Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Production of monoclonal antibodies to lactate dehydrogenase (LDH) isoenzymes for immunohistochemical study on fixed tissue section

Summary Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.     

The adaptation of enzymes to temperature: catalytic characterization of glucosephosphate isomerase homologues isolated from Mytilus edulis and Isognomon alatus, bivalve molluscs inhabiting different thermal environments

Homologues of glucosephosphate isomerase (GPI, EC 5.3.1.9) were purified to homogeneity and kinetically characterized from Mytilus edulis and Isognomon alatus, two bivalve molluscs experiencing contrasting thermal environments. The enzyme isolated from I. alatus functions at warmer temperatures (25-35 C) than GPI from M. edulis, a species that inhabits colder marine littoral habitats (5-20 C). The former exhibits apparent first-order (with respect to substrate) catalytic rate constants (Vmax/KM) in vitro that become progressively greater than the mussel enzyme as the assay temperature is raised. Apparent zero-order catalytic rate constants (Vmax) are relatively less differentiated. Catalytic efficiency, defined as the rate at which a catalytic event occurs in either reaction direction for reference standard states (substrate concentrations), is greater for the enzyme from the tropical species (I. alatus) at all realistic combinations of temperature and substrate concentration except for the lowest temperatures and highest substrate concentrations, where the GPI from the boreal/temperate M. edulis is more efficient. This pattern of catalytic divergence appears to be due primarily to differentiation in Vmax/KM. These results and other published data are reviewed and shown to be inconsistent with claims that adaptation of enzymes to higher cell temperatures requires a loss in catalytic efficiency.      

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

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The one-electron reduction potentials of NAD

The one-electron reduction potential (E¹₇) of NAD⁺ has been determined by pulse radiolysis to be ?0.94 V. E²₇ (E¹₇ for the free radical, NAD^.) is +0.30 V. E¹₇ for 1-methylisonicotinamide is ?0.77 V.     

Lactase polymorphism in adult British natives: estimating allele frequencies by enzyme assays in autopsy samples.

In an attempt to estimate allele frequencies of lactase persistence in adult British natives, sucrase was assayed simultaneously with lactase under conditions that gave optimal activities for both enzymes. A trimodal distribution in the ratios of enzyme activities was demonstrated. Circumstantial evidence and statistical analyses suggest that the trimodal distribution is due to the different levels of lactase activity in the three genotypes--homozygous persistent, heterozygous, and homozygous nonpersistent, and that it is possible to correct for "nongenetic" variation by using sucrase as an internal standard. The allele frequency for lactase persistence was estimated to be .747. The implications of our findings on the genetic mechanisms involved in lactase persistence are discussed.     

Human Mucin Genes Assigned to 11p15.5: Identification and Organization of a Cluster of Genes

Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC,andMUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster betweenHRASandINS,and more detailed analysis of recombinant breakpoints revealed thatMUC6is telomeric toMUC2.Using these recombinantsD11S150was mapped close toMUC2.Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of theMUCcluster and to integrate the physical and genetical maps. The gene order was determined to beHRAS–MUC6–MUC2–MUC5AC–MUC5B–IGF2.TheMUCgenes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of theMUCgenes on the map corresponds to the relative order of their expression along the anterior–posterior axis of the body, suggesting a possible functional significance to the gene order.     

Comparative cytotoxicity of fumonisin B1 in two cell lines derived from normal human bronchial epithelial cells using four distinct bioassay techniques

This study focuses on the cytotoxic effects of fumonisin B₁ (FB₁) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two related cell lines, the clonogenic assay, measuring cell survival and proliferation, indicated that FB₁ had a more toxic effect on the nontransfected cells. This kind ofin vitro approach using cells which retain many characteristics of normal cell growth and differentiation can go some way to developing evaluation models for food safety in the case of mycotoxin contamination without resorting totally to whole animal testing. Nevertheless, one or two cytotoxicity tests may be inadequate for a complete appraisal of toxic potential: rather, as wide a range of methodologies as feasible should be employed initially before meaningful conclusions may be drawn.