Molecular characterisation of the Stc mutation of Escherichia coli K-12

The previously described Stc ^- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF⁻ to OmpF⁺. Restriction mapping of chromosomal DNA from Stc ⁺ and Stc⁻ strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc⁻ phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc⁻ phenotype involves increase in ompF expression.     

A new strategy to create ordered deletions for rapid nucleotide sequencing

A method is described for generating ordered deletions using previously published techniques but a new strategy. This method is simpler than the published ones and has many advantages. Target DNA is cloned in both orientations into one of the unique restriction enzyme sites adjacent to the complementary region of the commercially available primers in bacteriophage M13. Ordered unidirectional deletions are created using BAL 31 nuclease and religating into M13 vector DNA without the need of purifying BAL 31-digested DNA from a gel.     

Distribution of stresses in the left ventricular wall of the intact heart

The left ventricle is modelled as a prolate spheroid of viscoelastic material with an aim to demonstrate the qualitative effects of anistropy and nonhomogeneity in the calculation of intact ventricular wall stresses. The pericardial pressure is accounted for in the analysis and an attempt is made to examine to what extent this influences the ventricular stresses. Numerical results are also obtained by computing the analytical expressions derived through the analysis.     

Analysis of epidermal proteins for DNA-binding activity

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.     

In vivo spin trapping of free radicals generated in brain, spleen, and liver during gamma radiation of mice

Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur.     

Magnetic resonance studies of fredericamycin A: evidence for O2-dependent free-radical formation

Fredericamycin A, a newly described potent antitumor antibiotic, exhibits unusual spectroscopic and physical properties. The drug shows a striking color change from red to blue on exposure to O2, with the appearance of an optical absorption band at 675 nm; on addition of acid these changes are readily reversed. 1H and 13C NMR spectra of fredericamycin A show that the resonances from the quininoid half of the molecule disappear after exposure to O2 but reappear on acidification in parallel with the observed optical spectral shift. These unusual NMR data are explained by electron spin resonance studies which demonstrate that fredericamycin A spontaneously forms an oxidized free radical with electron transfer to O2. The observed hyperfine structure of this radical is consistent with one-electron oxidation of the quininoid group. After fredericamycin A is exposed to O2, an EPR signal is observed with axial symmetry with temperature and power saturation behavior suggestive of .O2-. Spin-trapping EPR studies demonstrate that the drug reduces O2 to .O2- and H2O2 to .OH. This spontaneous mechanism of O2 reduction with the generation of oxidized drug free radicals and reduced oxygen free radicals is unprecedented among anticancer drugs, suggesting that fredericamycin A could be the forerunner of a new class of anticancer drug.     

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be “linked” together on multiple resistance plasmids. For Cd²⁺, AsO₂ ^?, AsO₄ ^3?, Hg²⁺, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. ThecadA ATPase ofS.aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd²⁺ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K⁺ ATPases ofEscherichia coli andStreptococcus faecalis, the H⁺ ATPases of yeast andNeurospora, the Na⁺/K⁺ ATPases of vertebrate animals, and the Ca²⁺ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in themer system include a DNA-binding regulatory protein from themerR gene and a Hg²⁺ transport system consisting of a periplasmic Hg²⁺-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.     

Mixed function oxidases in response to quality and quantity of dietary protein

Feeding of rice diet reduced the food consumption and growth of rats. Hepatic Cytochrome P-450, NADPH Cytochrome c reductase and the activity of cytochrome P-450 dependent enzymes (Aniline hydroxylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase) were also decreased by feeding rice diet. Supplementation of lysine and threonine to rice diet improved the activity of these enzymes. NADPH regeneration and microsomal phosphatidylcholine were reduced by feeding rice diet. The phenobarbitone induced sleeping time was decreased by supplementing rice diet with lysine and threonine. The effect of protein is probably partly attributed to changes in membrane phosphatidylcholine content and NADPH regeneration rate.     

Neural differentiation of melanocytes in vitiliginous skin

Biopsies from 74 cases of vitiligo were examined to study the reactions of marginal melanocytes. In 35 cases, the melanocytes at the edge of the patch appeared highly dendritic and large with marked arborization of the dendrites between the surrounding epidermal cells. These cells showed low pigmentation but a high enzyme activity which extended along the dendrites. The enzymes include tyrosinase/dopa oxidase, dopamine oxidase and noradrenaline activity, indicating that these cells have a biphasic melanogenic/adrenergic differentiation. This similarity between the dendritic melanocytes and the amelanotic melanoma cell line HT-18 is of interest.