Integrated cultivation of salmonids and seaweeds in open systems

Bacterial abundance and production in a vertical profile in Lake Kariba (17dgS), Zimbabwe, were affected by solar irradiance. At the surface, 1.87 × 10⁹ bacteria 1^–1 were found and abundance peaked at 10 m (2.5 × 10⁹ bacteria l^-1), then decreasing with depth. Bacterial reproduction at the surface(0.145 µg C1^–1 h^–1) was nearly four times less than the production at 10 m although bacterial numbers were only 26% less. Thus, bacterial production per cell was lower at the surface than deeper down, suggesting that bacterial production is inhibited at the surface.Bacterial production in GF/F filtered lake water in Whirl Pack bags showed an exponential decrease down to 3 m depth. The inhibition was well in accordance with light extinction in the UV region. Phosphatase activity was low in light exposed bags compared to dark, indicating photolysis of extracellular enzymes, or phototransformation of recalcitrant DOM, which substitutes enzyme activity. Hypolimnetic enzyme activity was less affected by solar light than epilimnetic.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.     

A novel bioluminogenic assay for α-chymotrypsin

The use of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin as a novel substrate for α-chymotrypsin has been demonstrated. The kinetic parameters determined are K_M = 0.38mmol/L, k_cat = 6.5 s^?1 and k_cat/k_M = 17,100 (L/mols). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L -phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases.     

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Effect of support material and enzyme pretreatment on enantioselectivity of immobilized subtilisin in organic solvents

Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. (c) 1994 John Wiley & Sons, Inc.     

Symmetric enzyme distribution in asymmetric UF polysulfone membranes

Invertase as well as as amyloglucosidase were immobilized within asymmetyric ultrafiltration membranes that were prepared from polysulfone or homogeneously modified polysulfone. The chemical modification was carried out by sulfonation and halomethylation. This additional change of the surface properties of the capillaries within the membrane offers the possibilities for various types of enzyme fixation, namely adsorption, charge interactions, or covalent bonding. By variation of the immobilization conditions the distribution of the enzyme could be adjusted over the membrane's cross section. At a distinct enzyme concentration in the loading solution a homogeneous enzyme distribution within the membrane could be verified. This was shown by diffusion experiments. Under ultrafiltration conditions using a solution that contains membrane-impermeable macromolecules as well as a membrane-permeable solute like saccharose the residence time within the membrane was increased due to gel formation atop the membrane yet the kinetic was no affected. The nonpermeable soluble starch was not reacted by the amyloglucosidase membrane, indicating that the skin layer was free of enzymes. (c) 1994 John Wiley & Sons, Inc.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.     

Peroxisome proliferators as inducers and substrates of UDP-glucuronosyltransferases

Summary— Peroxisome proliferators, despite their chemically unrelated structures, share the common property of being able to stimulate the glucuronidation of bilirubin in rodents and, probably, also in man. The aryloxycarboxylic acids (clofibric acid, fenofibrate, bezafibrate, ciprofibrate), tiadenol and probucol, all of which have hypolipidemic properties, as well as the fatty acid-like perfluorodecanoic acid all enhanced the expression of the UDP-glucuronosyltransferase (UGT) form involved in the conjugation of the pigment. This induction is manifested by an increase in the mRNA species encoding the protein with a subsequent increase in the neosynthesis of the corresponding protein in the endoplasmic reticulum. The induction process is concomitant with that of cytochrome P-450-IVA1 and cytosolic epoxide hydrolase, which, like bilirubin UGT, are mainly involved in the metabolism of endogenous substrates. With a series of carboxylic acids related to clofibric acid, it was possible to demonstrate that induction was mediated via specific interactions based on the physicochemical properties of the inducers. Until now, the molecular basis of induction of bilirubin UGT is not known. The peroxisome proliferators that possess a carboxyl group are good substrates of UGT, especially in man. The acylglucuronides formed are known for their instability and reactivity which could contribute to the toxicity encountered in some patients treated with the drugs. There is convincing evidence that UGT bilirubin does not catalyze the glucuronidation of these substances even if the two types of substrate form acylglucuronides.