Combining proteomics and lipid analysis to unravel Confidor stress response in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model for studying the influence of different stress factors on eukaryotic cells. In this work we used the pesticide imidacloprid, in the Confidor formulation, as the stress factor and analyzed its influence on the metabolic activity, proteome and lipid content and composition of Saccharomyces cerevisiae yeast. During the cultivation of yeast, the lowest recommended application dose of Confidor (0.025%, v/v) was added to the growth media and its influence on the mitochondria, cytosol with microsomes, and the whole yeast cells was monitored. The results show that under the stress provoked by the toxic effects of Confidor, yeast cells density significantly decreased and the percentage of metabolically disturbed cells significantly increased comparing with untreated control. Also, there was a downregulation of majority of glycolytic, gluconeogenesis, and TCA cycle enzymes (Fba1, Adh1, Hxk2, Tal1, Tdh1,Tdh3, Eno1) thus providing enough acetyl‐CoA for the lipid restructuring and accumulation mechanism since we have found the changes in the cell and mitochondrial lipid content and FA composition. This data suggest that lipids could be the molecules that orchestrate the answer of the cells in the stress response to the Confidor treatment.     

Heteroepitaxy of Cerium Oxide Thin Films on Cu(111)

An important part of fundamental research in catalysis is based on theoretical and modeling foundations which are closely connected with studies of single-crystalline catalyst surfaces. These so-called model catalysts are often prepared in the form of epitaxial thin films, and characterized using advanced material characterization techniques. This concept provides the fundamental understanding and the knowledge base needed to tailor the design of new heterogeneous catalysts with improved catalytic properties. The present contribution is devoted to development of a model catalyst system of CeO₂ (ceria) on the Cu(111) substrate. We propose ways to experimentally characterize and control important parameters of the model catalyst—the coverage of the ceria layer, the influence of the Cu substrate, and the density of surface defects on ceria, particularly the density of step edges and the density and the ordering of the oxygen vacancies. The large spectrum of controlled parameters makes ceria on Cu(111) an interesting alternative to a more common model system ceria on Ru(0001) that has served numerous catalysis studies, mainly as a support for metal clusters.     

Reduced ACTH/cortisol responses to naloxone in men with Parkinson's disease

Summary In order to establish whether the inhibitory control exerted by endogenous opioid peptides on ACTH/cortisol secretion changes in patients affected by Parkinson's disease, ten parkinsonian male subjects and eight age matched normal controls were tested with naloxone (4 mg an i.v. bolus plus 10 mg infused in two hours). In a different occasion all subjects were tested with normal saline. Experiments started at 09.00 h. Plasma ACTH and cortisol concentrations showed a slight physiological decline during saline test in both groups. In the normal controls and in the parkinsonian patients both ACTH and cortisol levels were significantly higher after naloxone administration than during saline test. However, both naloxone induced ACTH and cortisol responses were significantly higher in normal than in parkinsonian subjects. In agreement with the well-known opioid deficiency characterizing the parkinsonian brain, these data show a reduced opioid inhibitory control of ACTH/cortisol secretion in patients with Parkinson's disease.     

Urinary mercapturic acid diastereoisomers in rats subchronically exposed to styrene and ethanol

