Determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

Sensitive high-performance liquid chromatographic assays have been develope to determine the levels of the lactone and lactone plus carboxylate (total) forms of the antitumor agent irinotecan (CPT-11) and its active metabolite SN-38, in human plasma. The related compound camptothecin was used as the internal standard. The selective sample pretreatment for the lactone forms involved a single solvent extraction with acetonitrile-n-butyl chloride (1:4,v/v), whereas the sample clean-up for the total forms was a simple protein precipitation with aqueous perchloric acid-methanol (1:1, v/v), which results in the conversion of the carboxylate to the lactone forms. Chromatography was carried out on a Hypersil ODS column, with detection performed fluorimetrically. The methods have been validated, and stability tests under various conditions have been performed. The lower limits of quantitation are 0.5 and 2.0 ng/ml for the lactone and total forms, respectively. The assays have been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.     

The determination of the binding constant of metalloenzymes for their active site metal ion from ligand inhibition data: Theoretical analysis and application to the inhibition of thermolysin by 1,10-phenanthroline

An equation is found relating the fractional activity, (v/v₀), of an enzyme assay mixture to the total concentrations of metalloenzyme, active site metal ion, metal-binding ligand and substrate and the stability constants of the complexes present. When (v/v₀) is measured as a function of the total ligand concentration, this equation offers a way of data-plotting which yields straight lines and permits the calculation of the metal-binding constant K_ME from either the slope or the intercept, provided that mixed complexes (enzyme-metal ion-ligand) do not contribute significantly to the change in (v/v₀). Since deviations from linearity occur in the latter case, the proposed inhibition plot serves as a diagnostic tool for the recognition of such complexes. Application to the inhibition of thermolysin by 1,10-phenanthroline gives a value of 2.1 × 10¹¹m⁻¹ for K_ZnE, the binding constant of the active site zinc ion, at pH 7.50, 25°C and ionic strength 0.1. The equation also allows the rapid calculation of the ligand concentration necessary to attain a desired degree of inhibition when the total enzyme and active site metal ion concentrations of the solution are known.     

Chromosome numbers of Costa Rican grasses

Chromosome counts are presented for 217 species of 103 genera of grasses from Costa Rica. Four of the genera and 90 of the species are reported for the first time. In addition, 26 counts differ from previous records for the same taxon. The generaGynerium, Lithachne, Luziola, andThrasya were heretofore unknown cytologically. First counts are also given for the following species:Agrostis bacillata, A. pittieri, A. sub patens, Andropogon angustatus, A. bicornis, A. virgatus, Aristida capillacea, A. jorullensis, Arundinella berteroniana, A. deppeana, Axonopus aureus, A. capillaris, A. chrysoblepharis, Bouteloua americana, B. disticha, Bromus exaltatus, Calamagrostis intermedia, C. nuda, C. pittieri, Chusquea lehmannii, C. tonduzii, Cinna poaeformis, Deschampsia pringlei, Digitaria panicea, Echinochloa polystachya, Echinolaena gracilis, Eragrostis simplicijlora, Eriochloa distachya, E. polystachya, Eriochrysis cayennensis, Festuca dolichophylla, Gymnopogon fastigiatus, Gynerium sagittatum, Hymenachne amplexicaulis, Imperata contracta, Isachne polygonoides, Ischaemum latijolium, Lithachne pauciflora, Luziola fragilis, Manisuris aurita, Panicum aquaticum, P. arundinariae, P. boliviense, P. cordovense, P. grande, P. haenkeanum, P. milleflorum, P. parviglume, P. pilosum, P. polygonatum, P. rudgei, P. stenodes, P. trichanthum, P. trichoides, P. viscidellum, Paspalum candidum, P. centrale, P. decumbens, P. fasciculatum, P. jimenezii, P. microstachyum, P. multicaule, P. nutans, P. parviflorum, P. pilosum, P. prostratum, P. pumilum, P. reclinatum, P. saccharoides, P. scabrum, P. serratum, P. squamulatum, P. standleyi, Pennisetum bambusiforme, P. distachyum, Pereilema beyrichianum, Pharus parvifolius, Sacciolepis myuros, Schizachyrium condensatum, Sorghastrum incompletum, Sporobolus ciliatus, S. purpurascens, Streptochaeta sodiroana, Thrasya gracilis, T. petrosa, T. robusta, Trisetum pringlei, T. tonduzii, Uniola pittieri, and Zeugites pittieri. In addition, the following counts are different from previous records for the same taxon:Aegopogon cenchroides, Axonopus poiophyllus, Bouteloua media, Brachiaria plantaginea, Chusquea subtessellata, Digitaria adscendens, Homolepis aturensis, Hymenachne donacifolia, Ichnanthus axillaris, I. pallens, I. tenuis, Ischaemum ciliare, Ixophorus unisetus, Oplismenus burmannii, O. hirtellus, Panicum geminatum, P. glutinosum, P. mertensii, P. parvifolium, P. sellowii, Paspalum convexum, Pennisetum nervosum, P. setosum, Polypogon elongalus, Raddia costaricensis, Schizachyrium hirtijlorum.     

