Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.     

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI₂) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI₂ (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical ₁-adrenoceptor densities and affinities (as calculated from [³H]-CGP binding studies), G_i and G_s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI₂, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI₂ is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.     

Hydrolysis of triglyceride by the whole cell of Pseudomonas putida 3SK in two-phase batch and continuous reactors systems

Batch and continuous hydrolysis of olive oil in an organic-aqueous two-phase system using the live whole cell of Pseudomonas putida 3SK as a source of a lipase is investigated. The strain was not only fully viable and grown well, but also produced extracellular lipase simultaneously. The degree of hydrolysis, depending on olive oil concentration in the solvents, was maximal at 13.5% (w/v) and decreased with the increase of the substrate concentration. At the optimal condition, a degree of hydrolysis higher than 95% was achieved with 24 h at 30 degrees C when the reaction was carried out in a two-phase batch stirred reactor. For long-term operation a continuous stirred reactor was designed. When the reaction was carried out in a continuous stirred reactor, the degree was hydrolysis reached 86% at a dilution rate of 0.2 h(-1). Satisfactory performance of a two-phase bioreactor was obtained in a long-term continous operation, which lasted for at least 30 days by feeding organic solvent containing olive oil and aqueous media separately. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic synthesis of alkyl beta-D-xylosides by transxylosylation and reverse hydrolysis

The Trichoderma reesei beta-xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl beta-D-xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl beta-D-xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium-controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium-controlled synthesis. The specificity of the beta linkage is confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. (c) 1994 John Wiley & Sons, Inc.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Polymeric derivatives of plant growth regulators: synthesis and properties

The polymeric formulations of plant growth regulators (PGRs) are high molecular weight systems in which the PGR unit is attached to the polymeric chain by a hydrolysable chemical bond. These polymeric derivatives (esters, ethers, or else) of PGRs are characterised by the ability to release the active compound (PGR) from their solutions (mainly aqueous) in certain conditions. The release of the PGR can be controlled by external factors (pH, temperature, enzymes, solution concentration), and inherent properties of the whole macrosystem chemical structure, such as the type of the hydrolysable bond between PGR unit and the main polymeric chain, the structure of the polymer chain (e.g. molecular weight, level of hydrophilicity, and the content of hydrophobic groups, macromolecular conformation in solution etc.). These controlled (slow) release PGRs display certain advantages over conventional PGR formulations due to their prolonged action, improved efficiency (e.g. wide range of effective concentrations) greater safety to non-target organisms and the applicators. In addition the ability of altering the solubility level and modifying the aplication form is of considerable interest. The biological activity efficiency of polymeric PGRs has been documented and the relation of this efficiency to the PGR unit hydrolytic release ability has been mentioned. Slow release polymeric PGRs are considered to solve certain problems in agriculture.Abbreviations PGR plant growth regulator - C(S)RF controlled (slow) release form - PD polymeric derivative - ACC 1-amino-cyclopropane-1-carboxylic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BAP N⁶-benzylaminopurine - ABA abscisic acid - GA gibberellin - LMW low molecular weight - HMW high molecular weight     

Disruption of Potential α-Helix in the G Loop of the Guinea: Pig 5-Hydroxytryptamine2 Receptor Does Not Prevent Receptor Coupling to Phosphoinositide Hydrolysis

Abstract: Heterogeneity of the 5-hydroxytryptamine₂ (5-HT₂) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT₂ receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT₂ receptors with Pl hydrolysis, the second messenger generally linked with 5-HT₂receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT₂ receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HT_a receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT₂ receptor were 97% homologous. However, the guinea pig 5-HT₂ receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT₂ receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT₂ receptor from guinea pig. 5-HT and the 5-HT₂ receptor agonist α-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT_1c receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT₂ receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5-HT₂ receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT₂ receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT₂ receptors were also coupled to PI hydrolysis.     

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.