Determination of doxazosin and verapamil in human serum by fast LC-MS/MS: application to document non-compliance of patients

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of doxazosin and verapamil in human serum has been developed. Trimipramine-d₃ as an isotopic labelled internal standard was used for quantification. Serum samples were prepared by simple liquid–liquid extraction with mixture of tert butyl methyl ether and ethyl acetate (1:1, v:v). The analytes and internal standard were separated on C18 column using an isocratic elution with 5 mM ammonium formate with 0.02% formic acid and 0.02% formic acid in acetonitrile (55:45, v:v) at a flow rate of 1.1 mL/min. Positive TurboIonSpray mass spectrometry was used with multiple reaction monitoring of the transitions at: m/z 455.3 → 165.2 and 150.2 for verapamil, m/z 452.2 → 344.4 and 247.4 for doxazosin, m/z 298.2 → 103.1 for trimipramine-d₃. Linearity was achieved between 1 and 500 ng/mL (R² ≥ 0.997) for both analytes. An extensive pre-study method validation was carried out in accordance with FDA guidelines. This assay was successfully applied to determine the serum concentrations of doxazosin and verapamil in suspect non-compliance patients.     

Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol

Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and preconditioning- related proteins were identified that were all upregulated by resveratrol: MAPKK, two different alphaB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxiredoxin- 2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -alphaB-crystallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofilament regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins.     

Rapid redistribution of synaptic PSD-95 in the neocortex in vivo

Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10–21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times τ_r ~ 22–63 min from P10–P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (τ_r ~ 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size.     

Synthesis and mass spectrometric fragmentation characteristics of imidazole ribosides-analogs of intermediates of purine de novo synthetic pathway

Two inherited deficiencies have been described in purine de novo synthesis pathway. Both the defects are diagnosed by detecting ribosides--dephosphorylated substrates of the enzymes--in patient's urine. We describe here a synthesis and mass spectrometric fragmentation of ribosides potentially of diagnostic importance for defects in the second part of the pathway. All the species, except 5-amino-4-imidazolesuccinocarboxamideriboside can be synthesized from the commercially available 5-amino-4-imidazolecarboxamideriboside by chemical methods. Fragmentation spectra of the compounds were obtained by the ion trap mass spectrometry. During fragmentation an opening of the imidazole ring was not observed for any of the compounds but loss of its substituents in the form of small molecules (NH3, CO2, CO) is the major route of fragmentation. The ribose moiety cleaves off molecule(s) of water, undergoes a cross-ring cleavage or breaks away as a whole.     

Analysis of intracellular nucleotides by capillary electrophoresis-mass spectrometry

A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism--AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.     

The effects of feed restriction on plasma biochemistry in growing meat type chickens (Gallus gallus)

The effect of feed restriction on plasma hormones (triiodothyronine - T(3), thyroxine - T(4), and corticosterone), protein, lipid, carbohydrate, and mineral metabolism and activity of plasma enzymes (creatine kinase, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase) were studied in meat type female chickens (Gallus gallus). Ad libitum fed birds were compared with those subjected to severe and moderate quantitative feed restriction from 16 to 100 days of age. Feed restriction elevated plasma T(4) and corticosterone levels and reduced T(3). A feed restriction-induced decrease was observed for plasma protein and albumin concentrations, but not for uric acid and creatinine. Total plasma lipids, triacylglycerols, cholesterol, high density lipids, and calcium were lower for the feed restricted chickens, in particular during the latter phase of the experiment. Concentrations of glucose and phosphorus were not altered by feeding treatment. Activity of alkaline phosphatase was significantly increased in restricted chicks from day 58. Significant changes of plasma biochemical parameters induced by severe and moderate quantitative feed restriction illustrate that limiting feed intake poses an intensive stress on meat type chickens during the rapid growth period. However, activities of creatine kinase, aspartate aminotransferase, and alanine aminotransferase were significantly higher in ad libitum fed chickens during this period. This elevation in enzymatic activity may be in response to tissue damage, indicating potential health and welfare problems also in ad libitum fed meat type chickens, resulting from selection for intensive growth.     

