Influence of various biological matrices (plasma, blood microdialysate) on chromatographic performance in the determination of β-blockers using an alkyl-diol silica precolumn for sample clean-up

A HPLC column-switching system with LiChrospher RP-8 ADS precolumn was applied for the determination of beta-blockers (atenolol, pindolol, propranolol) in human plasma. The influence of biological matrices on the changes of the chromatographic parameters such as retention time, peak symmetry, area and selectivity were investigated. After injection of 5 ml plasma a decrease of retention times of the analytes was observed of up to 25% and an increase of asymmetry factors of up to 5%. Peak areas and selectivities were not changed. The observed effect could indicate changes of chromatographic performance caused by contributions of the analytical column or the ADS precolumn. The experiments with microdialysis excluded the contribution of the analytical column. A detailed investigation of experiments have been discussed in this paper.     

Synthesis and reactivity of early-late heterobimetallic complexes displaying a η-tantalum-alkylidene to palladium interaction

Tantalaalkylidene compounds, CHR=TaCl₃L₂ (R=^tBu or CMe₂Ph, L=THF or 1/2dimethoxyethane) mixed with the cyclopalladated dimer [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)]₂, 1, afford good yields of heterodimetallic complexes [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CHR=TaCl₃L], 3a, 3b, in which the Ta=C unit is η²-interacting with the palladium atom, while a chloride ligand is bridging the tantalum and the palladium atoms. These compounds are fairly stable in air in the solid state and also in solution at RT. The interaction of the Ta=C unit with Pd in these bimetallic compounds is weak as shown by the ready formation of [Pd(2-C₆H₄CH₂NMe₂)PyCl] and CHR=TaCl₃Py₂ upon treatement with pyridine. Compounds analogous to 3a, b can also be obtained with 12 electrons tantalum complexes. Thus treating the same cyclopalladated dimer 1 with CHR=Ta(OAr)₃ (OAr=2,6-diisopropylphenyloxy) led to a much more stable though electron deficient species: [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CH^tBu=Ta(OAr)₃], 3c. Substitution in 3a of one chloride ion by an alkyl group occurred at the tantalum metal via reaction with ZnR₂ (R=---CH₂CMe₂Ph) leading to [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CH^tBu=TaCl₃(CH₂CMe₂Ph)], 4 for which there is no free rotation around the new Ta---C bond and in which one of the methylene protons is strongly interacting with the palladium centre. This compound is believed to mimic an intermediate to the formation of tantalacarbyne derivative, which was obtained earlier via reaction of the uncomplexed tantalacarbene compound with dialkylzinc compounds.     

NATURAL SELECTION FOR GRAZER RESISTANCE TO TOXIC CYANOBACTERIA: EVOLUTION OF PHENOTYPIC PLASTICITY?

Abstract We studied the selection response of the freshwater grazing zooplankter, Daphnia galeata, to increased abundance of cyanobacteria in its environment. Cyanobacteria are a poor‐quality and often toxic food. Distinct genotypes of D. galeata were hatched from diapausing eggs extracted from three time horizons in the sediments of Lake Constance, Europe, covering the period 1962 to 1997, a time of change in both the prevalence of planktonic cyanobacteria and levels of phosphorus pollution. We assessed whether the grazers evolved to become more resistant to dietary cyanobacteria by exposing genetically distinct clones to two diets, one composed only of the nutritious green alga, Scenedesmus obliquus (good food), and the other a mixture of S. obliquus and the toxic cyanobacterium Microcystis aeruginosa (poor food). Genotype performance was measured as the specific rate of weight gain from neonate to maturity (g_j). We evaluated evolutionary change in the Daphnia population using an analysis of reaction norms based on relative (log‐transformed) changes in g_j. Log(g_j) is a measure of the proportional effect of dietary cyanobacteria on other fitness components of the Daphnia phenotype. For comparison, we also analyze absolute (i.e., nontransformed) changes in g_j and discuss the interpretations of the two approaches. Statistical results using a general linear model demonstrate a significant effect of genotype (showing differences in g_j among genotypes), a significant genotype X food‐type interaction (showing differences in phenotypic plasticity among genotypes), and, in the case of log‐transformed data, a significant sediment‐genotype‐age X food‐type interaction. The latter shows that phenotypic plasticity evolved over the period studied. Two constraints act on response to selection in the D. galeata‐Lake Constance system. First, g_j on a diet containing poor food is highly correlated with g_j on a diet of good food, thus evolving resistance also meant evolving an increase in g_j on both diets. Second, because genotypes with a high g_j also grow to a large adult body size, which in turn increases Daphnia vulnerability to fish predation, we suggest that selection only acted to favor genotypes possessing a high potential g_j after cyanobacteria became prevalent. The presence of cyanobacteria depressed realized g_j and led to animals of small adult body size even if their genotypes had the potential for high g_j and large size. With realized g_j reduced, genotypes with an inherently high value could be selected even in the presence of predatory fish. The joint action of selection by dietary cyanobacteria and vulnerability to fish predation provides an explanation for the observed evolution of resistance to poor food through reduced phenotypic plasticity.     

