Evaluating fermentation characteristics of Kazachstania spp. and their potential influence on wine quality

World Journal of Microbiology and Biotechnology - The current study is the first one to demonstrate the wine fermentation potential of members of several species of the genus Kazachstania including...     

Structure–activity relationship of etomidate derivatives at the GABAA receptor: Comparison with binding to 11β-hydroxylase

At the GABA_A receptor, low concentrations of etomidate potentiate the inhibitory effect of GABA on specific binding of the closed channel ligand [³H]ethynylpropylbicycloorthobenzoate ([³H]EBOB). Here, we present SARs for etomidate and structurally related compounds inducing this effect. In the absence of GABA, similar SARs, but 14–20 times weaker potencies were observed. We discuss these SARs in comparison to the much higher potencies of these compounds as inhibitors of 11β-hydroxylase.     

Activation of the particulate and not the soluble guanylate cyclase leads to the inhibition of Ca2+ extrusion through localized elevation of cGMP

We examined whether localized increases in cytosolic cGMP have distinct regulatory effects on the concentration of cytosolic free Ca(2+) in ECV304 cells. Stimulation of the particulate guanylate cyclase by brain-type natriuretic peptide in fura-2-loaded cells caused a profound potentiation of the ATP-stimulated and thapsigargin-stimulated rise in cytosolic free Ca(2+). This effect is mediated by the inhibition of Ca(2+) extrusion via the plasma membrane Ca(2+)-ATPase pump. Furthermore, the addition of brain-type natriuretic peptide caused the partial inhibition of cation influx in ATP-stimulated cells. In contrast, elevation of cytosolic cGMP by activation of the soluble guanylate cyclase induced by the addition of sodium nitroprusside causes an increased reuptake of Ca(2+) into the intracellular stores without affecting cation influx or Ca(2+) efflux. Thus, localized pools of cGMP play distinct regulatory roles in the regulation of Ca(2+) homeostasis within individual cells. We define a new role for natriuretic peptides in the inhibition of Ca(2+) efflux that leads to the potentiation of agonist-evoked increases in cytosolic free Ca(2+).     

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.     

A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle.

We describe here a fluorescence assay for nitric oxide synthase activity in skeletal muscle based on a new indicator, 4,5-diaminofluorescein (DAF-2). The rapid and irreversible binding of DAF-2 to oxidized NO allows real-time measurement of NO production. The method is safer and more convenient than the usual citrulline radioassay and can be used with crude muscle extracts. Rabbit fast tibialis anterior (TA) muscle had a nitric oxide synthase (NOS) activity of 44.3 +/- 3.5 pmol/min/mg muscle. Addition of NOS blocker N(G)-allyl-L-arginine reduced this activity by 43%. Slow soleus muscle displayed NOS activity of 7.3 +/- 2.5 pmol/min/mg muscle, 16% that of the TA muscle. Continuous stimulation of TA muscle at 10 Hz for 3 weeks reduced NOS activity by 47% to an intermediate value consistent with the associated conversion of the muscle phenotype from fast to slow.     

Simultaneous analysis of 2-methoxyphenylmetyrapone and its seven potential metabolites by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic (HPLC) assay has been developed for the quantification of 2-methoxyphenylmetyrapone (2-MPMP) and its seven potential metabolites in rat urine and whole blood. 2-MPMP, 2-hydroxyphenylmetyrapone and their N-oxides, together with 2-methoxyphenylmetyrapol, 2-hydroxyphenylmetyrapol and their N-oxides were separated on an Isco Spherisorb ODS-2 reversed-phase column (250×4.6 mm, I.D., 5 μm), with an Isco Spherisorb ODS-2 guard cartridge (10×4.6 mm I.D.). A gradient elution was employed using solvent system A (acetonitrile–water–triethylamine–acetic acid, 27.3:69.1:0.9:2.7%, v/v) and solvent system B (methanol), the gradient program being as follows: initial 0–4 min A:B=74:26; 4–10 min linear change to A:B=50:50; 10–16 min maintain A:B=50:50; 16 min return to initial conditions (A:B=74:26). Flow-rate was maintained at 1.25 ml/min, and the eluent monitored using a diode array multiple wavelength UV detector set at 260 nm. Most of the analytes were baseline resolved, and analysis of samples recovered from blood or urine (pH 12, 3×5 ml of dichloromethane, recovery 20–95%) revealed no interference from any co-extracted endogenous compounds in the biological matrices, except for 2-hydroxyphenylmetyrapol N-oxide (2-OHPMPOL-NO) at low concentrations. The calibrations (n=6) were linear (r≥0.996) for all analytes (0.5–100 μg/ml), with acceptable inter- and intra-day variability. Subsequent validation of the assay revealed acceptable precision, as measured by coefficient of variation (C.V.) at the low (0.5 mg/ml), medium (50 μg/ml) and high (100 μg/ml) concentrations. The limits of detection for 2-MPMP and their available potential metabolites, except 2-OHPMPOL-NO, in rat urine and blood were both 0.5 μg/ml, respectively.