒸馏得到一种淡黄色的精油,前者出油率为0.18％。后者为0.08％。我们应用毛细管气相色谱,气相色谱/质谱联用,红外光谱和紫外光谱等方法,对两种精油进行化学分成分析,分别鉴定出35个和27个已知化学成分。两者相同的化学成分有:β-罗勒烯(Z)、β-罗勒烯(E)、α-蒎烯、β-蒎烯、月桂烯、小茴香烯、香叶醇、顺式-丁香烯、橙花叔醇等23个化学成分,分别占花营精油全油的90％和叶精油95％以上。