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In this work we report on the capacitive response of interdigitated electrodes (IDEs) covered with a thin (~50 μm) polydimethyl siloxane (PDMS; Sylgard ® 184) film to (1) the deposition and evaporation of drops of pure volatile organic compounds (VOCs) and to (2) the direct exposure to aqueous solutions of these VOCs (up to 1 mM) under continuous flow conditions. The investigated VOCs were chloroform, methyl tert -butyl ether, 1-hexanol, toluene, m-xylene, and n-hexane. It is shown that the capacitive response changes upon absorption of the VOCs are in both cases in line with their polymer affinity and their relative dielectric constants as compared to the one of thin PDMS layers. In addition, the response changes were fully reversible. Chloroform, methyl tert -butyl ether, and 1-hexanol were measured reproducibly with a detection limit of ~0.1 mM. For toluene, m-xylene, and n-hexane the response reproducibility was strongly impaired due to their low water solubility and the lowest measured concentrations were in the order of ~0.2 mM for the aromatics and ~0.009 mM for n-hexane. Within a closed flow cell system, the presence of a bypass circuit and/or a parasitic parallel impedance to the environment (third electrode effect) was observed. In the case of n-hexane and m-xylene additional features of the response profile were observed before reaching equilibrium, revealing the contribution of additional response mechanisms. This could be rationalized by an overshoot of the VOC concentration in the PDMS or by swelling effects. To conclude, this work reports on the capability of direct detection of organic pollutants in water by capacitive PDMS-coated IDE platforms. It points out the challenges in system design and response interpretation, which facilitates the further development of water pollutant sensoring systems.

Aims: To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria from traditional fermented foods. Methods and results: A total of 119 strains of LAB from fermented meat (nem chua) were analyzed with both (GTG)5-PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)5-PCR and in MALDI-TOF MS; five species grouped alike for (GTG)5-PCR and for MALDI-TOF MS, however, differences in minimal similarity between the delineated (GTG)5-PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)5-PCR profiles; two species, each represented by a single MALDI-TOF cluster were subdivided in the corresponding (GTG)5-PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analyzed using MALDI- TOF MS. PheS gene sequencing was used for validation. Conclusions: MALDI-TOF MS is a powerful, fast, reliable, and cost effective technique for the identification of lactic acid bacteria (LAB) associated with the production of fermented foods. Significance and Impact of the Study: Food LAB can be identified using MALDI-TOF MS and its application could possibly be extended to other food matrices and/or other food derived micro-organisms.

Saccharomyces cerevisiae dihydroceramidase Ydc1p hydrolyzes ceramide, resulting in accumulation of free long-chain bases and their phosphates. Yeast mutants lacking YDC1 are characterized by increased chronological lifespan. Moreover, we found YDC1 up-regulated in a yeast mutant displaying reduced chronological lifespan. These data suggest an important role for Ydc1p in chronological lifespan determination in yeast. Mitochondria are known to play an important role in chronological lifespan and apoptosis. In this study we demonstrated that overexpression of YDC1 results in reduced chronological lifespan and increased apoptotic cell death. We found YDC1 overexpression to result in mitochondrial fragmentation and dysfunction. Interestingly, vacuoles also appeared to be fragmented and dysfunctional upon YDC1 overexpressing. Exogenous addition of ceramide to YDC1-overexpressing cultures increased chronological lifespan and restored organelle function. In conclusion, this study describes a direct link between ceramide metabolism in yeast and mitochondrial and vacuolar fragmentation and function, with consequences for chronological lifespan in yeast.

RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant ΔgcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.

The matured technology of electronic noses and tongues makes it promising to utilize the technique of combining chemically diverse sensor multi-arrays with multi-component analysis tools and pattern recognition techniques for a wider range of medical and environmental applications. With a selfdesigned sensor system and flow cell setup-up we studied the capacitive response of a polydimethyl siloxane (PDMS) covered interdigitated electrode (IDE) sensor upon exposure to methyl tert-butyl ether (MtBE) contaminated MilliQ water and plain mineral water. The sensor responded to MtBE concentrations as low as 2 ppm for both aqueous solutions with full reversibility. Signal drifts due to temperature effects can be remedied by sensor calibration. State-of-the-art sensor interfaces, system minimization and integration, and smart sensing layer design promise further significant optimization possibilities for system sensitivity, stability and response time.

