Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

Coexistence of natural enemies in a multitrophic host–parasitoid system

Abstract.  1. This study explored the temporal and spatial aspects of coexistence over many generations in a multispecies host–parasitoid assemblage.
2. The long-term interaction between the cabbage root fly, Delia radicum (Diptera: Anthomyiidae), and two of its natural enemies, Trybliographa rapae (Hymenoptera: Fitigidae) and Aleochara bilineata (Coleoptera: Staphylinidae), in a cultivated field at Silwood Park over 19 years was explored.
3. Although time series showed that the populations were regulated, the impact of the natural enemies was highly variable. Within-year determinants showed that the spatial response of the specialist parasitoid, T. rapae , was predominantly independent of host density while A. bilineata acted simply as a randomly foraging generalist parasitoid.
4. These findings are compared and contrasted with an earlier investigation of the same system when only the first 9 years of the time series were available. This study demonstrated the potential of long-term field studies for exploring hypotheses on population regulation, persistence, and coexistence.     

Association of viral and plasmid DNA with the nuclear matrix during productive infection

The association of simian virus 40 (SV40) DNA or plasmid DNA in subcellular fractions from either infected or transfected cells was examined. In lytically infected cells, approx. 25% of viral specific DNA during the infection cycle was retained in nuclei after washing with low ionic strength buffer and 1% Triton X-100. Viral replicating DNA found in the nuclear matrix was capable of performing limited DNA synthesis by the endogenous DNA polymerase in vitro. Viral DNA synthesized in vitro hybridized preferentially to SV40 Hind-III B and C fragments which are in proximity to the origin of replication. In plasmid-transfected COS-7 cells (SV40-transformed cells), the amount of plasmid DNA found in the nuclear matrix was related to its replication efficiency in cells. More than 80% of the plasmid DNA was tightly associated with subnuclear structures. Little or no plasmid DNA was found in the cytoplasmic fraction. The results suggest that, in extrachromosomal model systems, the association of DNA with nuclear matrix is important for the regulation of DNA replication.     

Characterization of aminoalkylimidazol-4-one mutagens from liquid-reflux models

A new class of low molecular weight, aminomethylimidazol-4-one (IQ-"like") mutagens have been produced by the reaction of creatinine with the amino acid L-threonine, in liquid-reflux models, mimicking cooking, of diethylene glycol:5% distilled water (2 h at 150 degrees C). Two mutagens, 2-amino-1-methyl-5-propylideneimidazol-4-one (AMPI) and 2-amino-5-ethylidene-1-methylimidazol-4-one (AEMI) were isolated and characterized by UV absorption spectra, mass spectra, and 1H-NMR. The mutagen AEMI was identical to that obtained from the reaction of creatinine with acetaldehyde. These mutagens were positive in all IQ-sensitive Ames tester strains and were not inactivated by acidic nitrosation at pH 1.0. Products displaying mutagenicity were also obtained by refluxing creatinine with other hydroxyamino acids such as L-serine, L-homoserine, and L-4-amino-3-hydroxybutyric acid, and aldehydes such as glyoxal, methylglyoxal, glycolaldehyde, but not formaldehyde. Simple model systems such as creatinine and acetaldehyde may be useful in more clearly defining the exact mechanism of formation of IQ-type mutagens (aminomethylimidazo-quinolines and -quinoxalines) produced during cooking, as well as in screening for potential inhibitors of IQ-type mutagen formation, and elucidating the mechanism of such inhibition.     

A Monte Carlo investigation of homogeneity tests of the odds ratio under various sample size configurations

Epidemiologic data for case-control studies are often summarized into K 2 x 2 tables. Given a fixed number of cases and controls, the degree of sparseness in the data depends on the number of strata, K. The effect of increasing stratification on size and power of seven tests of homogeneity of the odds ratio is studied using Monte Carlo methods. In all the designs considered here, the numbers of cases and controls per stratum are the same. Considering both size and power in non-sparse-data settings, we recommend the Breslow-Day statistic (1980, Statistical Methods in Cancer Research, 1. The Analysis of Case-Control Studies, p. 142; Lyon: International Agency for Research on Cancer) for general use. In sparse-data settings the T4 statistic of Liang and Self (1985, Biometrika 72, 353-358) performs the best when all tables, regardless of sample size, have odds ratios generated from the same distribution. In sparse-data settings characterized by a large table with an odds ratio of 1 and many small tables with odds ratios greater than 1, the T5 statistic of Liang and Self (1985) performs the best. One of the most important results of this study is the generally low power for all homogeneity tests especially when the data are sparse.     

Effect of fatty acid composition of dietary fat on energy balance and expenditure in hamsters

The comparative effects of feeding diets containing corn, olive, coconut, or menhaden fish oil on efficiency of energy deposition and on short term energy expenditure were examined in growing hamsters. Diets comprising oils mixed with laboratory diets at 10% oil w/w were fed ad libitum for 3 weeks. Animals fed laboratory diets were used as controls. Body composition was determined before and after the feeding period using 3H2O distribution space. Oxygen consumption was measured in each animal during the final week. Weight gains of groups fed corn and olive oil diets exceeded those of the group fed laboratory diet alone (p less than 0.05), although metabolizable energy intakes were similar across groups. Corn oil fed animals demonstrated higher carcass energy gains as fat compared with laboratory diet fed or menhaden oil fed groups. This was reflected in an increased fractional deposition of metabolizable energy intake in the group fed corn oil diet compared with the latter two groups. Fecal energy losses were lower in the group fed corn oil diet, and higher in the group fed laboratory diet alone, compared with other groups. Oxygen consumption did not differ between groups. These findings indicate that feeding dietary fish oil, compared with corn oil, favours energy substrate oxidation reducing the fraction of metabolizable energy partitioned for storage.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.