Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads

 Saccharomyces cerevisiae cells were immobilized on preformed cellulose beads by adsorption. The fermentation capacity of the immobilized yeast cells was found to be practically independent of the hydrogen ion concentration between pH 3.1 and 6.25. The fermentation capacity was maximal at 30 °C. The immobilized yeast cells were used for continuous production of ethanol in a fluidized-bead reactor. The average values characteristic for the process were an ethanol concentration of 41.9±0.1 g l^-1, a fermentation efficiency of 82.9±2.1% and a volumetric productivity of 3.94±0.52 g l^-1 h^-1. Received: 9 October 1995/Accepted: 22 April 1996     

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

 Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X₂₄ xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)^-1. In this culture condition, X₂₄ is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X₂₄ enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X₂₄ xylanase had a Michaelis constant, K _m, of 12.43 mg oat spelt xylan ml^-1 and a V _max of 1639 μmol min^-1 (mg protein)^-1. Received: 17 May 1995/Received last revision: 25 September 1995/Accepted: 29 September 1995     

Production of 13C polyunsaturated fatty acids from the microalga Phaeodactylum tricornutum

An integrated process for the indoor production of ¹³C labelled PUFA from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor from which the exhaust gas from the reactor is returned to the culture by a low pressure compressor. To avoid accumulation of dissolved oxygen in the culture medium, the exhaust gas is bubbled through a sodium sulphite solution before returning it to the reactor. Carbon is removed from the medium before inoculating the alga, then labelled ¹³CO₂ is injected for pH control and carbon supply. The reactor has been operated in semicontinuous mode at a dilution rate of 0.01 h^–1, a biomass productivity of 0.1 g L^–1 d^–1 being obtained. Under this conditions both pH and dissolved oxygen were correctly controlled and the adequacy of the system for autotrophic production of labelled biomass was demonstrated. Analysis by GC-MS revealed that the fatty acids content of the biomass obtained was 10% d.wt., the content of eicosapentaenoic acid was 2.5% d.wt. All the fatty acids were labelled, more that 90% of the carbon present in these fatty acids was ¹³C. Element analysis of biomass and supernatant showed that 59.5% of injected carbon was assimilated into the biomass whereas 33% remained in the supernatant, and 7.5% remained undetected. Due to the high cost of ¹³CO₂ different strategies for the optimisation of labelled carbon use are proposed.     

Spatial patterns of trees and juveniles in a laurel forest of Tenerife, Canary Islands

Spatial patterns are important characteristics of the forest and theycan reveal such things as successional status and ecological characteristics ofthe species. We tested the hypothesis that spatial distribution will bedifferent, depending on whether the species is intolerant or tolerant to shade.We assessed the spatial distribution of trees (> 4 cm dbh) andjuveniles in eight laurel forest plots. A univariate spatial analysis(performed with Ripley's K₁) showed that all tree species havesignificantaggregation at short distances (2 m). Nevertheless, two groups ofspecies could be differentiated: Erica scoparia,Myrica faya and Ilex canariensisshowed a tendency for aggregation at large distances (larger than 6m)while L. azorica and Prunuslusitanicashowed aggregation only at shorter distances. Ripley's BivariateK_1,2 analyses showed no significant differences in the spatialdistribution ofanalyzed species pairs from a null model. Only Laurusazoricahad a sufficient sample size for analysis of juvenile distribution. Aunivariateanalysis revealed thatL. azorica seedlings (stems < 50 cm high)were clumped in some plots up to 5 m, but this was not consistent.Saplings (stems > 50 cm high and < 4 cm dbh)didnot show strong clumping even at short distances. L.azoricasaplings had no significant aggregation with, nor repulsionfrom, adults of the same or different species. Spatial patterns of the speciesshould be considered in the development of restoration plans of the laurelforest 90%of which has disappeared or been intensively disturbed on Tenerife Island.     

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

The content of four immunomodulatory steroids and major androgens in human semen

Seminal fluid fulfils a dual role: it provides optimal conditions for fertilization and protects male germ cells from infections. Besides both major sexual hormones and cortisol it contains a considerable amounts of dehydroepiandrosterone (DHEA), known to counteract the excessive actions of glucocorticoids. From this point of view of importance may be our recent finding of both 7-hydroxy-dehydroepiandrosterone epimers (7-OH-DHEA) in semen, believed to be in some instances the locally active immunoprotective agents. The concentrations of these steroids were of the same range or even higher than in blood. Here further data on 7-OH-DHEA in semen, along with other relevant steroid hormones, are given in 79 samples, either from healthy males or from patients with various sexual disorders. A method has been developed enabling us a simultaneous determination of DHEA, 7-OH-DHEA epimers, testosterone, dihydrotestosterone and cortisol in seminal fluid. It was based on ether extraction, solvent partition and HPLC separation, followed by specific radioimmunoassays in the respective fractions. In addition, the steroids were measured in serum and the concentrations in both fluids were compared. The concentrations of 7-OH-DHEA in seminal fluid varied from 1.8 to 15.7 nmol/l, while those of DHEA were about five times higher.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.