Molecular evolution of cyclooxygenase and lipoxygenase

Four oxygenases of the arachidonic acid cascade (cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase and 15-lipoxygenase) were investigated by the method of computer-assisted sequence comparison. From the calculations, some aspects of evolution and function of these enzymes were revealed. (1) The evolutionary origin of cyclooxygenases was different from that of lipoxygenases. (2) Cyclooxygenase was a distantly related member of a peroxidase family. (3) Enzymes with 12-lipoxygenase activity were created independently twice by gene duplication.     

Molecular cloning and expression of human arachidonate 12-lipoxygenase

The cDNA for a 12-lipoxygenase was isolated from cDNA library of human erythroleukemia cells. The cDNA had an open reading frame encoding 663 amino acids with a calculated molecular weight of 75,513. The deduced amino acid sequence of human 12-lipoxygenase exhibited 41.5%, 65.3% and 65.4% identity with human 5-lipoxygenase, human 15-lipoxygenase and porcine 12-lipoxygenase, respectively. Blot hybridization analysis of RNA from human erythroleukemia cells demonstrated a single species (3.1 kb) of mRNA with the cDNA probe for 12-lipoxygenase of these cells, but not with the cDNA for porcine leukocyte enzyme. The cytosol of Escherichia coli transformed with a recombinant pUC19 plasmid oxygenated the position 12 of arachidonic acid.     

Carbohydrate fermentation by Clostridium difficile

Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01% L-cysteine.HCl and 0.1% agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.     

Electrophoretically estimated genetic distance and divergence time between chimpanzee and man

Amount of genetic differentiation between chimpanzee and man was estimated from the result of comparative electrophoretic screening of blood protein variations at 32 independent genetic loci. TheNei's genetic distance (D) was calculated as 0.4514, and from this value the divergence time between the two species was estimated as 2.26 million years; considering the variation among amino-acid substitution rate in different proteins, the corrected figures were given as genetic distance of 0.5706 and divergence time of 2.85 million years. This genetic difference is considered too small the two species to be allocated in different families, in accordance with the results of the similar kind of analyses byKing andWilson (1975) and Bruce andAyala (1979). Discussions were made for a discrepancy between the divergence times estimated by using and not by using the splitting time recognized by paleoprimatologists as a reference, and for the difference in the estimations made in different laboratories.     

Population genetics of Japanese monkeys: II. Blood protein polymorphisms and population structure

Genetic variability in individual troops of the Japanese macaque (Macaca fuscata fuscata) was quantified by the proportion of polymorphic loci and the average heterozygosity per individual from the results of starch-gel electrophoreses of blood proteins controlled by 32 independent genetic loci. The former averaged 9.2% and the latter 1.3%, the values being remarkably lower than those estimated for other animal populations. Geographical distribution of the genetic variations was not uniform in the whole species but the variants occurred only in limited areas. Assuming the selective neutrality of segregating alleles and the two-dimensional stepping-stone model of population structure, the genetic migration rate between the local demes per generation could be estimated to average less than inverse of average effective deme size. Here, the local deme is not a troop itself, but it consists of several troops tightly connected with each other by frequent exchanges of reproductive males. Analyses of correlation between geographic and genetic distances between troops revealed that the gene constitutions of two troops apart more than 100 km on an island could be regarded as practically independent of each other. These results suggest that the population structure of the Japanese macaque species has a tendency to split into a number of local subpopulations in which the effect of random genetic drift is prevailing.     

Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.     

Unique requirements for template primers of DNA polymerase beta from rat ascites hepatoma AH130 cells.

The optimal condition for the rat DNA polymerase beta activity with (rA)n . (dT)12-18 as a template-primer was determined. The activity was remarkably affected by the concentration of the primer, (dT)12-18' and the mixing ratio of (dT)12-18 to (rA)n. DNA polymerase beta requires higher primer concentration (Km = 11.1 microM with respect to 3'-OH of the primer) than DNA polymerase gamma (Km = 0.04 microM) or oncornaviral DNA polymerase (Km = 0.08 microM) and the enzyme represented the maximum activity in the base ratio of 2:1 with (dT)12-18 and (rA)n suggesting the difference in reaction mechanisms of these enzymes. Under the optimized conditions, the specific activity of the near homogeneous preparation of DNA polymerase beta was 1,000,000 units per mg protein.     

Effect of disinfectants on variola virus in cell culture.

Twenty kinds of disinfectants were examined for ability to inactivate variola virus. Cytopathic effect and plaque formation on monolayer cultures of an established monkey kidney cell line were used as indicators of virus inactivation. A micromethod using microplate cultures, and not requiring a CO2 incubator, was adopted. The procedures were straightforward, showing good reproducibility. Among the compounds tested, several were found to be superior because of the minimum concentrations required for complete inactivation of virus. The purified viruses were shown to be more sensitive to the compounds than were the crude samples. The virus inactivation kinetics curves were determined by plaque counting. The usefulness of this method for quantitative analysis of disinfecting effect is suggested.