N-demethylation of lergotrile by Streptomyces platensis.

Thirty-eight microorganisms were screened for their ability to produce metabolites of the semisynthetic alkaloid, lergotrile. A total of five microorganisms were found to biotransform lergotrile, and N-desmethyl lergotrile was detected as the principal metabolite with most organisms. Streptomyces platensis (NRRL 2364) appeared to form the metabolite in highest yield, and a preparative-scale conversion was accomplished with a recovered yield of 50%. Structure proof was accomplished with comparative thin-layer chromatography, mixed melting point, mass spectrometry, and remethylation to lergotrile.     

Microbial models of mammalian metabolism: microbial reduction and oxidation of pentoxifylline.

Fourteen microorganisms, including fungi, yeasts, and bacteria, were screened for their ability to metabolize the xanthine drug pentoxifylline. Thirteen cultures either reduced the drug to the alcohol metabolite or oxidatively cleaved the ketonic side chain to homologous carboxylic acid metabolites. The alcohol metabolite was the predominant or sole metabolite in all organisms, with conversions ranging from 6 to 91%. Preparative-scale production of the alcohol metabolite with Rhodotorula rubra (ATCC 20129) allowed for the isolation of this product with a 40% yield. Two organisms also produced the carboxylic acid metabolites at low levels (2 to 10%). The routes of metabolism in microbial cultures are the same as those reported in mammalian systems.     

Microbial models of mammalian metabolism: microbial reduction and oxidation of pentoxifylline

Fourteen microorganisms, including fungi, yeasts, and bacteria, were screened for their ability to metabolize the xanthine drug pentoxifylline. Thirteen cultures either reduced the drug to the alcohol metabolite or oxidatively cleaved the ketonic side chain to homologous carboxylic acid metabolites. The alcohol metabolite was the predominant or sole metabolite in all organisms, with conversions ranging from 6 to 91%. Preparative-scale production of the alcohol metabolite with Rhodotorula rubra (ATCC 20129) allowed for the isolation of this product with a 40% yield. Two organisms also produced the carboxylic acid metabolites at low levels (2 to 10%). The routes of metabolism in microbial cultures are the same as those reported in mammalian systems.     

A method for the selection of production media for actinomycete strains based on their metabolite HPLC profiles

The manipulation of growth conditions of microorganisms is a common strategy used by pharmaceutical companies to improve the quantities and spectra of secondary metabolites with potential therapeutic interest. In this work, the effects of fermentation media on secondary metabolite production from a set of Actinomycetes was statistically compared. For this purpose, we created an automated method for comparing the ability of microorganisms to produce different secondary metabolites. HPLC analyses guided the selection of those media in which a wider chemical diversity was obtained from microorganisms inoculated in a wide spectrum of production media. Fermented media yielding a better secondary metabolite profile were included in subsequent drug discovery screening.     

Inhibition of peripheral adrenergic neurotransmission by lergotrile in spontaneously hypertensive rats

Intraperitoneal injection of lergotrile (0.5 mg/kg) produced arterial hypotension and bradycardia for 120 and 90 minutes, respectively, in anesthesized spontaneously hypertensive rats (SHR). During this time frame, lergotrile (0.5 mg/kg, i.p.) greatly attenuated diastolic blood pressure and cardiac rate responses to electrical stimulation (0.062-4 Hz) of the sympathetic outflow in pithed SHR, but had no significant effect on comparable increments in pressure and rate produced by exogenous norepinephrine (0.01–10 μg/kg, i.v.). Pretreatment of SHR with haloperidol (2 mg/kg, i.p.) prevented lergotrile-induced hypotension and partially reversed its inhibitory effect on neurogenic vasoconstrictor responses. Haloperidol alone had no significant effect on baseline arterial blood pressure or responses to sympathetic nerve stimulation. Administration of hexamethonium (20 mg/kg, i.v.) to SHR antagonized the hypotensive response to lergotrile (0.5 mg/kg, i.p.), although hydralazine (2 mg/kg, i.p.) still produced a marked reduction in pressure.These results suggest that lergotrile produces arterial hypotension and bradycardia primarily by inhibiting peripheral sympathetic nerve function through a dopaminergic mechanism. The probable site of action of lergotrile is at presynaptic (neuronal) dopamine receptors which are known to be inhibitory to neurogenic release of norepinephrine.     

