Induction of phenylpropanoid and tyramine metabolism in pectinase- or pronase-elicited cell suspension cultures of tobacco (Nicotiana tabacum)

The induction of the phenylpropanoid pathway and of tyramine metabolism was monitored in cell suspension cultures of Nicotiana tabacum treated with cell wall-degrading enzymes, in an attempt to correlate the synthesis of hydroxycinnamic acid amides of tyramine with the formation of wall-bound phenolic polymers. Treatment with commercial pectinase (from Penicilium occitanis ) induced a rapid rise in phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate:CoA ligase (EC 6.2.1.12), tyramine hydroxycinnamoyltransferase (EC 2.3.1.110) and peroxidase (EC 1.11.1.7) activities, and a concomitant decline in cinnamyl alcohol dehydrogenase (EC 1.1.1.195) activity. The induction of the phenylpropanoid pathway and of the synthesis of cinnamoyl-tyramines preceded the death of a large proportion of the elicited cells. When the cultures were treated with pronase (from Streptomyces griseus ), most cells remained alive and the induction of enzymes of the phenylpropanoid pathway lasted for several days, resulting in an accumulation of cinnamoyltyramines in the cells and in the culture medium. Treatment with pronase induced an increase in the activity of moderately anionic isoperoxidases which were also induced in pectinase-treated cells. Cinnamyl alcohol dehydrogenase activity remained stable in pronase-elicited cells, which rapidly accumulated thioglycolic acid-extractable phenolic polymers in their cell walls. The accumulation of these polymers coincided with the induction of 4-coumarate:CoA ligase but preceded the rise in tyramine hydroxycinnamoyltransferase and peroxidase activities.     

Characterization of the Rhodococcus sp. NI86/21 gene encoding alcohol: N,N′-dimethyl-4-nitrosoaniline oxidoreductase inducible by atrazine and thiocarbamate herbicides

A protein with a mol. mass of 51,000 (ThcE) that was induced in Rhodococcus sp. N186/21 during assimilation of thiocarbamate herbicides, atrazine, ethanol, propanol, glycerol, propionaldehyde or ethanolamine was identified by two-dimensional electrophoresis. The thcE gene was cloned and sequenced. The deduced amino acid sequence revealed ThcE as a member of group III alcohol dehydrogenases. ThcE displayed strong homology with sequenced subunit fragments of the homodecameric N,N-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductases (MNO) of Amycolatopsis methanolica and Mycobacterium gastri. N-Terminal sequence analysis of purified MNO from Rhodococcus sp. NI86/21 confirmed the identity with ThcE. When overproduced in Escherichia coli, ThcE was insoluble and no MNO activity was detected.Abbreviations BSM Basal salt medium - EPTC S-ethyl dipropylthiocarbamate - MDH methanol dehydrogenase - MNO methanol - NDMA oxidoreductase - NDMA N,N-dimethyl-4-nitrosoaniline     

Effect of veratryl alcohol and manganese (IV) oxide on ligninolytic activity in semi solid cultures of Phanerochaete chrysosporium

An inert carrier (nylon sponge), a non-inert carrier (barley straw) and the addition of veratryl alcohol or manganese (IV) oxide to the cultures were used to study the production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during semi solid state fermentation conditions. By supplementing the medium with these compounds we could stimulate the ligninolytic system of this fungus. The different carriers employed and the effect of adding veratryl alcohol or manganese (IV) oxide to the cultures were compared in order to determine the best system to produce high activities of ligninolytic enzymes. Lignin peroxidase (LiP) activities higher than 500 U/L and manganese-dependent peroxidase (MnP) activities about 1100 U/L were achieved.     