Das Wachstum beim absoluten Hungern

Ohne Zusammenfassung     

The role of endogenous growth regulators in the differentiation processes of walnut (Juglans regia L.)

The character of endogenous growth substances was investigated in developing buds, young fruits and mature walnut leaves. The relatively high content of auxins and gibberellin-like substances was found by means of bioassays in the youngest primordia of vegetative buds. The level of auxins drops in the further course of primordia transformation into the staminate catkins. The development of leaf-buds is characterized by the accumulation of inhibitory activity as revealed by theAvena bioassay, whereas the data obtained from the lettuce bioassay indicate a pronounced stimulation. The onset of terminal bud development is also accompanied by inhibitions and it is only with pistillate flower differentiation that the temporary rise in auxin level is observed. An inhibitory activity was found in these extracts using lettuce bioassay. There is a relatively high auxin level in young fruits, mature leaves and resting buds during the mid-summer period whereas the accumulation of clearcut inhibitions is signalled by the results of lettuce bioassay. The regulatory role of growth substances in differentiation may be better understood during the second year as many leaf-abnormities appear only with the outgrowing of the bud. Abnormal catkins differ in the number of florets and stamens and some even bear pistillate flowers. Fruit development is liable to deviations in the early stages of differentiation. Abnormal fruits enable us to elucidate many structural peculiarities.     

Tetrachloroethene-dehalogenating bacteria

Tetrachloroethene is a frequent groundwater contaminant often persisting in the subsurface environments. It is recalcitrant under aerobic conditions because it is in a highly oxidized state and is not readily susceptible to oxidation. Nevertheless, at least 15 organisms from different metabolic groups, viz. halorespirators (9), acetogens (2), methanogens (3) and facultative anaerobes (2), that are able to metabolize tetrachloroethene have been isolated as axenic cultures to-date. Some of these organisms couple dehalo-genation to energy conservation and utilize tetrachloroethene as the only source of energy while others dehalogenate tetrachloroethene fortuitously. Halorespiring organisms (halorespirators) utilize halogenated organic compounds as electron acceptors in an anaerobic respiratory process. Different organisms exhibit differences in the final products of tetrachloroethene dehalogenation, some strains convert tetrachloroethene to trichloroethene only, while others also carry out consecutive dehalogenation to dichloroethenes and vinyl chloride. Thus far, only a single organism, 'Dehalococcoides ethenogenes' strain 195, has been isolated which dechlorinates tetrachloroethene all the way down to ethylene. The majority of tetrachloroethene-dehalogenating organisms have been isolated only in the past few years and several of them, i.e., Dehalobacter restrictus, Desulfitobacterium dehalogenans, 'Dehalococcoides ethenogenes', 'Dehalospirillum multivorans', Desulfuromonas chloroethenica, and Desulfomonile tiedjei, are representatives of new taxonomic groups. This contribution summarizes the available information regarding the axenic cultures of the tetrachloroethene-dehalogenating bacteria. The present knowledge about the isolation of these organisms, their physiological characteristics, morphology, taxonomy and their ability to dechlorinate tetrachloroethene is presented to facilitate a comprehensive comparison.     

Thyrotropin-releasing hormone-induced depletion of G(q)alpha/G(11)alpha proteins from detergent-insensitive membrane domains

Na+ transport through the F0 domain of Na(+)-F1F0-ATPases involves the combined action of subunits c and a but the residues involved in Na+ liganding in subunit a are unknown. As a first step towards the identification of these residues, we have cloned and sequenced the gene encoding subunit a of the Na(+)-F1F0-ATPase of Acetobacterium woodii. This is the second sequence available now for this subunit from Na(+)-F1F0-ATPases. A comparison of subunit a from Na(+)-F1F0-ATPases with those from H(+)-translocating enzymes unraveled structural similarity in a C-terminal segment including the ultimate and penultimate transmembrane helix. Seven residues are conserved in this region and, therefore, likely to be involved in Na+ liganding.