Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

The composition of the murein of Escherichia coli

Escherichia coli murein, the polymer from which the shape-maintaining structure of the cell envelope is made, shows unexpected complexity. The separation of murein building blocks with high performance liquid chromatography reveals about 80 different types of muropeptides. Their behavior in high performance liquid chromatography and their chemical structure are described. The complexity of E. coli murein is due to the free combination of seven different types of side chains (L-Ala-D-Glu-R with R = -OH, -m-A2pm, -m-A2pm-D-Ala, -m-A2 pm-Gly, -m-A2pm-D-Ala-D-Ala, -m-A2pm-D-Ala-Gly, -m-A2pm-Lys-Arg) with two types of cross-bridges (D-Ala-m-A2pm, -m-A2pm-m-A2pm). The novel type of cross-bridge, A2pm-A2pm, contains an L,D-peptide bond, as shown by Edman degradation and chemical analysis of the reaction products. The A2pm-A2pm cross-bridge is assumed to play a role in the adaptation of the cross-linkage of murein to different growth conditions of the cell. The structural data of E. coli murein agree best with a model of a thin, however multilayered, murein sacculus.     

In vitro fusion of newt macrophages

Spontaneous formation of multinucleate giant cells is often observed in in vitro cultures of peritoneal adherent macrophages from the newts, Notophthalmus viridescens and Taricha granulosa (urodele amphibians). The frequency of such giant cells in these cultures is increased by the addition of phorbol myristic acetate at the initiation of the cultures. This high frequency of multinucleate cells permitted us to evaluate whether multinucleate giant cells arise by cell fusion and/or by repeated nuclear division without cytokinesis. Cell fusion is readily detectable by scanning electron microscopy. To determine whether nuclear division without cytokinesis also occurs, some cultures were treated with colchicine to arrest mitotic figures; others were pulsed with tritiated thymidine to detect DNA synthesis. Mitotic figures were not seen in acridine orange-stained samples. In monolayers that were processed for autoradiography, only a few nuclei were marked with tritium. These observations suggest that nuclear division does not contribute significantly, if at all, to the formation of multinucleate giant cells from cultured newt peritoneal macrophages.     

Quantitation of the guanine nucleotide binding regulatory protein Gs in S49 cell membranes using antipeptide antibodies to alpha s

Polyclonal antibodies reactive against the guanine nucleotide binding stimulatory protein, Gs, were affinity-purified from two rabbits immunized with a synthetic peptide corresponding to amino acids 28-42 in the alpha-subunit, alpha s. On immunoblots, these antibodies recognized alpha s, but not alpha-subunits from two other guanine nucleotide binding regulatory proteins, Gi and Go. A competitive enzyme-linked immunosorbent assay was developed in which inhibition of antibody binding to peptide-coated microtiter plates was used to quantitate purified Gs or Gs in cholate extracts of cell membranes. Plasma membranes derived from wild type S49 lymphoma cells contained 18.9 +/- 2.3 pmol/mg of membrane protein of alpha s. The same membranes bound 169 +/- 12 fmol/mg of protein of [125I]iodocyanopindolol to beta-adrenergic receptors, indicating that the amount of Gs is far in excess of the amount of beta-adrenergic receptors. Thus, even if every beta-adrenergic receptor molecule were to activate 10 Gs molecules, in order for Gs to be limiting for the receptors to reach their high affinity state, it is likely that compartmentation exists for target cell membrane receptors and Gs. Moreover, a comparison of beta-adrenergic receptor number and Gs levels in several different S49 lymphoma cell mutants having lesions in receptors or Gs argues against a coordinate regulation of beta-adrenergic receptors and Gs.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase

Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.     

Synthetic oligodeoxynucleotide probes for the rapid detection of bacteria associated with human periodontitis

Analysis of the subgingival microflora has recently implicated Actinobacillus (Haemophilus) actinomycetemcomitans and several black Bacteroides species in the aetiology of juvenile, adult and rapidly progressing periodontitis. Rapid bacteriological diagnosis has been hampered by the slow growth and fastidious nature of these bacteria. To construct diagnostic probes, dideoxy sequencing of the 16S rRNA molecules from A. (H.) actinomycetemcomitans, Haemophilus aphrophilus, Bacteroides gingivalis, Bacteroides intermedius subgroup II, Bacteroides asaccharolyticus and several closely related species was performed. Next, oligodeoxynucleotides, complementary to defined regions of the 16S rRNA exhibiting considerable evolutionary divergence, were synthesized for use as molecular probes. In a dot-blot hybridization assay, all strains from each of the species for which probes were constructed were correctly identified, with a detection limit of less than 5 x 10(3) organisms. No cross-hybridization to closely related species (except for H. aphrophilus and Haemophilus paraphrophilus) or contaminating bacteria was observed. Using a modified DNA/RNA hybridization technique, the detection could be performed in less than 12 h, as compared to 2-3 weeks using conventional bacteriological procedures.     

The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface Ig

EBV-transformed B lymphocyte cell lines (EBV-BLCL) produce superoxide after stimulation with phorbol ester, a capacity unique among nonmyeloid cells. The superoxide producing system of EBV-BLCL (B cell oxidase) was compared with the phagocytic NADPH-oxidase and the relationship of the capacity to produce superoxide to the presence of the EBV-genome was analyzed. The two EBV-transformed B cell lines F1 and HELL generated superoxide in response to PMA (2.3 nmol/10(6) F1 cells x 1 h and 6.27 nmol/10(6) HELL cells x 1 h with 1 microgram/ml of PMA), whereas no superoxide release was detected with the EBV-positive Burkitt lymphoma line WIL-2 and the EBV-negative plasmocytoma line U-266. Also, F1 and HELL showed lucigenin-dependent chemiluminescence (CL) after PMA-treatment, whereas no CL responses were detected from WIL-2 or U-266. Further, F1 and HELL cells contained a low potential cytochrome b-245 (10.9 and 61.0 pmol/mg protein, respectively) and also a 45 kDa diphenylene-iodonium (DPI)-binding peptide, both components of the phagocytic NADPH-oxidase. In contrast, neither the cytochrome b-245 nor the 45 kDa DPI-binding peptide were detected in WIL-2 and U-266. In addition, DPI inhibited O2- production by PMA-stimulated EBV-BLCL and polymorphonuclear granulocytes. Further, F1 line cells showed superoxide dismutase-inhibitable lucigenin-dependent CL when triggered by protein A-bearing staphylococci (Cowan strain I) or by a mAb directed against human IgG in the presence of solid-phase goat anti-mouse-Ig antibody. From a panel of eight EBV-BLCL, only five responded with CL when exposed to protein A-bearing staphylococci, whereas all showed CL when treated with phorbol ester. Inasmuch as all eight EBV-BLCL possessed surface Ig and a "functional" oxidase, their differential response to cross-linking of surface Ig may be determined by differences in signal transduction. Superoxide production by EBV-BLCL appears thus related to expression of an electron transport chain structurally homologous, if not identical, with the "phagocytic" NADPH-oxidase. Apparently, the presence of EBV-genome in B cell lines does not per se lead to expression of this oxidase. This suggests that nontransformed B cells may, at a certain differentiation stage, also express a superoxide-generating chain. From the finding of stimulation of superoxide production of EBV-BLCL via surface Ig it appears possible that also Ag may be able to trigger such B cells to production of superoxide which might have an important role in the physiology of B cells.