Development of a capillary electrophoretic separation of an N-(substituted)-glycine-peptoid combinatorial mixture

Capillary electrophoresis was used for the separation of a combinatorially synthesized N-(substituted)-glycine (NSG) peptoid mixture. This mixture consisted of 24 trimeric compounds sharing a common backbone structure but differing in the side chain attached at the N-terminal residue. Standards of the individual components were unavailable so that development of the separation was based on the mixture. A variety of buffer additives were investigated to enhance the CE resolution of this diverse mixture. Ion-pairing agents, cyclodextrins and organic modifiers were all evaluated as buffer additives. The best separations were achieved using a combination of buffer additives, each serving a different purpose in the separation. Heptane sulphonic acid (HSA) was used to reduce hydrophobic intramolecular interactions. Methyl-β-cyclodextrin was used to provide host–guest interactions in order to resolve the very hydrophobic components of the NSG-peptoid mixture. The optimized run buffer consisted of 250 mM sodium phosphate buffer, pH 2.0, with 25 mM HSA and 40 mg/ml BCD and resulted in the resolution of 21 peaks for the 24 peptoids in the combinatorial mixture.     

Shaping of Drosophila Alcohol Dehydrogenase Through Evolution: Relationship with Enzyme Functionality

Drosophilidae is a large, widely distributed family of Diptera including 61 genera, of which Drosophila is the most representative. Drosophila feeding is part of the saprophytic trophic chain, because of its dependence upon decomposing organic matter. Many species have adapted to fermenting fruit feeding or to artificial (man-made) fermentation habitats, such as cellars and breweries. Actually, the efficient exploitation of niches with alcohols is considered one of the reasons for the worldwide success of this genus. Drosophila alcohol dehydrogenase (ADH), a member of the short-chain dehydrogenase/reductase family (SDR), is responsible for the oxidation of alcohols, but its direct involvement in fitness, including alcohol tolerance and utilization, gives rise to much controversy. Thus, it remains unclear whether ADH differentiation through evolution is somehow associated with natural adaptation to new feeding niches, and thus maybe to Drosophila speciation, or if it is a simple reflection of neutral divergence correlated with time separation between species. To build a hypothesis which could shed light on this dilemma, we analyzed the amino acid variability found in the 57 protein ADH sequences reported up to now, identified the taxon-specific residues, and localized them in a three-dimensional ADH model. Our results define three regions whose shaping has been crucial for ADH differentiation and would be compatible with a contribution of ADH to Drosophila speciation. Received: 11 August 1997 / Accepted: 30 December 1997     

Glial Fibrillary Acidic Protein Expression in Rat Brain and in Radial Glia Culture Is Delayed by Prenatal Ethanol Exposure

Abstract: The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glial, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glial. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.     

Enzymatic Routes for the Synthesis of Rhododendrin and Epi-Rhododendrin

The synthesis of (R)- and (S)-3-(4-hydroxyphenyO-1-methylpropyl-β-D-glucopyranosides has been achieved by two enzymatic steps, namely an oxido-reduction step involving alcohol dehydrogenases from different origin for the preparation of both aglycones in enantiomeric pure form, and a transglycosidation step involving a thermophilic β-glucosidase from the archaeon Sulfolobus solfataricus.     

Enzymatic measurement of ethanol or NAD in acid extracts of biological samples

An enzymatic method for the measurement of ethanol has been developed to permit analyses with unneutralized acid extracts of blood, liver, cell suspensions, or other biological materials. Components of the assay mixture include NAD, yeast alcohol dehydrogenase, tris(hydroxymethyl)aminomethane (Tris), and lysine. Tris is a trapping agent for the reaction product, acetaldehyde. Lysine is used to maintain the pH at 9.7 where oxidation of ethanol is quantitative and most rapid, even when as much as 0.2 ml of 0.5 n HClO₄ is added. Lysine also causes the reaction to be 2 to 4 times faster than it is when either glycine or 2-amino-2-methyl-1-propanol is used as the buffer. The assay is linear up to an ethanol concentration of 0.125 mm in the reaction mixture and is complete by 4 min. By substituting ethanol for NAD in the reagents, the assay performs equally well in measuring NAD.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.