ACCURACY OF PHYLOGENETIC-ESTIMATION METHODS UNDER UNEQUAL EVOLUTIONARY RATES

A comparative study of the accuracy of three different approaches to phylogenetic estimation was made on simulated data with differing rates of change in different lineages. The three approaches were maximum likelihood, maximum parsimony, and phenetic clustering. The data were generated by simulating genetic drift with different population sizes over a simple four-species tree topology. Although the accuracy of all three approaches was found to be dependent on the number of loci (characters), maximum likelihood was found to perform considerably and consistently better than maximum parsimony or phenetic clustering.     

Affinity purification of human deoxycytidine kinase: avoidance of structural and kinetic artifacts arising from limited proteolysis

Homogeneous deoxycytidine kinase has been isolated from leukemic human T-lymphoblasts by affinity chromatography based on a multisubstrate analog, deoxycytidine 5'-adenosine 5"'-P1,P4-tetraphosphate (dCp4A). Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the Mr of the native protein, 59,300, is comparable to the value of 52,000 from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric pH was 6.1. But, enzyme isolated without protease inhibitors exhibited two fragments of Mr = 30,000 and 33,000, suggesting that proteolytic cleavage of the parental polypeptide had occurred during affinity chromatography. Both the parental and proteolyzed enzymes phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. However, the proteolyzed enzyme had an increased apparent Km for deoxycytidine. In consequence of this, a mixture of the two forms produced bimodal kinetic plots, whereas linear kinetics were displayed by each form alone.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.     

Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Bangham's report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).     

Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.     

中国人ε珠蛋白基因5′侧序列中多态性Hinc Ⅱ位点的进一步研究

本文进一步研究了我国不同民族的正常个体以及β地中海贫血患者θ珠蛋白基因5′侧序列中的多态性HincⅡ位点及其遗传性质。在广西壮族正常个体和β地中海贫血纯合子中,该多态性位点的发生频率均为75％,与正常汉族人测得值相近。家系分析资料表明,该多态性位点完全按照孟德尔规律进行遗传。     

A species difference between hamster and rat in the effect of oestrogens on growth of large preantral follicles

Intact or hypophysectomized 23-day-old hamsters and rats were injected s.c. with 2 mg diethylstilboestrol (DES) or 1 mg oestradiol cyclopentylpropionate (OECP) on Days 23-25 and killed on Day 26. Although serum oestradiol was elevated to the same high levels by OECP, ovarian and uterine weights were increased in the rat by OECP or DES whereas only the uterus responded in the hamster. This correlated with the ability of the oestrogens to increase significantly the number of large preantral and antral follicles in the intact rat but only the number of follicles with 2-3 layers of granulosa cells in the immature hamster. Qualitative study revealed that DES and OECP increased the number of large preantral follicles in the adult hypophysectomized rat but were ineffective in the adult hamster. It is concluded that for the immature and adult hamster oestrogens do not play a major role in the recruitment of large preantral follicles.     

Do protozoa conceal a high potency of nucleoside triphosphate hydrolysis present in Toxoplasma gondii?

ATP hydrolytic activity in whole cell homogenates of some protozoa was assayed in the presence or absence of dithiothreitol. The activities in all protozoan cell homogenates, except Toxoplasma gondii, ranged from 0.6 to 32 mumol/mg protein/hr, irrespective of the presence or absence of dithiothreitol. A remarkably higher activity, 11,690 mumol/mg protein/hr, was observed for T. gondii in the presence of dithiothreitol. These results indicate that the higher ATP hydrolytic potency observed for T. gondii is not universal to protozoa, rather it is unique to T. gondii.