An ecological characterization of Coastal Maine (north and east of Cape Elizabeth) / Stewart I. Fefer and Patricia S. Schettig: principal investigators.

1

An ecological characterization of Coastal Maine (north and east of Cape Elizabeth) / Stewart I. Fefer and Patricia S. Schettig: principal investigators.

5

An ecological characterization of Coastal Maine (north and east of Cape Elizabeth) / Stewart I. Fefer and Patricia S. Schettig: principal investigators.

2

An ecological characterization of Coastal Maine (north and east of Cape Elizabeth) / Stewart I. Fefer and Patricia S. Schettig: principal investigators.

3

An ecological characterization of Coastal Maine (north and east of Cape Elizabeth) / Stewart I. Fefer and Patricia S. Schettig: principal investigators.

4

Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing Tetrahymena thermophila cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in Tetrahymena, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli. Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a β-lyase activity that nicks DNA 3′ to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7,8-dihydroxy-8-oxo-2′-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix–hairpin–helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

Kinetic characterization of brush border myosin-I ATPase

Brush border myosin-I (BBM-I) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells. We used stopped-flow kinetic analysis to measure the rate and equilibrium constants for several steps in the BBM-I ATPase cycle. We determined the rates for ATP binding to BBM-I and brush border actomyosin-I (actoBBM-I), the rate of actoBBM-I dissociation by ATP, and the rates for the steps in ADP dissociation from actoBBM-I. The rate and equilibrium constants for several of the steps in the actoBBM-I ATPase are significantly different from those of other members of the myosin superfamily. Most notably, dissociation of the actoBBM-I complex by ATP and release of ADP from actoBBM-I are both very slow. The slow rates of these steps may play a role in lengthening the time spent in force-generating states and in limiting the maximal rate of BBM-I motility. In addition, release of ADP from the actoBBM-I complex occurs in at least two steps. This study provides evidence for a member of the myosin superfamily with markedly divergent kinetic behavior.

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of α-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.