Synthesis and biological activity of (D)Ala2, Leu5-enkephalins containing hydrophilic or hydrophobic moieties

The synthesis in solution of some modified (D)Ala²,Leu⁵-enkephalins has been carried out. The lipophilic properties of the parent compound have been modified by amidation of the carboxyl function with alkylamines of increasing hydrophilicity to increase permeability of the blood-brain barrier. Attempts to reduce enzymatic degradation have been carried out either by reductive glucosamination or by amidation of the carboxyl function with 2-amino-2-deoxy-β-D-glucopyranose. Esterification of the carboxyl function of (D)Ala²,Leu⁵-enkephalin with polyethylenglycole 1000 has also been carried out. The effects induced by these modifications have been evaluated by in vitro and in vivo tests.     

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×10⁸ N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.     

Effect of oxygen pressure on glycogen synthesis by rat-liver slices

1. Glycogen synthesized by rat-liver slices 0.5mm. thick incubated at 1atm. oxygen pressure in Hastings medium with glucose was localized in the cells of the periphery of the slice. Cells of the interior of this slice do not synthesize glycogen. 2. Inner cells of thin slices (about 0.3mm. thick) can synthesize glycogen when such slices are incubated under the same conditions, but oxygen pressures higher than 1atm. are required if inner cells of slices 0.5mm. or more thick are to be able to synthesize glycogen. 3. Localization of newly synthesized glycogen in rat-liver slices incubated in Hastings medium with glucose does not depend on glucose concentration. 4. Calculation of the minimum oxygen pressure required to synthesize glycogen gives values between 0.09 and 0.17atm. 5. The advantages of high oxygen pressures for the study of the synthesis of glycogen and other compounds that require ATP are discussed.     

Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase.

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.     

Presence of terminal N-acetylgalactosamine residues in subregions of the endoplasmic reticulum is influenced by cell differentiation in culture.

Using Helix pomatia lectin as a specific probe for terminal, nonreducing N-acetylgalactosamine residues, glycoprotein precursors bearing newly initiated O-linked oligosaccharides have been localized in the lumen of the endoplasmic reticulum and cis-Golgi cisternae. This pattern contrasts with the detection of the terminal disaccharide galactose beta-1,3-N-acetylgalactosamine by Arachis hypogaea lectin in middle and trans-Golgi compartments, which are considered elongation sites for O-glycosylation. Distribution of H. pomatia ligands in the endoplasmic reticulum is confined to specialized regions or subcompartments in both human colonic adenocarcinoma cells and cultured chicken chondrocytes. Since in cartilage, chondrocytes contain H. pomatia-binding sites exclusively concentrated in cis-Golgi cisternae, primary cultures of this cell type have been used to study those conditions that promote initiation of O-glycosylation in the endoplasmic reticulum. A correlation has been found between the age of the culture and the extent of reactivity of the endoplasmic reticulum with either H. pomatia lectin or antibody against the sequence GalNAc alpha-serine/threonine (Tn antigen). Cells showing an extensive reaction are not hindered in their secretory activity and still maintain the chondrocyte phenotype. Taken together the results suggest that the intracellular distribution of the glycosylation enzymes is not only cell type-specific as previously shown (Roth, J., Taatjes, D. J., Weinstein, J., Paulson, J. C., Greenwell, P., and Watkins, W. M. (1986) J. Biol. Chem. 261, 14307-14312) but it might also vary depending on the stage of cell differentiation.     

Complexes of bivalent cations with neryl and geranyl pyrophosphate: Their role in terpene biosynthesis

Kinetic analysis of the nonenzymic solvolysis of neryl and geranyl pyrophosphate (NPP and GPP, respectively) showed that the dissociation constants of the bis-metallic complexes with Mg²⁺ and Mn²⁺ were larger for NPP than for GPP by approximately one order of magnitude. Rate constants for reaction of the bis-metallic complexes were larger for NPP than for GPP. Qualitatively similar behavior was observed with complexes of Co²⁺. Extents of elimination and cyclization were increased by metal ions. Carbocyclase-catalyzed formation of cyclic monoterpene hydrocarbons in the presence of Mg²⁺ involved bis-metallic complexes as the “true” substrates.     

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major β-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the β-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrodrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.     

Effects of long-term in vitro exposure to aluminum,cadmium or lead on differentiation and cholinergic receptor expression in a human neuroblastoma cell line

Neurotoxicity of long-term exposure to lead, aluminum and cadmium has been studied in vitro on the human neuroblastoma cell line IMR32 by measuring cytotoxicity, and the effects on neuronal-specific characteristics such as nitrite outgrowth and expression of cholinergic receptors as parameters of toxicity. Cytotoxicity was highest with cadmium, intermediate with lead and lowest with aluminum exposure. Lead, but not cadmium and aluminum, interfered with neurite growth. The expression of a-bungarotoxin binding sites and muscarinic receptors was markedly increased by cadmium and not affected by aluminum exposure. Lead induced only an increase of toxin binding sites. These in vitro modifications are discussed in relation to the possible use of neuronal cell lines for detecting neurotoxic effects of heavy metals.Abbreviations ACh acetylcholine - -Bgtx -bungarotoxin - BrdUr 5'-bromodeoxyuridine - CNS central nervous system