In Vivo and in Vitro Activity And Mechanism Of Action of the Multidrug Cytarabine-L-Glycerylyl-Fluorodeoxyuridine

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 μM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 μ M), but no activity in FM3A/TK^? (IC50: 18.3 μM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK^? (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7–70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.     

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.     

Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris.

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.     

Structure and electrophysiological responses of gustatory organs on the ovipositor of the parasitoid Leptopilina heterotoma

Location, structure and histology of chemosensilla on the tip of the ovipositor of the parasitoid Leptopilina heterotoma are described based on SEM and TEM studies. Furthermore, we developed a method for recording extracellular action potentials from the gustatory neurons in response to host haemolymph. This method allowed us to record multi-unit recordings from a sensillum occurring singly on the unpaired ovipositor valve. The TEM study of the ovipositor tip revealed the presence of six dendrites, the electrophysiological recordings provided evidence for the activity of three or possibly four gustatory neurons in response to the complex stimulus offered, leaving other taste functions or a mechanoreceptor function open for the remaining neurons.     

Physiological variations in plasma testosterone in normal adult male subjects measured by a competitive protein-binding technique

A method of estimating plasma testosterone utilizing a single paper chromatographic procedure and competitive protein-binding is described. The influence of some conditions of sample collection on plasma testosterone has been investigated. Delay in the separation of plasma from cells and the type of anticoagulant used were both without significant effect. There was an approx. two-fold variability in plasma testosterone concn. in samples withdrawn daily, weekly and monthly from four young, normal adult males.     

Antioxidative properties of Lactobacillus sake upon exposure to elevated oxygen concentrations

The ability of bacteria to overcome oxidative stress is related to the levels and types of antioxidative mechanisms which they possess. In this study, the antioxidative properties in Lactobacillus sake strains from different food origins were determined at low temperature (8 degrees C) and upon exposure to oxygen levels between 20 and 90% O(2). The L. sake strains tested grew well at 8 degrees C and in the presence of 20% O(2), however, most of the strains could not grow at O(2) levels as high as 50 and/or 90%. Cell-free extracts of all strains possessed certain levels of hydroxyl radical scavenging, metal chelating and reducing capacities essential for growth of cells at ambient O(2). At elevated O(2) concentrations, a high H(2)O(2) splitting capacity and low specific rates of H(2)O(2) production were demonstrated in the O(2)-insensitive strain L. sake NCFB 2813, which could grow at elevated O(2) conditions. Although H(2)O(2) was generated in the O(2)-sensitive L. sake DSM 6333 at levels which were not directly toxic to the cells (<0.2 mM), we can conclude that its removal is essential for cell protection at elevated O(2) conditions.     

Enantiomers of 2-methyl- and 2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid: Preparation by resolution,determination of absolute configuration,and Curtius rearrangement

Both enantiomers of 2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid 2 and 2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid 3 were prepared via resolution of the corresponding racemic carboxylic acids with (R)- and (S)-1-phenylethylamine, respectively. Absolute configuration of (−)-(R)-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid was determined by X-ray crystallography. Curtius rearrangement of acyl azides prepared from enantiomers of these heterocyclic carboxylic acids carried out in benzyl alcohol afforded enantiomers of the corresponding benzyl carbamates, which upon hydrogenolysis gave racemic 2-amino-2-methyl-3,4-dihydro-2H-1,4-benzoxazin-3-one 4 and 2-amino-2,4-dimethyl-3,4-dihydro-2H-1,4h-benzoxazin-3-one 5. Chirality 10:791–799, 1998. © 1998 Wiley-Liss, Inc.     

Propionibacterium freudenreichii thrives in microaerobic conditions by complete oxidation of lactate to CO2

In this study we show increased biomass formation for four species of food-grade propionic acid bacteria (Acidipropionibacterium acidipropionici, Acidipropionibacterium jensenii, Acidipropionibacterium thoenii and Propionibacterium freudenreichii) when exposed to oxygen, implicating functional respiratory systems. Using an optimal microaerobic condition, P. freudenreichii DSM 20271 consumed lactate to produce propionate and acetate initially. When lactate was depleted propionate was oxidized to acetate. We propose to name the switch from propionate production to consumption in microaerobic conditions the ‘propionate switch’. When propionate was depleted the ‘acetate switch’ occurred, resulting in complete consumption of acetate. Both growth rate on lactate (0.100 versus 0.078 h⁻¹) and biomass yield (20.5 versus 8.6 g* mol⁻¹ lactate) increased compared to anaerobic conditions. Proteome analysis revealed that the abundance of proteins involved in the aerobic and anaerobic electron transport chains and major metabolic pathways did not significantly differ between anaerobic and microaerobic conditions. This implicates that P. freudenreichii is prepared for utilizing O₂ when it comes available in anaerobic conditions. The ecological niche of propionic acid bacteria can conceivably be extended to environments with oxygen gradients from oxic to anoxic, so-called microoxic environments, as found in the rumen, gut and soils, where they can thrive by utilizing low concentrations of oxygen.