Distribution of conjugated linoleic acid and metabolites in different lipid fractions in the rat liver.

Conjugated linoleic acid (CLA) is known to provide certain health benefits in experimental animal models. The major CLA isomer in food is c 9,t11-CLA. A primary objective of this study was to investigate the uptake of c 9,t11-CLA and its downstream metabolites into various lipid fractions in the liver of rats fed either a high or low CLA diet (containing 0.1 or 0.8 g CLA/100 g diet, respectively). As expected, the levels of all conjugated diene (CD) fatty acids (CD 18:2 + CD 18:3 + CD 20:3 + CD 20:4) were elevated about 8-fold in the high CLA diet group. However, there was no change in the distribution of CLA and CLA metabolites into various lipid fractions due to CLA intake. Unlike linoleic acid or gamma-linolenic acid, which were distributed mainly in phospholipids, CD 18:2, CD 18:3, and CD 20:3 were incorporated primarily in neutral lipid. Furthermore, the incorporation of all nonconjugated unsaturated fatty acids was not perturbed by CLA. Regardless of the level of CLA in the diet, CD 20:4 was predominantly enriched in phosphatidylserine and phosphatidylinositol. In contrast, arachidonic acid was primarily enriched in phosphatidylcholine and less so in phosphatidylethanolamine. The above findings may have potential implication regarding the role of CLA in modulating eicosanoid metabolism.     

Distinct Combinations of Borrelia burgdorferi Sensu Lato Genospecies Found in Individual Questing Ticks from Europe

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.     

A general cloning strategy for divergent plant cytochrome P450 genes and its application in Lolium rigidum and Ocimum basilicum

The investigation of plant cytochrome P450 genes and enzymes is a field of growing interest. Apparently, an even greater diversity of cytochrome P450 genes exists in plants in comparison to other eukaryotes. This may be due to their role in the biosynthesis of secondary metabolites that are present in plants in an enormous variety. Most cloning approaches are hampered by the large sequence diversity of plant cytochrome P450 genes. We present a method to clone divergent cytochrome P450 ESTs by a nested RT-PCR-strategy. These ESTs were used for the subsequent cloning of the corresponding full-size cDNAs of divergent families via cDNA-library screening. Sixteen cytochrome P450 genes belonging to different cytochrome P450-families have been identified in this way, proving the efficacy of the strategy. Received: 1 December 2000 / Accepted: 26 February 2001     

On the evolution of clonal plant life histories

Clonal plant life histories are special in at least four respects: (1) Clonal plants can also reproduce vegetatively, (2) vegetative reproduction can be realised with short or long spacers, (3) and it may allow to plastically place vegetative offspring in benign patches. (4) Moreover, ramets of clonal plants may remain physically and physiologically integrated. Because of the apparent utility of such traits and because ecological patterns of distribution of clonal and non-clonal plants differ, adaptation is a tempting explanation of observed clonal life-history variation. However, adaptive evolution requires (1) heritable genetic variation and (2) a trait effect on fitness, and (3) it may be constrained if other evolutionary forces are overriding selection or by constraints, costs and trade-offs. (1) The few studies undertaken so far reported broad-sense heritability for clonal traits. Variation in selectively neutral genetic markers appears as pronounced in populations of clonal as non-clonal plants. However, neutral markers may not reflect heritable variation of life-history traits. Moreover, clonal plants may have been sampled at larger spatial scales. Empirical information on the contribution of somatic mutations to heritable variation is lacking. (2) Clonal life-history traits were found to affect fitness. However, much of this evidence stems from artificial rather than natural environments. (3) The relative importance of gene flow, inbreeding, and genetic drift, compared with selection, in the evolution of clonal life histories is hardly explored. Benefits of clonal life-history traits were frequently studied and found. However, there is also evidence for constraints, trade-offs, and costs. In conclusion, though it is very likely, that clonal life-history traits are adaptive, it is neither clear to which degree this is the case, nor which clonal life-history traits constitute adaptations to which environmental factors. Moreover, evolutionary interactions among clonal life-history traits and between clonal and non-clonal ones, such as the mating system, are not well explored. There remains much interesting work to be done in this field – which will be particularly interesting if it is done in the field.     

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS.

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13C] and [U-13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.     

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.