Propachlor degradation by a soil bacterial community.

Soil from a pesticide disposal site was used to enrich for microorganisms that degraded the acylanilide herbicide propachlor (2-chloro-N-isopropylacetanilide). After seven transfers of the enrichment, the culture contained about six strains. The highest yield of microbial biomass occurred if just two of these isolates, strains DAK3 and MAB2, were inoculated into a mineral salts medium containing propachlor. When only strain DAK3 was grown on propachlor, a metabolite (2-chloro-N-isopropylacetamide) was released into the medium. Strain MAB2 could grow on this metabolite. The results of morphological and physiological tests suggest that strains DAK3 and MAB2 most closely resemble species belonging to the genera Moraxella and Xanthobacter, respectively. Strain DAK3 can respire and grow on N-substituted acylanilides containing methyl, ethyl, or isopropyl substitutions, but is incapable of respiration or growth on acetanilide, aniline, or the acylanilide herbicides alachlor and metolachlor. Strain DAK3 appears to use the aromatic C atoms of propachlor for growth, as suggested by the growth yield on propachlor and the induction of catechol 2,3-oxygenase activity in acylanilide-grown cells.     

Propachlor degradation by a soil bacterial community.

Soil from a pesticide disposal site was used to enrich for microorganisms that degraded the acylanilide herbicide propachlor (2-chloro-N-isopropylacetanilide). After seven transfers of the enrichment, the culture contained about six strains. The highest yield of microbial biomass occurred if just two of these isolates, strains DAK3 and MAB2, were inoculated into a mineral salts medium containing propachlor. When only strain DAK3 was grown on propachlor, a metabolite (2-chloro-N-isopropylacetamide) was released into the medium. Strain MAB2 could grow on this metabolite. The results of morphological and physiological tests suggest that strains DAK3 and MAB2 most closely resemble species belonging to the genera Moraxella and Xanthobacter, respectively. Strain DAK3 can respire and grow on N-substituted acylanilides containing methyl, ethyl, or isopropyl substitutions, but is incapable of respiration or growth on acetanilide, aniline, or the acylanilide herbicides alachlor and metolachlor. Strain DAK3 appears to use the aromatic C atoms of propachlor for growth, as suggested by the growth yield on propachlor and the induction of catechol 2,3-oxygenase activity in acylanilide-grown cells.     

Microbial transformations of galanthamin-precursors

Summary Galanthamin is a medical important alkaloid. Its chemical synthesis gives a racemic product in low yields. Starting with a belladinderivative an enzymatic ring closure should lead exclusively to a chiral product possibly with the native structure. Although this reactions type is unknown in preparative biotransformations a large number of microorganisms were tested, unfortunately without success. On the other hand in the screen transformation products were found resulting from specific dealkylations of the subtrate. The type of metabolite formed was dependent on the fungi utilized for the transformation. Additionally two N-oxides were formed by Septomyxa affinis, one in good yield. It is possible that the chirality of this compound can direct the ring closure preferentially or exclusively to the desired stereoisomer of narwedine.     

Bioconversion of oleuropein to hydroxytyrosol by lactic acid bacteria

The aim of this work is to study the conversion of oleuropein-a polyphenol present in olives and olive oil by-products-into hydroxytyrosol, a polyphenol with antioxidant and antibacterial properties. The hydrolysis reaction is performed by lactic acid bacteria. Six bacterial strains (Lactobacillus plantarum 6907, Lactobacillus paracasei 9192, Lactobacillus casei, Bifidobacterium lactis BO, Enterococcus faecium 32, Lactobacillus LAFTI 10) were tested under aerobic and anaerobic conditions. The oleuropein degradation and hydroxytyrosol formation were monitored by HPLC. Results showed that oleuropein could be successfully converted into hydroxytyrosol. The most effective strain was Lactobacillus plantarum 6907, with a reaction yield of hydroxytyrosol of about 30 %. Different reaction mechanisms were observed for different microorganisms; a different yield was observed for Lactobacillus paracasei 9192 under aerobic or anaerobic conditions and an intermediate metabolite (oleuropein aglycone) was detected for Lactobacillus paracasei 9192 and Lactobacillus plantarum 6907 only. This study could have significant applications, as this reaction can be used to increase the value of olive oil by-products and/or to improve the taste of unripe olives.     

