Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.     

Lack of Physiological Depth Patterns in Conspecifics of Endemic Antarctic Brown Algae: A Trade-Off between UV Stress Tolerance and Shade Adaptation?

A striking characteristic of endemic Antarctic brown algae is their broad vertical distribution. This feature is largely determined by the shade adaptation in order to cope with the seasonal variation in light availability. However, during spring-summer months, when light penetrates deep in the water column these organisms have to withstand high levels of solar radiation, including UV. In the present study we examine the light use characteristics in parallel to a potential for UV tolerance (measured as content of phenolic compounds, antioxidant activity and maximum quantum yield of fluorescence) in conspecific populations of four Antarctic brown algae (Ascoseira mirabilis, Desmarestia menziesii, D. anceps and Himantothallus grandifolius) distributed over a depth gradient between 5 and 30 m. The main results indicated that a) photosynthetic efficiency was uniform along the depth gradient in all the studied species, and b) short-term (6 h) exposure to UV radiation revealed a high tolerance measured as chlorophyll fluorescence, phlorotannin content and antioxidant capacity. Multivariate analysis of similarity indicated that light requirements for photosynthesis, soluble phlorotannins and antioxidant capacity are the variables determining the responses along the depth gradient in all the studied species. The suite of physiological responses of algae with a shallower distribution (A. mirabilis and D. menziesii) differed from those with deeper vertical range (D. anceps and H. grandifolius). These patterns are consistent with the underwater light penetration that defines two zones: 0–15 m, with influence of UV radiation (1% of UV-B and UV-A at 9 m and 15 m respectively) and a zone below 15 m marked by PAR incidence (1% up to 30 m). These results support the prediction that algae show a UV stress tolerance capacity along a broad depth range according to their marked shade adaptation. The high contents of phlorotannins and antioxidant potential appear to be strongly responsible for the lack of clear depth patterns in light demand characteristics and UV tolerance.     

Ubiquitination-dependent quality control of hERG K+ channel with acquired and inherited conformational defect at the plasma membrane

Membrane trafficking in concert with the peripheral quality control machinery plays a critical role in preserving plasma membrane (PM) protein homeostasis. Unfortunately, the peripheral quality control may also dispose of partially or transiently unfolded polypeptides and thereby contribute to the loss-of-expression phenotype of conformational diseases. Defective functional PM expression of the human ether-a-go-go–related gene (hERG) K⁺ channel leads to the prolongation of the ventricular action potential that causes long QT syndrome 2 (LQT2), with increased propensity for arrhythmia and sudden cardiac arrest. LQT2 syndrome is attributed to channel biosynthetic processing defects due to mutation, drug-induced misfolding, or direct channel blockade. Here we provide evidence that a peripheral quality control mechanism can contribute to development of the LQT2 syndrome. We show that PM hERG structural and metabolic stability is compromised by the reduction of extracellular or intracellular K⁺ concentration. Cardiac glycoside–induced intracellular K⁺ depletion conformationally impairs the complex-glycosylated channel, which provokes chaperone- and C-terminal Hsp70-interacting protein–dependent polyubiquitination, accelerated internalization, and endosomal sorting complex required for transport–dependent lysosomal degradation. A similar mechanism contributes to the down-regulation of PM hERG harboring LQT2 missense mutations, with incomplete secretion defect. These results suggest that PM quality control plays a determining role in the loss-of-expression phenotype of hERG in certain hereditary and acquired LTQ2 syndromes.     

Prevalence and antifungal drug sensitivity of non‐albicans Candida in oral rinse samples of self‐caring elderly

