sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence

The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. beta-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.     

Neuronal and Nonneuronal Contributions to Renal Catecholamine Content in the Dog

Endogenous noradrenaline and 3,4-dihydroxyphenylethylamine (dopamine) levels were measured in different zones of the dog kidney following chronic unilateral renal denervation. In outer and inner renal cortex, and in outer medulla, greater than 95% of the tissue content of both catecholamines was contributed by renal nerves, whereas in inner medulla only nonneuronal catecholamines were found. The amounts of neuronal dopamine present in outer renal cortex were greater than would be expected for a population of solely noradrenergic nerves.     

Effect of 9.6-GHz pulsed microwaves on the orb web spinning ability of the cross spider (Araneus diadematus)

Eight cross spiders (Araneus diadematus) were exposed overnight (16 h) during web-building activity to pulsed 9.6-GHz microwaves at average power densities of 10, 1, and 0.1 mW/cm2 (estimated SARs 40, 4, and 0.4 mW/g). Under these conditions, 9.6-GHz pulsed microwaves did not affect the web-spinning ability of the cross spider.     

Abelson transformed fibroblasts lacking the EGF receptor are not tumourigenic in nude mice.

Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor alpha TGF. alpha TGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by alpha TGF as opposed to the v-abl-encoded protein-tyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild-type fibroblast lines. However, in contrast to the wild-type lines, v-abl-transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for alpha TGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas.     

Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus.

A human cytomegalovirus (HCMV) glycoprotein gene with homology to glycoprotein B (gB) of herpes simplex virus and Epstein-Barr virus and gpII of varicella zoster virus has been identified by nucleotide sequencing. The gene has been expressed in recombinant vaccinia virus and the gene product recognized by monoclonal antibodies and human immune sera. Rabbits immunized with the recombinant vaccinia virus produced antibodies that immunoprecipitate gB from HCMV-infected cells and neutralize HCMV infectivity in vitro. These data demonstrate a role for this protein in future HCMV vaccines.     

Further Verification of the Isotope Dilution Approach for Estimating the Degree of Participation of [3H]thymidine in DNA Synthesis in Studies of Aquatic Bacterial Production

The optimal concentration of [³H]thymidine (i.e., the maximal degree of participation in DNA synthesis) as determined by adding increasing amounts of labeled thymidine at the same specific activity was similar to the concentration of thymidine inhibiting the de novo pathway as determined by isotope dilution plots. These experiments provide further verification of the isotope dilution approach for determining the degree of participation of [³H]thymidine in DNA synthesis.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Atlas of foliar surface features in woody plants,IX. Betulaceae of eastern United States

Scanning electron microscopy was used to examine foliar surface features such as cuticular patterns, epicuticular wax, and trichome types in species ofAlnus, Betula, Carpinus, Corylus, andOstrya. Six trichome types are recognized in this survey, four non-glandular (acicular, filiform, aduncate, subulate) and two glandular (stipitate gland and peltate scale). The distribution of these trichomes among the genera supports the recognition of two tribes, Betuleae (Alnus andBetula) and Coryleae (Carpinus, Corylus, andOstrya). Trichome morphology can be an important supportive taxonomic character in determining evolutionary relationships.     

Analysis of the length of S-phase required to show sister chromatid differential staining

In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only approximately 50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

Demonstration of the existence, and partial characterization, of a cell-surface beta-hexosaminidase from rat splenocytes

Rat splenocytes are shown to exhibit cell-surface located beta-N-acetylglucosaminidase and beta-galactosidase activities. Preincubation experiments, solubilization experiments and chemical cross-linking experiments show that these enzymatic activities are indeed cell-surface localized. The solubilization and partial purification of the beta-N-acetylglucosaminidase activity is reported. Kinetic studies of the partially purified material with a variety of competitive inhibitors at several pH values suggest that at physiological pH the cell surface beta-N-acetylglucosaminidase may function as a carbohydrate binding protein rather than as a glycosidase.