Esterification of arylhydroxylamines: Evidence for a specific gene product in mutagenesis

Characterization of a $\text{Salmonella}\text{typhmurium}$ mutant strain (TA98/1,8-DNP₆) resistant to the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitroarenes) revealed that it was also non-responsive to the mutagenic action of nitroso- and N-hydroxylaminoarenes. The mutant strain was fully sensitive to the mutagenic action of the corresponding hydroxamic acid ester. These results suggest that TA98/1,8-DNP₆ is deficient in a specific esterifying enzyme and that esterification of the penultimate mutagenic metabolites of nitro- and aminoarenes ($\text{e.g.}$, arylhydroxylamines) to form potent electrophiles is controlled by a specific gene.     

The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture.

Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0.5 M-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Critical fields in cell fusion and dielectrophoresis

It is increasingly recognized that exposure to electrical fields can reversibly increase the electrical permeability and conductivity of the cellular membrane. It is therefore worthwhile to determine under what circumstances the critical fields capable of affecting the cellular membrane can be reached. We have evaluated the field intensities E and the gradients of E² in the neighborhood of the more common field electrode shapes, the sphere-sphere and cylinder-cylinder electrodes, and put these in terms of the values useful for the practical electro-fusion and dielectrophoresis (DEP) of cells. The calculations were performed using the charge-image technique. From this it was observed that gross errors (underestimations of up to 1000-fold) of the field would be made if neglect of the electrode polarization were made as in simple electrostatic calculation. The dielectric forces (in terms of (E)²) show interesting spatial character.The electrical breakdown of cellular membranes affects the exchange of information and materials between the cell and its environment, and can be used to fuse cells of the same type or of differing types. It is moreover of importance in handling cells during DEP while obtaining their spectral characteristics or in cell-sorting. It is expected that the results here will aid in the more proper use of electrical fields for such ends.     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.     

Chronic Hepatitis Associated with Drug Abuse: Significance of Hepatitis B Virus

One hundred and seventy-seven former heroin addicts, consisting of 85 who were newly admitted to a methadone maintenance program and 92 who had received methadone for a mean period of 30 months, were prospectively studied for up to 2 years in order to determine: (1) the effect of heroin withdrawal on the hepatic abnormalities, and (2) the incidence of HBsAg, anti-HBs, and anti-HCc as indices of the frequency of hepatitis B virus infection. Our study indicates that (1) hepatic abnormalities persist when heroin is discontinued and are not temporally related to drug and/or needle usage, and (2) that 71% of subjects had either HBsAg or anti-HBs; anti-HBc was tested for in 16 patients and was present in 100%, although 9 of the 16 were both HBsAg- and anti-HBs-negative. This study suggests that hepatitis B is largely responsible for the liver dysfunction. It is proposed that an abnormality in immune function, induced by heroin, is responsible for the high incidence of chronic hepatitis. Attention is drawn to the similarity between former drug addicts and hemophiliacs, since both develop chronic hepatitis in spite of anti-HBs in the serum.     

Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose.

Over a wide range of growth rates, two strains of Escherichia coli growing aerobically in continuous culture under glucose limitation utilized glucose at rates identical with those at which cells harvested from the chemostats transported [14C]glucose.