doi: 10.1111/j.1741‐2358.2010.00407.x
Prevalence and antifungal drug sensitivity of non‐albicans Candida in oral rinse samples of self‐caring elderly Aim: To assess the prevalence and antifungal drug sensitivity of non‐albicans Candida (NAC) species in elderly outpatients. Materials and methods: We investigated oral rinse samples of 194 self‐caring elderly population (mean age 83 years) with emphasis on background factors for harbouring NAC. Susceptibility of Candida species to antifungal drugs was determined using standard methodology. Multiple logistic regression analysis was performed taking positive NAC count as the dependent variable and a number of known Candida risk factors as independent variables. Results: Prevalence of candidal carriage of the population was 78.4%, of which 0.5% of the subjects were NAC positive. Candida dubliniensis was the most prevalent NAC species, followed by Candida glabrata and Candida parapsilosis. The NAC positive elderly were more often edentulous with dental prostheses or had fewer teeth than Candida albicans‐positive or yeast‐negative subjects. Dental caries slightly increased the risk for having NAC strains (odds ratio 1.08), whilst greater age appeared to lower the risk (odds ratio 0.77). Candida species were susceptible to the commonly used antifungal agents in general, but with considerable variation among species. Occasionally, some NAC exhibited lower antifungal susceptibility. Conclusion: The possibility of oral reservoirs of NAC strains which are resistant to common antifungals should be noted in elderly outpatients.     

Protein quality control at the plasma membrane

Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined.     

Improved embryogenesis from anther culture and plant regeneration in timothy

Timothy (Phleum pratense L.) is an important forage grass grown in northern temperate areas. Development of haploid cell culture techniques for timothy has been limited due to the recalcitrance of timothy in tissue culture. In this study, timothy anther culture techniques were established. Liquid PG-96 (Pulli and Guo, 1996) induction medium significantly promoted embryo yield; the best result was 800–1000 embryos (calli) per 100 anthers. Genotype was an important factor in androgenetic embryogenesis of timothy. Embryos were obtained from 16 genotypes out of the 28 genotypes tested. The optimum stage for microspore development was between the very late uninucleate stage and the binucleate stage. Cold pretreatment applied to the donor plants (spikes) increased embryo yield. Despite a high embryo induction rate, green plant regeneration rate was relatively low. The frequency of albinos was reduced by use of low light intensity conditions during regeneration. Over 300 green plants were recovered. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mechanisms of D-Aspartate Release Under Ischemic Conditions in Mouse Hippocampal Slices

The release of preloaded D-[³H]aspartate, an unmetabolizable analogue of L-glutamate, was studied in superfused hippocampal slices from 7-day-old and 3-month-old (adult) mice under various cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals and metabolic poisons. The release was generally markedly enhanced in most of the above conditions, the responses being greater in adults than in developing mice. The presence of dinitrophenol had the most pronounced effect at both ages, followed by NaCN- and free-radical-containing media and ischemia. Hypoxia did not affect release in the immature hippocampus. Under most conditions K⁺ stimulation (50 mM) was still able markedly to enhance D-aspartate release. This potentiation under cell-damaging conditions in both adult and developing hippocampus signifies that increased L-glutamate release contributes to excitotoxicity and subsequent cell death. The mechanisms of ischemia-induced release of D-aspartate were analyzed in the adult hippocampus using ion channel inhibitors and modified superfusion media. The induced release proved to be partly Ca²⁺-dependent and partly Ca²⁺-independent. The results obtained with Na⁺ omission and homo- and heteroexchange with D-aspartate and L-glutamate demonstrated that a part of the release in normoxia and ischemia is mediated by the reversal of Na⁺-dependent glutamate transporters. The Na⁺ channel blockers amiloride and riluzole reduced the ischemia-induced release, also indicating the involvement of Na⁺ channels. In addition to this, the enhanced release of D-aspartate may comprise a swelling-induced component through chloride channels.     

Lamotrigine and Carbamazepine Affect Differently the Release of D-[3H]Aspartate from Mouse Cerebral Cortex Slices: Involvement of NO

The effects of lamotrigine and carbamazepine on the release of preloaded D-[³H]aspartate and the involvement of nitric oxide were studied with mouse cerebral cortical slices in a superfusion system. Lamotrigine inhibited the veratridine-evoked release, whereas the K⁺-stimulated release was attenuated more strongly by carbamazepine than by lamotrigine. These effects were accentuated by the N-methyl-D-aspartate receptor antagonist L-2-amino-5-phosphonovalerate and the nitric oxide synthase inhibitor L-nitroarginine, but diminished by the nitric oxide donor sodium nitroprusside. The results show that in addition to the blockade of voltage-sensitive Na⁺ (and Ca²⁺) channels, NO-mediated mechanisms are probably involved in the anticonvulsant actions of carbamazepine and, in particular, those of lamotrigine.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.