Turnover of cholesterol-4-14-C and cholic acid-24-14-C by rabbits fed a diet containing lactose

Rabbits fed 0.35% of cholesterol in diets containing either 29.35% of lactose or sucrose were studied for 14 weeks. The rabbits fed lactose had higher plasma and liver cholesterol concentrations than those fed sucrose. The half-life of cholesterol was 19.0 days and 35.0 days for rabbits fed sucrose and lactose, respectively. The half-life, pool size, and daily production of deoxycholic acid were 9.7 days, 1.29 g, and 74.1 mg for rabbits fed sucrose; and 14.2 days, 1.40 g, and 49.1 mg, for those fed lactose. Cholesterol was the major neutral sterol in the feces of the rabbits fed lactose, whereas coprostanol (5 Beta-cholestan-3 Beta-ol) dominated the corresponding fraction in those fed sucrose. The fecal steroid composition did not vary between day and night collections. No sterol esters were detected in the feces. Urinary elimination of radioactivity was less than 10% of that injected. The "lactose effect" seems to be due to enhanced retention of steroids, the mechanism of which has not been elucidated.     

Lactoperoxidase-catalyzed oxidation of [4-14C]estradiol

The conversion of [4-14C]estradiol to water-soluble products by lactoperoxidase (EC 1.11.1.7) in the presence of added or generated H2O2 was studied using albumin or tyrosine as acceptor. The enzyme was able to catalyze the oxidation and binding of estradiol to albumin even in the absence of 2,4-dichlorophenol at very low concentrations of hydrogen peroxide. Other systems in which H2O2 was replaced by oxygen and Mn2+, light-sensitized riboflavin or glutathione was also shown to be active in the conversion of estradiol to water-soluble products and the effect of inhibitors on these reactions was investigated. Possible mechanisms for the peroxidase-catalyzed formation of these estradio metabolites are discussed.     

Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.     

乙脑/登革4型嵌合病毒的构建及其小鼠神经毒力测定

目的构建以乙型脑炎病毒(Japanese encephalitis virus,JEV)疫苗株SA14-14-2为基因骨架的乙脑/登革4型嵌合病毒,并分析该嵌合病毒对小鼠的神经毒力。方法通过重叠PCR方法扩增含有登革病毒4型(DENV-4)H241株pr ME基因序列和乙型脑炎病毒疫苗株SA14-14-2的NS1蛋白前177个核苷酸的融合片段,用Nar I和Bgl II双酶切后替换乙型脑炎病毒疫苗株SA14-14-2全长克隆中的相应区域,构建成乙脑/登革4型嵌合全长克隆,通过体外转录和转染BHK21细胞获得嵌合病毒(JEV/DENV-4 chimeric virus,JD4)。通过测定嵌合病毒JD4和2个母本株JEV SA14-14-2株及DENV-4 H241株蚀斑大小、小鼠脑内神经毒力和皮下感染入脑能力、乳鼠脑内神经毒力,比较JD4和母本株之间的差异。通过将JD4在原代地鼠肾(primary hamster kidney,PHK)细胞传代30次,分析传代后嵌合病毒的神经毒力是否减弱及减弱的程度。结果测序结果表明,构建的嵌合病毒JD4基因组序列和预期一致,没有产生新的位点突变。JD4蚀斑较SA14-14-2明显偏小,但和DENV-4 H241株没有明显区别。JD4对3周龄小鼠具有较强的脑内神经毒力,和母本株DENV-4 H241没有差异,对小鼠没有神经侵袭力。乳鼠实验结果表明,嵌合病毒JD4脑内神经毒力虽然略低于母本株DENV-4 H241,但两者之间没有明显差异,都明显强于乙脑疫苗株SA14-14-2。在PHK细胞传代30次后,小鼠神经毒力虽然有所减低,但并不明显。结论成功构建了嵌合病毒JD4,通过测定并比较JD4与母本株的蚀斑特征、小鼠及乳鼠神经毒力等试验,为分析登革疫苗候选株安全性研究奠定了基础。     

Preparation of 3-deoxy-3-deuteroestrone and 3-deoxy-4-14C-estrone

3-Deoxy-3-deuteroestrone (1,3,5(10)-estratrien-17-one-3-d) and 3-deoxy-4-¹⁴C-estrone (1 ,3 ,5(10)-estratrien-17-one-¹⁴C) have been prepared. The mechanism of reductive dehalogenation of aryl iodides with lithium aluminum deuteride is discussed. The ¹³C-NMR spectrum of 3-deoxy-3-deuteroestrone is discussed.