Rapid synthesis of oligodeoxyribonucleotides VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support

A new kieselguhr-polydimethylacrylamide support has been used in a continuous flow, column system for solid phase synthesis of oligodeoxyribonucleotides by a phosphotriester procedure. Using only protected mononucleotides a 14-mer, 20-mer and 27-mer were assembled in high repetitive yield using a simple manually operated, bench top apparatus.     

Influence of environmental temperature on mitochondrial membranes

Mitochondrial phospholipids from goldfish lateral line muscle were analysed with respect to polar and apolar groups. Groups of 20 goldfish, acclimated to 5, 20 and 30°C, were used. Temperature-induced shifts of both polar and apolar groups of the mitochondrial phospholipids were observed. The fatty acid composition of mitochondrial phospholipids is characterized by a large amount of polyenoic acids, dominated by docosahexaenoic acid and by octadecadienoic acid. At the higher acclimation temperatures, a significant decrease in docosahexaenoic acid is found. However, the resultant effect of environmental temperature on the degree of unsaturation is small, in contrast to the marked effect on mean chain length. Pronounced changes in the molar ratio of phosphatidylcholine and phosphatidylethanolamine are seen; a decrease in mitochondrial phosphatidylcholine is observed at low acclimation temperature, which is compensated for by a nearly equal increase in phosphatidylethanolamine. The main phospholipids are, apparently, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, comprising 90% of the total pool of 12 species. It is found that the anionic nature of the phospholipids is increased at low acclimation temperatures. We discuss this effect and its probable importance in the stabilization of the surface potential of the mitochondrial membranes.     

Lysis of Lactococcus lactis subsp. cremoris SK110 and Its Nisin-Immune Transconjugant in Relation to Flavor Development in Cheese

To develop a nisin-producing cheese starter, Lactococcus lactis subsp. cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.     

The pH dependent substrate specificity of udp-glucose: isovitexin 2″-O-glucosyltransferase in Silene alba

In Silene alba plants the dominant allele of gene Fg controls an enzyme which catalyses the formation of isovitexin 2″-O-glucoside both in petals and green parts. Both isovitexin and isoorientin can act as substrate. K_mvalues for the isovitexin glucosylation are 0.09 mM for isovitexin and 0.3 mM for UDP-glucose, V_max 0.17 nmol min^?1 mg protein^?1. For the isoorientin glucosylation K_m values of 0.45 mM for isoorientin, of 0.75 mM for UDP-glucose and V_max of 0.27 nmol min^?1 mg protein^?1 are found. The pH optima for both substrates differ markedly. For the substrate with one hydroxyl in the B-ring, isovitexin, the pH optimum is pH 8.5. For isoorientin, which has two hydroxyls in the B-ring, a pH optimum of 7.5 is found. These results suggest that the B-ring hydroxylation pattern influences the pH at which the substrate has optimal affinity for the enzyme. The location of the carbon-carbon bound glucose on a the flavonoid skeleton is of importance for enzyme activity as well. Vitexin, which has glucose at the 8-position, was not a substrate. The glucosylation of vitexin could, however, be demonstrated in enzyme extracts of petals of plants, grown from seed collected in Armenia; in these petals apart from isovitexin glycosides, vitexin glycosides are found as well.     

Dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats

The hypothesis was tested that dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats, which in turn causes a decreased copper absorption. Male rats were fed adequate-copper (5 mg Cu/kg) diets containing either fructose or glucose (709.4 g monosaccharide/kg) for a period of 5 wk. Fructose vs glucose significantly lowered copper concentrations in plasma and the liver, but did not alter hepatic copper mass. Fructose feeding resulted in a significantly lesser intestinal solubility of copper as based on either a smaller soluble fraction of copper in the liquid phase of small intestinal contents or a lower copper concentration in the liquid phase. The latter fructose effect can be explained by the observed fructose-induced increase in volume of liquid phase of intestinal digesta. After administration of a restricted amount of diet extrinsically labeled with⁶⁴Cu, rats fed fructose also had significantly lower soluble⁶⁴Cu fraction in the digesta of the small intestine. Although this study shows that fructose lowered intestinal copper solubility, only a slight reduction of apparent copper absorption was observed. It is suggested that the fructose-induced lowering of copper status in part counteracted the fructose effect on copper absorption at the level of the intestinal lumen.     

Quantitative Reverse Sample Genome Probing of Microbial Communities and Its Application to Oil Field Production Waters

This paper presents a protocol for quantitative analysis of microbial communities by reverse sample genome probing is presented in which (i) whole community DNA is isolated and labeled in the presence of a known amount of an added internal standard and (ii) the resulting spiked reverse genome probe is hybridized with a master filter on which denatured genomic DNAs from bacterial standards isolated from the target environment were spotted in large amounts (up to 1,500 ng) in order to improve detection sensitivity. This protocol allowed reproducible fingerprinting of the microbial community in oil field production waters at 19 sites from which water and biofilm samples were collected. It appeared that selected sulfate-reducing bacteria were significantly enhanced in biofilms covering the metal surfaces in contact with the production waters.