Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Homologous adaptation to oxidative stress induced by the photosensitized Pd-bacteriochlorophyll derivative (WST11) in cultured endothelial cells

Various forms of cellular stress induce adaptive responses through poorly understood mechanisms. In maintaining homeostasis, endothelial cells respond and adapt to changes in oxidative stress that prevail in the circulation. Endothelial cells are also the target of many oxidative stress-based vascular therapies. The objectives of this study were to determine whether endothelial cells adapt to oxidative stress induced upon the photosensitization of WST11 (a water-soluble Pd-bacteriochlorophyll derivative being developed as a photodynamic agent) and to study possible cellular mechanisms involved. The hallmark of WST11-based photodynamic therapy is the in situ generation of cytotoxic reactive oxygen species causing vascular shutdown, hypoxia, and tumor eradication. Here we demonstrated that photodynamic therapy also induces adaptive responses and tolerance following a sublethal preconditioning of endothelial cells with the same (homologous) or different (heterologous) stressor. A link among p38 MAPK activity, expression of hsp70 and hsp27, and homologous adaptation to reactive oxygen species induced by photosensitized WST11 was established. In addition to characterization of some key proteins involved, our observations provide a beneficial new working tool for the studies of mechanisms involved in oxidative stress and adaptation using light-controlled photosensitization.     

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.     

Effect of imidacloprid soil treatments on occurrence of Formosan subterranean termites (Isoptera: Rhinotermitidae) in independent monitors

Periodic sampling of 30 independent monitors, initially active with the Formosan subterranean termite, Coptotermes formosanus Shiraki, was conducted to evaluate the effects of soil treated with imidacloprid on nearby termite activity. Monitors were located adjacent (1-3 m) to the buildings. Soil around and under the buildings was treated with 0.05% imidacloprid. None of the termites collected showed latent mortality attributed to imidacloprid intoxication. Imidacloprid soil treatments did not measurably reduce C. formosanus populations adjacent to the treatments. Imidacloprid does not seem to fit the liquid-bait model.     

Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade

The receptor tyrosine kinase RET functions as the signal transducing receptor for the GDNF (for "glial cell-derived neurotrophic factors") family of ligands. Mutations in the RET gene were implicated in Hirschsprung disease (HSCR), multiple endocrine neoplasia type 2 (MEN 2), and thyroid carcinomas. In this report we demonstrate that the docking protein FRS2 is tyrosine phosphorylated by ligand-stimulated and by constitutively activated oncogenic forms of RET. Complex formation between RET and FRS2 is mediated by binding of the phosphotyrosine-binding domain of FRS2 to pY1062, a residue in RET that also functions as a binding site for Shc. However, overexpression of FRS2 but not Shc potentiates mitogen-activated protein (MAP) kinase activation by RET oncoproteins. We demonstrate that oncogenic RET-PTC proteins are associated with FRS2 constitutively, leading to tyrosine phosphorylation of FRS2, MAP kinase stimulation, and cell proliferation. However, loss-of-function HSCR-associated RET mutants exhibit impaired FRS2 binding and reduced MAP kinase activation. These experiments demonstrate that FRS2 couples both ligand-regulated and oncogenic forms of RET, with the MAP kinase signaling cascade as part of the response of RET under normal biological conditions and pathological conditions, such as MEN 2 and papillary thyroid carcinomas.     

Synthetic peptide approach to the analysis of kinase activities of avian EGF receptor and v-erbB protein

Analysis of the structure and function of a protein such as the epidermal growth factor receptor is facilitated by the use of antibodies directed against discrete portions of the protein. Here, we describe the characterization and use of antibodies directed against synthetic peptides corresponding to specific portions of the epidermal growth factor receptor and/or v-erbB protein. In particular, one useful antiserum has allowed us to compare the protein kinase activities of the epidermal growth factor receptor and the v-erbB proteins and to conclude that the v-erbB protein is a protein-tyrosine specific kinase as is its homologue the avian epidermal growth factor receptor.     

Aberrant DNA Methylation in ES Cells

Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.     

Combined effect of microbial and chemical control agents on subterranean termites

Termite mortality was measured when fungi were combined with bacteria or a chemical termiticide to determine whether a synergistic effect occurred. The fungus Beauveria bassiana was combined with the non-repellant chemical termiticide imidacloprid. Of the three B. bassiana strains tested one, B. bassiana ATCC 90519, was sufficiently pathogenic on its own that the advantage of a supplementary chemical treatment was marginal. The mortality caused by another fungal strain, B. bassiana ATCC 26037, was improved in combination with imidacloprid at both of the tested chemical concentrations over the first 14 days. The remaining fungal strain, B. bassiana ATCC 90518, demonstrated an overall mortality rate in combination with imidacloprid of 82.5%, versus a rate of 65.0% for the fungus alone. The fungus Isaria fumosorosea (Ifr) was combined with the bacterium Bacillus thuringiensis (Bt). On day 5, Ifr, Bt, and the combined treatment at a 10⁶ spores or cells/ml dosage caused 8.8%, 22.5%, and 15.0% mortality, respectively. The Bt and combined mortality rates are not significantly different. Control mortality on day 5 was 5.0%. On day 13 the combined 10⁶ treatment mortality rate was 91.3%, which was significantly higher than all other treatments: control at 17.5%, Ifr at 36.3% and Bt at 35.0%. When Ifr and Bt were applied at a 10⁹ spores or cells/ml dosage, Ifr alone caused a mortality rate of 97.5% as early as day 5. The combination with Bt could not significantly increase the effectiveness of this dosage. These data demonstrate the potential for synergistic effects of fungal and chemical treatment methods, thereby broadening the use of microbial control agents and reducing the quantity of chemical agents necessary to effect control.