Generally applicable fed-batch culture concept based on the detection of metabolic state by on-line balancing

In many microorganisms, flux limitations in oxidative metabolism lead to the formation of overflow metabolites even under fully aerobic conditions. This can be avoided if the specific growth rate is controlled at a low enough value. This is usually accomplished by controlling the substrate feeding profile in a fed-batch process. The present work proposes a control concept which is based on the on-line detection of metabolic state by on-line calculation of mass and elemental balances. The advantages of this method are: 1) the check of measurement consistency based on all of the available measurements, 2) the minimum requirement of a priori knowledge of metabolism, and 3) the exclusive use of simple and established on-line techniques which do not require direct measurement of the metabolite in question. The control concept has been linked to a simple adaptive controller and applied to fed-batch cultures of S. cerevisiae and E. coli, organisms which express different overflow metabolites, ethanol and acetic acid, respectively. Oxidative and oxidoreductive states of S. cerevisiae and E. coli cultures were detected with high precision. As demonstrated by the formation of acetic acid in E. coli cultures, metabolic states could be correctly distinguished for systems for which traditional methods, such as respiratory quotient (RQ), are insensitive. Hence, it could be shown that the control concept allowed avoidance of overflow metabolite formation and operation at maximum oxidative biomass productivity and oxidative conversion of substrate into biomass. Based on mass and elemental balances, the proposed method additionally provides a richness of additional information, such as yield coefficients and estimation of concentrations and specific conversion rates. These data certainly help the operator to additionally evaluate the state of the process on-line.     

Prolactin and luteinizing hormone (LH) release throughout the postpartum period in the suckled first-calf beef cow

A study was performed to examine the release patterns of prolactin and LH of young beef cows with one (single calf) or two calves (double calf) throughout the postpartum interval. The effect on prolactin release of intramuscular and intra-carotid administration of lergotrile and intra-carotid administration of L-dopa was also examined. In approximately 50% and 65% of the cases, no prolactin release could be detected after the beginning of or during the suckling stimulus in cows with one or two calves respectively. LH plasma concentrations remained constant throughout the experiment in all animals. The chosen intramuscular lergotrile treatment lowered plasma prolactin concentrations to baseline levels but had no effect on the length of the postpartum interval. No effect on prolactin release was observed by the given intra-carotid treatments of both lergotrile and L-dopa. First postpartum estrus was observed on days 67 and 88 in the single and double calf cows respectively. The number of suckling periods did not change during the postpartum period but their duration decreased during the same period. These results demonstrate that in at least half of the cases the suckling stimulus does not cause a release of prolactin from the pituitary in the young beef cow. Also, the inhibitory effect of suckling on the resumption of ovarian cyclic function postpartum appears to be of a quantitative nature and mediated by a factor other than prolactin.     

Short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione a novel cytotoxic marine oxysterol

The first and short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione 1 a metabolite from marine algae, has been achieved in six steps from readily available diosgenin 5. Selective deoxygenation of primary alcohol of triol 6 has been accomplished in one step using Et(3)SiH and catalytic amount of B(C(6)F(5))(3) to produce compound 9 in high yield. Oxidation of 11 with PCC, allowed the introduction of 3,6-ene-dione functionality, and further catalytic hydrogenation and deprotection furnished the 3,6-diketo steroid 1.     

DIFFERENTIAL EFFECTS OF THREE DOPAMINE AGONISTS: APOMORPHINE, BROMOCRIPTINE AND LERGOTRILE

Abstract— The ergolines are a new class of proposed dopamine receptor agonists, whose efficacy in treatment of Parkinsonism is under investigation. In order to explore the mechanisms of their action. two ergolines (bromocriptine and lergotrile) were compared to apomorphine for in vivo effects on behavior and in vitro effects on uptake and release of [³H]dopamine by brain minces. Inhibition of dopamine synthesis in vivo significantly interfered with both bromocriptine- and lergotrile-induced stereotypy, while apomorphine-induced stereotypy was not affected. Significant differences among the compounds were also seen neurochemically: bromocriptine inhibited the release of [³H]dopamine. while lergotrile increased release. Apomorphine did not affect uptake or release of [³H]dopamine. The results, of both behavioral and neurochemical experiments, suggest that two ergolines enhance dopaminergic function by action on presynaptic dopaminergic sites in addition to receptor agonism.     