The antifungal plant defensin DmAMP1 interacts with fungal sphingolipids of mannosyldiinositolphosphorylceramide (M(IP)2C) class. We screened a Saccharomyces cerevisiae transposon (Tn) mutant library against DmAMP1 and identified one DmAMP1-resistant mutant with the Tn inserted in the M(IP)2C biosynthesis gene IPT1 (DmTn11) and one DmAMP1-hypersensitive mutant with the Tn inserted in rDNA (HsTnII). However, tetrad analysis pointed to HsTnII as a spontaneous mutant. Apparently, membranes of DmTn11 lack M(IP)2C, whereas membranes of HsTnII have increased M(IP)2C levels. In addition, DmTn11 and HsTnII are characterized by increased and reduced oxidative stress resistance/chronological life-span (CL), respectively. A putative involvement of M(IP)2C in oxidative stress and CL in yeast is discussed.

The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)(2)C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP)(2)C-biosynthesis gene IPT1, as a novel DmAMP1-sensitivity gene in Saccharomyces cerevisiae. SKN1 was previously shown to be a KRE6 homologue, which is involved in beta-1,6-glucan biosynthesis. We demonstrate that a Deltaskn1 mutant lacks M(IP)(2)C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP)(2)C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae.

The biosynthesis and maturation of glucocerebrosidase were studied in fibroblasts from patients with the neurological and non-neurological forms of Gaucher disease and in control cells. In control fibroblasts the precursor of glucocerebrosidase (62-63 kDa), observed after a short pulse with [35S]methionine, was converted during the chase period to a 66-kDa intermediate form and, finally, to the 59-kDa mature protein. In fibroblasts from patients with the non-neurological phenotype of Gaucher disease (type 1) the same biosynthetic forms were seen as in control fibroblasts. These biosynthetic forms correspond to the three-banded pattern seen in control and Gaucher type 1 fibroblast extracts analysed by the immunoblotting procedure, or after electrophoresis and fluorography of extracts of such fibroblasts cultured for 5 days with [14C]leucine. The 59-kDa protein seen in type 1 fibroblasts was unstable and disappeared after a prolonged chase; this disappearance was not observed when the cells were grown in the presence of leupeptin. In fibroblasts from patients with the neurological forms of Gaucher disease (types 2 and 3) the 62.5-kDa precursor of glucocerebrosidase was present in near-normal amounts after a short pulse, but the 59-kDa form was not detected even when cells were cultured with leupeptin. These results are in accordance with the absence of the 59-kDa band in immunoblots of types 2 and 3 fibroblast extracts. Culturing of type 1, type 2 and type 3 Gaucher fibroblasts in the presence of leupeptin led to an increase in the activity of glucocerebrosidase.

Summary The effect of aspartate-derived amino acids on growth of Arabidopsis and barley plants and callus was studied. In barley plandets a concerted inhibition due to the addition of lysine and threonine on the culture medium was shown. The effect is reversed by the addition of methionine or its precursor homoserine. No such effect could be demonstrated either on barley callus or in Arabidopsis plantlets and callus, until arginine was added to the medium. Lysine itself exerted a growth inhibition and this effect is specifically relieved by the addition of arginine. The origin of this relief of lysine inhibition by arginine is discussed. Taking into account the characteristics of the various plant systems, a selection scheme to obtain regulatory mutants is proposed.

The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.

The binding of series of alkyl and aryl β- d -glycopyranosides and their 1-thio analogues to the active site of β- d -glucosidase from Stachybotrys atra has been investigated. The binding constants for competitive and uncompetitive inhibition clearly demonstrated the existence of a hydrophobic aglycon-binding-site. The correlations found between competitive and uncompetitive inhibition suggest that the latter type of inhibition originates from the unspecific binding of the aglycon group to the aglycon binding-site of the intermediary enzyme-glycosyl complex.

