Numerical analysis of electrophoretic protein patterns of Providencia rustigianii strains from human diarrhoea and other sources

Twenty strains of Providencia rustigianii (including the type strain of Prov. friedericiana) have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 12 strains (almost exclusively associated with the intestinal tract) from humans, plus eight largely from the intestinal tract of pig, penguin and environmental sources. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 20 Prov. rustigianii strains formed six clusters at the 88% S level. One of these clusters included the type strains of both Prov. friedericiana and Prov. rustigianii, thereby confirming the synonymy of these two species. In the second analysis, the principal protein bands were excluded. At the 86% S level the 20 Prov. rustigianii strains formed a single cluster, whilst a field strain of Morganella morganii and the respective type strains of three other Providencia species remained unclustered. The total protein pattern of the type strain of Prov. alcalifaciens was very similar to that of Prov. rustigianii phenon 3 and the M. morganii field strain, which indicates that careful biochemical characterization may be necessary to ascribe strains to a species before typing by the PAGE technique. Alternatively, a selective analysis of the protein bands may be used to confirm the identity of the strains, as shown in this study.     

Binding of zinc(II) to beta-D-fructose 2,6-bisphosphate

The formation of a complex between Zn(II) and beta-D-fructose 2,6-bisphosphate was shown because the latter compound: activated bis(5'-guanosyl)tetraphosphatase (EC 3.6.1.17) and dinucleoside triphosphatase (EC 3.6.1.29) only to the extent that they could be inhibited by Zn(II); increased the consumption of Zn(II) necessary to titrate to an end point a solution of the metallochromic indicator eriochrome black T; coeluted with Zn(II) in a gel filtration column capable of resolving them if unbound. Neither of those effects was shown by D-fructose 1,6-bisphosphate under the same conditions.     

Ultrastructure and division behaviour of dinoflagellate chromosomes

Chromosomes of Prorocentrum triestinum and P. micans have similar substructural and morphometrical values as revealed by electron microscopy of thin sections. However, differences were found between the species in mean length, volume and numerical density of chromosomes, and the volume of the chromosome complement, the nuclear volume and the chromosome number. When examined by a whole-mount procedure both Prorocentrum species have left-handed screw-like chromosomes which end in differentiated telomeres. The chromosomes divide sequentially from one telomere towards the other, presenting a Y and finally a V configuration. At the region where each chromosome divides nascent sister chromatids are connected by two bridges. Sister chromatids have similar quantitative values when compared with each other and with the still undivided chromosome, which suggests that both replication and division take place as coupled events.Supported by CAICYT, grant 2409/83     

Molecular orbital studies of oxygen activation and mechanisms of cytochromes P-450-mediated oxidative metabolism of xenobiotics

Molecular dimensions and molecular orbital calculations of the electronic structures of 56 substrates, inhibitors and inducers of the cytochromes P-448 and other families of the cytochromes P-450 are reported. Substrates of the cytochromes P-448 are shown to be planar molecules with relatively large values of area/depth², and to have electronic structures with relatively low values for ΔE, the difference in energy between the frontier orbitals (E(LEMO) − E(HOMO)). Substrates of other families of the cytochromes P-450 are globular molecules, with relatively low values of area/depth² and relatively high values of ΔE. Molecular orbital calculations of the active oxygen species, singlet oxygen and superoxy anion, have also been made. Singlet oxygen is a poor electron donor (low values of E(HOMO)) but a good electron acceptor (low values of E(LEMO)), whereas superoxy anion is a good electron donor and a poor electron acceptor. Cytochrome P-448 substrates, which are good electron donors, would preferentially accept singlet oxygen, a good electron acceptor; substrates of the other families of cytochrome P-450, which are less effective electron donors, would preferentially accept superoxy anion, a good electron donor, although substrates of both cytochromes P-448 and other P-450s may accept both species of active oxygen. Together with recent published evidence, these data provide a greater understanding of the mode of activation of oxygen by the various families of the cytochromes P-450, and to the insertion of active oxygen into the substrates. Mechanisms are proposed for the oxygenation of substrates, namely, epoxidation involving singlet oxygen and hydroxylation by superoxy anion. Finally, a detailed explanation of the cytochrome P-450 cycle is discussed, and mechanisms of the different types of oxidative metabolism are presented.     

Organization of the murine Mx gene and characterization of its interferon- and virus-inducible promoter.