Metabolite identification of missile fuel JP-10 by gas chromatography mass spectrometry

The identification of the metabolites of the Air Force missile fuel JP-10 was accomplished. 5-Hydroxy-JP-10 was identified as the urinary metabolite of rats, mice and hamsters exposed to JP-10. 5-Keto-JP-10 was a major metabolite found in kidney extracts of male rats exposed to JP-10 but not found in kidney extracts from female rats or from other species. Sex-related differences in the formation of 5-keto-JP-10 in rats were studied.     

Systems approaches to succinic acid-producing microorganisms

Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly higher than that obtained using wild-type bacteria.     

Modeling of microorganisms transport in a cylindrical pore

A mathematical model accounting for key parameters as microbial propagation, metabolite formation, dispersion, microbial chemotaxis and water flooding has been proposed to simulate the transport of microorganisms and their metabolites in a cylindrical pore with oil adhered to its inside surface. The model focuses on the transport and the concentration distributions of microorganisms and their metabolites in the cylindrical pore, especially the concentrations that on the oil-water interface. Results from the present model indicate that microorganisms and their metabolites assembled on the oil-water interface during the water flooding process, and the concentration gradients of microorganisms and their metabolites from the pore center region up to the oil-water interface in radial direction of the cylindrical pore were consequently formed. Equilibrium concentrations of microorganisms and their metabolites in the cylindrical pore were obtained when water flow rate within a certain scope, and there existed a critical water flow rate at which the maximum equilibrium concentration of microorganisms on the oil-water interface was developed. Investigations carried out in this study may provide better understanding on the transport mechanism of microorganisms in porous media.     

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

Simultaneous boosting of source and sink capacities doubles tuber starch yield of potato plants

An important goal in biotechnological research is to improve the yield of crop plants. Here, we genetically modified simultaneously source and sink capacities in potato (Solanum tuberosum cv. Desirée) plants to improve starch yield. Source capacity was increased by mesophyll‐specific overexpression of a pyrophosphatase or, alternatively, by antisense expression of the ADP‐glucose pyrophosphorylase in leaves. Both approaches make use of re‐routing photoassimilates to sink organs at the expense of leaf starch accumulation. Simultaneous increase in sink capacity was accomplished by overexpression of two plastidic metabolite translocators, that is, a glucose 6‐phosphate/phosphate translocator and an adenylate translocator in tubers. Employing such a ‘pull’ approach, we have previously shown that potato starch content and yield can be increased when sink strength is elevated. In the current biotechnological approach, we successfully enhanced source and sink capacities by a combination of ‘pull’ and ‘push’ approaches using two different attempts. A doubling in tuber starch yield was achieved. This successful approach might be transferable to other crop plants in the future.     

Increased antimetabolite sensitivity with variation of carbon source during growth.

In Serratia marcescens, analogs of leucine (norleucine), methionine (alpha-methylmethionine), histidine (3-amino-1,2,4-triazolealanine), tyrosine (p-aminophenylalanine), and tryptophan (7-methylindole) are conditional inhibitors of growth; inhibition occurs during the metabolism of some carbon sources but not with others. A further increase in sensitivity to growth inhibition by these analogs can be accomplished through the use of particular combinations of carbon sources present in the inoculum and in the subsequent analog-containing culture medium. Variable sensitivity to analog-mediated inhibition of growth observed during growth on glucose, glycerol, fructose, or citrate correlated inversely with the intracellular pool sizes of the amino acids cognate to the analogs used. The above-cited results, in conjunction with previous results obtained with Pseudomonas aeruginosa and Bacillus subtilis, involve diverse biochemical pathways and suggest that nutritional manipulation to alter the pattern of carbon flow in microorganisms is a generally useful means to accomplish increased sensitivity to growth inhibition by metabolite analogs.     

Selective procedure for isolation of microorganisms producing pullulanase and isoamylase

Selective isolation of microorganisms producing pullulanase and isoamylase was accomplished using a two plate detection assay which distinguished both activities and excluded microorganisms producing other extracellular amylases. Over 115,000 colonies tested, 190 strains producing pullulanase and 57 strains producing isoamylase were isolated; extracellular activities of isolated strains were 0.2 to 4.2 and 0.5 to 2.1 nkat/ml of culture, respectively.This paper is dedicated to Prof. Dr. Raúl E. Trucco, in occasion of his 75th anniversary.