Tumours were induced on Arabidopsis thaliana by wounding the stem and infecting with Agrobacterium tumefaciens strains T37, C58 or Ach5. After about three weeks the tumours were excised and cultured in vitro on solidified medium without growth hormones. Both octopine (Ach5) and nopaline (T37 and C58) strains induce unorganised tumours on the plant. After excision and in vitro culture the T37 and C58 tumours immediately differentiate into teratomata and normal plants. The Ach5 tumours grow mainly as undifferentiated tissue, but also give rise spontaneously to teratomata and to a lesser extent to normal plants. The tumourous state of the callus tissue and teratomata was confirmed by the presence of octopine and nopaline.

The effect of phenols on the hydrolysis of substituted phenyl β- d -gluco- and β- d -xylo-pyranosides by β- d -glucosidase from Stachybotrys atra has been investigated. Depending on the glycon part of the substrate and on the phenol substituent, the hydrolysis is either inhibited or activated. With aryl β- d -xylopyranosides, transfer of the xylosyl residue to the phenol, with the formation of new phenyl β- d -xylopyranosides, is observed. With aryl β- d -glucopyranosides, such transfer does not occur when phenols are used as acceptors, but it does occur with anilines. A two-step mechanism, in which the first step is partially reversible, is proposed to explain these observations. A qualitative analysis of the various factors determining the overall effect of the phenol is given.

We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits of mitochondrial electron transport chain and ATP synthase is significantly downregulated in HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the importance of functional mitochondria and respiration in determining yeast chronological lifespan and apoptosis.

Plasma disposition of sulphadimidine (SDM) and its metabolites was studied in laying hens after 100 mg SDM kg-1 doses were administered as a single intravenous dose, a single oral dose and multiple oral doses once daily for five consecutive days. SDM was extensively metabolised by acetylation and hydroxylation. In plasma, the metabolite observed with the highest concentration was N4-acetylsulphadimidine (N4-SDM) followed by hydroxymethylsulphadimidine (CH2OH) and 5-hydroxysulphadimidine. Following intravenous administration a biphasic elimination (as seen for a capacity limited reaction) pattern for SDM and its metabolites was observed. Multiple (5x) SDM dosing revealed plasma SDM concentrations ranging between 7 and 108 micrograms ml-1; within 96 hours of termination of the multiple SDM dosing, the plasma SDM concentration was below 0.01 micrograms ml-1. The renal clearances of N4-SDM and the hydroxy metabolites were approximately 10 times greater than that of SDM. The SDM mass balance (faecal/urinary recovery) showed a loss of 56 per cent after intravenous dosage and of 67 per cent after a single oral dosage; the hydroxy metabolites accounted for the highest percentage in faeces/urine. Thus additional metabolic pathways must exist in laying hens.

We have purified an induced beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra to apparent homogeneity. The induced enzyme was clearly different from the constitutive beta-D-glucosidase. The molecular weight was 65 500-69 000, the pH optimum was at 6.7 and the isoelectric point at 4.8. Carbohydrate content (related to glucose) was 14.4%. The enzyme showed beta-D-glucosidase, beta-D-xylosidase and beta-D-thioglucosidase activity. These three activities sppear to be due to the same enzyme. The enzyme was strongly inhibited by D-glucono-(1 leads to 5)-lactone and nojirimycin and was very sensitive to sulfhydryl reagents.

The interaction of alcohols in the hydrolysis of aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra has been investigated. The results constitute support for the presence of a glycosyl-enzyme intermediate, formed during the first step (glycosylation) of the proposed two-step mechanism. Transfer of the glycosyl group to an alcohol, with the formation of an alkyl glycopyranoside, can take place in parallel to the transfer to a water molecule (second or deglycosylation step). The alcohol binds to the free enzyme and to the glycosyl-enzyme intermediate. The glycosyl-enzyme-alcohol complex undergoes hydrolysis in addition to the alcoholysis. For aryl beta-D-glucopyranosides the deglycosylation step is rate-limiting. For aryl beta-D-xylopyranosides two kinds of substrate behaviour can be observed. Depending on the substituent group on the phenyl ring, either both steps are rate-controlling or the first step is rate-limiting. Electron-withdrawing substituents increase the rate at which the substrate aglycon group is released.