 Styrene is stereoselectively oxidized by cytochrome P450 to its reactive metabolite, styrene oxide. The (R)- and (S)-enantiomers of styrene oxide can be conjugated with glutathione (GSH) to both (R)- and (S)-diastereoisomers of the specific mercapturic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). Several investigations have indicated different toxic potential of the (R)- and (S)-configurations of styrene oxide and its GSH- and N-acetyl-conjugates. In this study the mercapturic acid diastereoisomers were measured in the urine of rats exposed to styrene in combination with ethanol, a good inducer of styrene metabolism. Male Sprague-Dawley rats were given an isocaloric liquid diet containing ethanol (5% w/v) for 3 weeks. Starting from the 2nd week, the animals were also exposed to styrene vapours (300 ppm, 6 h/day, 5 days/week) in a dynamic exposure chamber. Both the (R)- and (S)-diastereoisomers of the M1 and M2 as well as the conventional biomarkers, mandelic acid (MA) and phenylglyoxylic acid (PGA) were measured in urinary samples. Approximately 30 and 25% reduction of the levels of brain non-protein sulfhydryls (NPS) was observed in the animals given styrene and ethanol, respectively, while the combined ethanol and styrene treatment resulted in a 60% decrease. Ethanol consumption also resulted in higher urinary levels of the M1-R, M1-S and M2 metabolites associated with increased M1-R/S ratio and higher urinary MA excretion compared to animals treated with styrene. These results suggest that the urinary mercapturic acid diastereoisomers may be used as a noninvasive tool to examine stereoselective patterns of styrene metabolism in vivo, as well as their alterations caused by ethanol. These compound-specific mercapturic acids may also be valuable indicators of styrene-induced disorders of GSH homeostasis in nonaccessible organs. Received: 19 December 1995/Accepted: 10 May 1996     

Sphaerospora renicola n.sp., a myxosporean from carp kidney,and its pathogenicity

Sphaerospora renicola n.sp. is a common parasite of carp in Czechoslovakia. Its life cycle involves intracellular stages in the epithelial cells of renal tubuli and trophozoite stages proliferating in the tubular lumen, transforming ultimately into pansporoblasts, each having one pansporoblast nucleus and producing two spores. The spores are almost globular with an average size of 7.3×7.2 , with polar capsules of equal size, and may have two slightly protruding tubercles on their shell valves. Differential diagnosis from otherSphaerospora species infecting carp, as well as fromMitraspora cyprini Fujita, is made. Intracellular stages ofS. renicola cause swelling and hyperplasia of the epithelium in renal tubuli followed by dystrophic changes. Accumulation of developmental stages in the tubular lumen provokes pronounced regressive changes of the epithelium, which may be followed by necrosis.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-microl volume of urine is added to a carbonate-hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70 degrees C for 10 min. The 'dabsylated' product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C18 column (250 x 4.6 mm, 5 microm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5-40.8 mg/m3), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.     

Trans,trans-muconic acid, a biological indicator to low levels of environmental benzene: some aspects of its specificity

BACKGROUND: The specificity of trans,trans-muconic acid (MA) as a biomarker of exposure to low benzene levels and the role of sorbic acid (SA) as a confounding factor were evaluated. MA, a urinary ring-opened metabolite of benzene, has been recently proposed for the biological monitoring of populations exposed to low levels of this chemical. The usual presence of MA in urine of non-occupationally exposed people is generally attributed to benzene world-wide contamination (mainly by smoking habits, urban pollution, and maybe by food contamination). However, the scientific literature reveals that the common food preservative and fungistatic agent SA is converted into MA though in trace amounts. METHODS: Urinary benzene and MA before and after administration of SA were measured in smokers and non-smokers. Benzene dissolved in urine was analyzed injecting a headspace sample in a gas-chromatografic system. Urinary MA was measured by means of a HPLC apparatus. RESULTS: The mean background values of MA were about 60 mg/L (or 50 mg/g creat.); after experimental administration of SA (447 mg), the mean urinary MA concentration became more than 20 times higher. The biotransformation rates of SA into MA after ingestion of 447 mg of SA ranged from 0.05 to 0.51%. The ratio between unmetabolized benzene in the two groups of smokers and non-smokers was significantly different from the ratio between MA in the same two groups. DISCUSSION: Other sources of MA excretion, different from benzene, influence the urinary concentration of the metabolite: only 25% of MA background values can be attributed to benzene. The urinary MA induced by 100 mg of ingested MA is 77% of that expected after an 8-hour benzene exposure to 0.5 ppm (current threshold limit value according to ACGIH). In conclusion, MA is not a sufficiently specific biomarker of low benzene exposure; a significant effect of SA ingestion is predictable.