Inhibition of leukocyte adhesion by the in vivo and in vitro administration of cannabinoids

Cannabinoids have been shown to affect various aspects of arachidonic acid metabolism both in vivo and in vitro. Eicosanoid metabolites of arachidonate and related octadecanoate are believed to be involved in cell adhesion processes as agonists in some instances and as antagonists in other cases. This report shows data in which cannabinoids exhibit marked inhibitory effects on the adhesion of mouse peritoneal cells to polystyrene culture dishes. The effects could be seen by in vivo administration of the drugs as well as by direct exposure of the cells in vitro. The data suggest that this inhibition of adhesion is mediated by one or more products generated by stimulation of a lipoxygenase pathway.     

Hepatocidol toxicity of Listeria species

A novel rat hepatocidal test, based on morphological changes in monolayer culture and the percentage of lactate dehydrogenase (LDH) released into the medium after exposure to culture filtrates of Listeria spp. was used to determine listerial toxicity and pathogenicity. Primary cultures of rat hepatocytes exposed to brain heart infusion (BHI) culture filtrates from ATCC strains of Listeria monocytogenes and L. ivanovii, released 91-92% and 95% of LDH after 3 h and 18.5 h, respectively. Cultured monolayers changed from normal hepatocytes into nonviable round forms. Brain heart infusion broth and BHI culture filtrates of other Listeria spp. were nontoxic to hepatocytes. The rat hepatocidal test is a quantitative and rapid system for studying listerial toxicity and pathogenicity.     

Proposed solution structure of endothelin

A model is proposed for the 3-dimensional structure of endothelin, a potent vasoconstrictor and pressor peptide from vascular endothelium. The model is derived through protein structure prediction and circular dichroism studies, and is based on the atomic coordinates for the bee-venom peptide apamin. The model derived shows the same turn-helix motif as observed for apamin and mast-cell degranulating peptide. On the basis of this model we suggest possible strategies for endothelin antagonist design, and note that this motif may be common in a number of peptides acting on channel proteins.     

The formation of a test system for assessing the safety of different types of pertussis preparations: their cytotoxic action on mouse thymocytes in vitro

The test for the evaluation of the toxicity of different types of pertussis preparations as manifested by their in vitro influence on mouse thymic cells (T test) has been finally worked out. The use of the T test has made it possible to reveal the nonstandard character of the production lots of adsorbed diphtheria-pertussis-tetanus vaccines, both whole-cell vaccine and Japanese acellular vaccine. The degree of the in vitro damaging action of pertussis preparations on mouse thymic cells greatly depends on the residual content of Bordetella pertussis nontoxoidized toxin which, in contrast to B. pertussis lipopolysaccharide and filamentous hemagglutinin, produces pronounced cytotoxic action on mouse thymic cells.     

Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor

The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.     

High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. Revised subunit composition

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.