Specific resistance of Mx+ mice to influenza virus is due to the interferon (IFN)-induced protein Mx. The Mx gene consists of 14 exons that are spread over at least 55 kilobase pairs of DNA. Surprisingly, the Mx gene promoter is induced as efficiently by Newcastle disease virus as it is by IFN. The 5' boundary of the region required for maximal induction by both IFN and Newcastle disease virus is located about 140 base pairs upstream of the cap site. This region contains five elements of the type GAAANN, which occurs in all IFN- and virus-inducible promoters. The consensus sequence purine-GAAAN(N/-)GAAA(C/G)-pyrimidine is found in all IFN-inducible promoters.     

Loss of urease activity in Helicobacter mustelae

Urease-negative variants of Helicobacter mustelae were isolated after spontaneous loss of activity during sub-culture. The whole-cell protein patterns showed that the loss of urease activity was linked to the absence of two polypeptides of 29·1 and 65·4 kDa. Restriction endonuclease analysis of chromosomal DNA indicated no substantial differences between the urease negative and positive variants. It is likely that a change in the expression of the gene for urease was responsible for these observations.     

Occurrence of dinucleosidetriphosphatase in the cytosol and particulate fractions from rat liver

Dinucleosidetriphosphatase (EC 3.6.1.29) is present in both the 37,000 g rat liver supernatant and precipitate (50 mU/g each fraction). These two activities show matching molecular weights, isoelectric points, substrate specificities, Km values, bivalent cation requirements and inhibition by zinc (II). The particulate triphosphatase and a residual dinucleosidetetraphosphatase (EC 3.6.1.17) are solubilized by freeze-thawing or by Triton X-100. Detergent treatment also extracts an unspecific phosphodiesterase I activity (EC 3.1.4.1) which also splits dinucleoside polyphosphates. The above findings suggest the occurrence of cytosolic and particulate degradative pathways for dinucleoside polyphosphates.     

Dinucleosidetriphosphatase from rat brain

Rat brain P1,P3-bis(5'-adenosyl)-triphosphate adenylohydrolase (dinucleosidetriphosphatase, EC 3.6.1.29) was purified 1000-fold. The enzyme hydrolyzed diadenosine and diguanosine triphosphates (Km values 14 and 40 microM, and relative V 100 and 40, respectively) to the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was hydrolyzed at a residual rate. Diadenosine di and tetraphosphates, NAD+, and artificial phosphodiesterase substrates were not hydrolyzed. Bivalent cations [Mg(II), Mn(II) or Ca(II)] were required for activity, but Zn(II) was a competitive inhibitor (Ki = 5 microM). The optimum pH value was about 7.5. A molecular mass of 34 kdalton (gel filtration) and an isoelectric point of 5.5 (chromatofocusing) were found.     

The genetics of Drosophila subobscura populations. 3. Inversion polymorphism and climatic factors

Summary The natural population of Drosophila subobscura of Mt. Parnes has been followed for two successive years and the frequency of the gene arrangements determined. Some changes occured in the first year but do not explain the existence of the geographical clines for these frequencies. Within the second year no changes were observed. For chromosomes J and U there is evidence for a greater fitness of the heterozygotes.Experiments on temperature effect on viability of adult male flies, on their temporal sterility, on the viability of flies during all their biological cycle and experiments on the effect of dryness on the viability of adult male flies did not show an important change of gene arrangement frequencies in the resistant individuals. Nor did diapausing females have very different gene arrangements, except for chromosome O. Cage experiments with temperature as a selective agent did not elucidate the action of this factor. It is concluded that temperature or dryness do not affect very much the polymorphic system, nor explain the existence of these clines.This polymorphic system is much more stable in D. subobscura than in D. pseudoobscura, its nearctic relative. It is also richer. It is concluded that it is historically older and helps the populations to cope with many environmental changes.     

Numerical analysis of electrophoretic protein patterns of 'Achromobacter'group B, E and F strains from human blood

Thirty-two clinical strains representing ' Achromobacter 'groups B, E and F were characterized by one-dimensional SDS-PAGE of cellular proteins. All the strains were isolated from blood samples from hospital patients in the United Kingdom. The protein patterns, which contained 40 to 45 discrete bands, were highly reproducible and were used as the basis for a numerical analysis which included all the protein bands. The 32 ' Achromobacter ' strains formed two clusters at the 77% S level. The first, phenon 1, included the 28 group B and the two group E strains and the second, phenon 2, contained the two strains of group F. The strains in each phenon were characterized by a clearly distinct pattern of protein bands. Phenon 1 could be further divided at the 87% S level into three subphenons which correlated with differences in the principal bands found between 40.0 and 42.5 kD. Strains of group E clustered with group B strains from which they could not be distinguished by protein patterns. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides a useful method for the classification of this group of bacteria. Reference strains of each of the PAGE types identified are available from NCTC for inclusion in future studies.