Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

Summary Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with N^G-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

Surfactant effects on adrenergic responses in the gills of the flounder (Platichthys flesus L.)

Summary The interactions between catecholamines and surfactants was investigated in perfused gills of the marine teleostPlatichthys flesus L. The activity of the branchial ion pumps was monitored via the electrogenic transepithelial potential (inside positive) measured in gills bathed and perfused with identical saline. Vascular resistance of the arterio-arterial and arterio-venous pathway was also recorded simultaneously by measuring respectively the afferent perfusion pressure and venous flow in gills perfused at constant flow and at constant efferent pressure. The specific effects of respective - and -adrenergic receptor stimulation was investigated by the administration of discrete doses of either adrenaline in the presence of 10 mol l^–1 propranolol or isoprenaline in the perfusate. In the absence of surfactants the -adrenergic effects were an inhibition of electrogenic ion transport, a decrease in venous flow and an increase in the vascular resistance of the arterioarterial vascular pathway. In contrast the -adrenergic effects consisted of a stimulation of electrogenic ion transport and a vasodilation of the arterio-arterial pathway. Both anionic (linear alkyl sulphonate; sodium lauryl sulphate) and non-ionic (nonyl phenol ethoxylate; synthetic alcohol ethoxylate) surfactants were administered in the perfusate at nominal concentrations of 1 mol l^–1 (0.3–0.5 mg l^–1). None of these compounds had any effect on the affinity or the efficacy of the -adrenergic responses. In contrast there was a significant reduction in the efficacy of isoprenaline in the presence of all of the surfactants used but only in the case of the synthetic alcohol ethoxylate was there an effect on the affinity of this agonist for the -adrenergic receptor. The results are discussed in the context of the mechanism of action of these environmental contaminants and the nature of adrenergic receptors in the gill.     

Analysis of vertebrate gap junction protein

A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X-100. The major band on SDS-polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent mol. wt. of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of four different experimental approaches. (i) The junctions form a sharp band in potassium iodide density gradients at 1.195 g/cm3 and the 16 K protein is the only detectable band in fractions of this bouyant density. (ii) The junctions are progressively solubilised by increasing concentrations of SDS (in the range 0.1-0.5%) and the dissolution of the junctional structure, observed by electron microscopy, parallels the release of the 16 K protein. (iii) Glutaraldehyde fixation of intact junctions cross-links the 16 K protein. (iv) The recoverable amount of the 16 K protein correlates with known changes in gap junctional area in the regenerating weanling rat liver after partial hepatectomy and in V79 cell cultures exposed to 4beta-phorbol 12-myristate 13-acetate.     

p40MO15, a cdc2-related protein kinase involved in negative regulation of meiotic maturation of Xenopus oocytes.

The clone MO15 which codes for a 40 kd protein (p40MO15) with 40% amino acid identity to the human cdc2 protein kinase has been isolated from a Xenopus cDNA library using a synthetic oligonucleotide probe. MO15 mRNA is accumulated during oogenesis, becomes de-adenylated during meiotic maturation, and is degraded after the mid-blastula-transition stage of embryogenesis. Translation of p40MO15 is restricted to non-mature oocytes. Specific inhibition of p40MO15 synthesis in stage VI oocytes by antisense oligonucleotide depletion of MO15 mRNA increases the rate of progesterone induced H1 kinase activation and oocyte maturation. This effect can be reversed by subsequent injection of synthetic MO15 mRNA. These results suggest that p40MO15 is involved in negatively regulating meiosis.     

Bacterial degradation of low concentrations of phenanthrene and inhibition by naphthalene

Phenanthrene-degrading bacteria were isolated from enrichment cultures of soils contaminated with creosote and jet fuel. The isolates from the creosote enrichments were classified by fatty acid methyl ester profiles as Acidovorax delafieldii and Sphingomonas paucimobilis; the bacterium from the jet fuel-contaminated soil was not identified and was designated strain JFD 11. All three isolates used phenanthrene as a sole carbon and energy source, and two of the isolates used fluoranthene as a sole carbon and energy source. Anthracene and fluorene were cometabolized by all three strains, but pyrene was not transformed. Naphthalene inhibited all of the strains, and 28-h cultures of A. delafieldii were inhibited by naphthalene concentrations as low as 5 ppm. Short-term degradation experiments were undertaken with center-well flasks and concentrations of phenanthrene ranging from 1.2 to 12.0 m. Since initial degradation rates were not a function of phenanthrene concentration, it was inferred that the half-saturation constants were less than the lowest phenanthrene concentration tested. Correspondence to: C.E. Cemiglia.     

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl₂. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.     

The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).