Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

Topography of the subunits of Micrococcus lysodeikticus F1-ATPase

Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [¹²⁵I] applied to membrane-bound or soluble pure F₁-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F₁-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F₀ component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry _{3 3 2 2} (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.     

Lipid-bound sugars in malignant human breast tumors

Microsomal preparations from malignant human breast tumors catalyzed the transfer of mannose and glucose from GDP-[14C]-Man and UDP-[14C]-Glc into lipid-linked sugars and glycoprotein-like substances. As judged by several criteria the obtained lipid-linked monosaccharides behaved as dolichyl phosphate mannose and dolichyl phosphate glucose whereas lipid-linked oligosaccharides behaved as polyprenyl diphosphate derivatives. The optimum conditions for mannosyl- and glucosyl-transfer reactions and the effect of dolichyl phosphate, detergent and EDTA on incubation mixture were described.     

Ultrastructure of Dimorphic Transformation in Paracoccidioides brasiliensis

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.     

Development of Competence in Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

Tryptophan- and thymine-requiring cells of Bacillus subtilis, emerging from an amino acid starvation treatment which causes arrest of the chromosomes at the terminus, were not transformable. During subsequent incubation in a thymineless medium supplemented with amino acids, the cultures developed competence while retaining chromosome arrest. The competent subpopulation apparently shares the synchronous chromosome arrest of the bulk population. This was shown by different methods. The principal method was marker frequency analysis of the deoxyribonucleic acid extracted from a population enriched for competent cells by a column-chromatographic method. It is concluded that development of the competent state can occur in nondividing cells, and that the presence of a replication fork actively engaged in synthesis of deoxyribonucleic acid is not required for the development of this state.     

Metabolic Changes in Nicotinamide Adenine Dinucleotide in Response to Anthrax Toxin

Bacillus anthracis produces a toxin both in vitro and in vivo which, when injected intravenously into rats, brings about the death of the animals accompanied by gross pulmonary edema. Lung tissue removed prior to death showed, in vitro, a 30% reduction in overall oxidative metabolism (Q(o2)), whereas the nicotinamide adenine dinucleotide (NAD)-independent succinic dehydrogenase remained unaffected. The NAD concentration in the lungs of injected animals was reduced by 50%. Upon addition of NAD, the Q(o2) of lung tissue from injected animals rose to control values. At 45 min after toxin injection, the serum lactate concentration began to rise, showing about a 3.5-fold increase over controls after 75 min. No changes occurred in the pyruvate concentration. These changes may be explained by increased use of the pyruvate for glycolytic energy production with further loss of NAD. Additional experiments with liver, spleen, kidney, and brain tissues showed that the toxin-induced reduction of Q(o2) is an effect specific for lung tissue. Brain tissue showed a significant increase in oxidative metabolism upon the addition of the toxin, whereas the other tissues remained unaffected. It is suggested that a principal effect of the toxin is to inhibit, in lung tissue, the regeneration of NAD in the respiratory chain.     

Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.