Lett Appl Microbiol, 2005, 40(2), 133 - 7 The effect of Saccharomyces cerevisiae on the stability of the herbicide glyphosate during bread leavening; Low FL et al.; Abstract f.l . low, i.c . shaw and j.a . gerrard . 2004.Aims: To investigate the ability of baker's yeast (Saccharomyces cerevisiae) to degrade the herbicide glyphosate during the fermentation cycle of the breadmaking process . Methods and Results: Aqueous glyphosate was added to bread ingredients and kneaded by commercially available breadmaking equipment into dough cultures . Cultures were incubated in the breadmaker throughout the fermentation cycle . The recovery of glyphosate levels following fermentation was determined, thus allowing an estimation of glyphosate degradation by yeast . Conclusions: It was shown, for the first time, that S . cerevisiae plays a role in metabolizing glyphosate during the fermentation stages of breadmaking . Approximately 21% was degraded within 1 h . Significance and Impact of the Study: As a result of projected increases in the glyphosate use on wheat and the role of bread as a dietary staple, this may contribute to more informed decisions being made relating to the use of glyphosate on glyphosate-resistant wheat, from a public health/regulatory perspective. J Org Chem, 2005 Jan 7, 70(1), 342 - 5 Stereoselective, Biocatalytic Reductions of alpha-Chloro-beta-keto Esters; Kaluzna IA et al.; Eighteen known and putative reductases from baker's yeast (Saccharomyces cerevisiae) were tested for the ability to reduce a series of alpha-chloro-beta-keto esters . In nearly all cases, it was possible to produce at least two of the four possible alpha-chloro-beta-hydroxy ester diastereomers with high optical purities . The utility of this approach was demonstrated by reducing ethyl 2-chloroacetoacetate to the corresponding syn-(2R,3S)-alcohol on a multigram scale using whole cells of an Escherichia coli strain overexpressing a single yeast reductase identified from the screening studies. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D378 - 82 The Yeast Resource Center Public Data Repository; Riffle M et al.; The Yeast Resource Center Public Data Repository (YRC PDR) serves as a single point of access for the experimental data produced from many collaborations typically studying Saccharomyces cerevisiae (baker's yeast) . The experimental data include large amounts of mass spectrometry results from protein co-purification experiments, yeast two-hybrid interaction experiments, fluorescence microscopy images and protein structure predictions . All of the data are accessible via searching by gene or protein name, and are available on the Web at http://www.yeastrc.org/pdr/. Phys Rev E Stat Nonlin Soft Matter Phys . 2004 Nov;70(5 Pt 1):052901 . Epub 2004 Nov 03. Water-network percolation transitions in hydrated yeast; Sokolowska D et al.; We discovered two percolation processes in succession in dc conductivity of bulk baker's yeast in the course of dehydration . Critical exponents characteristic for the three-dimensional network for heavily hydrated system, and two dimensions in the light hydration limit, evidenced a dramatic change of the water network dimensionality in the dehydration process. Bioprocess Biosyst Eng, 2004 Dec, 26(6), 377 - 83 Epub 2004 Oct 05. Control of yeast fed-batch process through regulation of extracellular ethanol concentration; Cannizzaro C et al.; At high growth rates, the biomass yield of baker's yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol . For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, mu, is fixed at a level below the point of ethanol production, i.e., mu(crit) . Optimally, growth should be maintained at mu(crit), but in practice, this is difficult because mu(crit) is dependent upon strain and culture conditions . In this work, growth was maintained at a point just above mu(crit) by regulating ethanol concentration in the bioreactor . The models used for control design are shown, as are the experimental results obtained when this strategy was implemented . This technique should be applicable to all microorganisms that exhibit an "overflow" type metabolism. Anal Biochem, 2005 Jan 1, 336(1), 11 - 9 A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors; Li H et al.; We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake . The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway . The mmFATP2 was expressed in a S . cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta . To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate . The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue . Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate . Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C . This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening . The assay basis and limitations are discussed. Arch Biochem Biophys, 2005 Jan 15, 433(2), 454 - 65 Relevance of Frank's solvent classification as typically aqueous and typically non-aqueous to activities of firefly luciferase, alcohol dehydrogenase, and alpha-chymotrypsin in aqueous binaries; Fadnavis NW et al.; Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109) . The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP . The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents. J Enzyme Inhib Med Chem, 2004 Aug, 19(4), 313 - 6 Inhibitory activity of cyanidin-3-rutinoside on alpha-glucosidase; Adisakwattana S et al.; Cyanidin-3-rutinoside, a natural anthocyanin, inhibited alpha-glucosidase from baker's yeast in dose-responsive manner . The IC50 value was 19.7 microM +/- 0.24 microM, compared with the IC50 value of voglibose (IC50 = 23.4 +/- 0.30 microM) . Cyanidin-3-rutinoside was found to be a non-competitive inhibitor for yeast alpha-glucosidase with a Ki value in the range of 1.31-1.56 x 10(-5)M . These results indicated that cyanidin-3-rutinoside could be classed as a new alpha-glucosidase inhibitor. Mol Cell Biochem, 2004 Oct, 265(1-2), 11 - 8 Effect of conjugated linoleic acid on Delta-5 desaturase activity in yeast transformed with fungal Delta-5 desaturase gene; Chuang LT et al.; Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers derived from linoleic acid (LA: delta9, 12-18:2), has been shown to exhibit various biological functions based on studies using cell culture and animal models . It was postulated that the beneficial effects of CLA were exerted through suppression of production of arachidonic acid (AA; delta5,8,11,14-20:4) and consequently, production of pro-inflammatory eicosanoids . In this study, we used the baker's yeast, Saccharomyces cerevisiae, transformed with fungal delta5-desaturase gene as a model, to study whether CLA affects the activity of delta5-desaturase, a rate-limiting step which converts dihomo-gamma-linolenic acid (DGLA; delta8,11, 14-20:3) to AA . The activity of delta5-desaturase was examined in the transformed yeast incubated in a medium supplemented with DGLA and one of four different CLA isomers (c9, t11-, t10, c12-, c9, c11- and t9, t11) . Results show that all four isomers were taken up readily by the yeast, and all of them suppressed the conversion of DGLA to AA . The degree of suppression, which varied significantly among four isomers was modulated by the level of CLA isomers added in the medium . Since portions of these CLA isomers could be converted to form delta5-CLA metabolites (delta5, c9, t11-, delta5, t10, c12-, delta5, c9, c11- and delta5, t9, t11-18:3), it is suggested that CLA suppressed the delta5-desaturation of DGLA to AA through substrate competition between DGLA and CLA isomers. FEMS Microbiol Lett, 2004 Nov 1, 240(1), 7 - 14 Freeze tolerance of the yeast Torulaspora delbrueckii: cellular and biochemical basis; Alves-Araujo C et al.; The freeze stress responses to prolonged storage at -20 degrees C in Torulaspora delbrueckii PYCC5323 were investigated . In this yeast, no loss of cell viability was observed for at least 120 days during freezing at -20 degrees C, whereas a loss of 80% was observed in a commercial baker's yeast after 15 days . In the former strain, freeze resistance was dependent on an adaptation process . The primary cell target of freeze stress was the plasma membrane, preservation of its integrity being related with a lower increase of lipid peroxidation and with a higher resistance to H(2)O(2), but not with the intracellular trehalose concentration. J Org Chem, 2004 Oct 29, 69(22), 7577 - 81 Synthesis of Chiral beta 3-aminoxy peptides; Yang D et al.; A series of chiral beta(3)-aminoxy acids or amides with various side chains have been synthesized via two different approaches . One is the Arndt-Eistert homologation approach, using chiral alpha-aminoxy acids as starting materials . The other approach, utilizing the enantioselective reduction of beta-keto esters catalyzed by baker's yeast or chiral Ru(II) complexes, produces chiral beta(3)-aminoxy acids with nonproteinaceous side chains . The oligomers of beta(3)-aminoxy acids can be readily prepared using EDCI/HOAt as the coupling reagent. J Biochem (Tokyo), 2004 Aug, 136(2), 177 - 82 Anaerobic treatment of antibiotic production wastewater and kinetic evaluations; Degirmentas I et al.; In this study, the anaerobic treatment of high-strength antibiotic production wastewater and the development of a mathematical model for the treatment were attempted . Anaerobic treatability was investigated using synthetic solutions and original wastewater of which the initial chemical oxygen demand (COD) was determined . Initial COD of solutions was increased from 3,000 to 43,000 mg O(2)/liter in an anaerobic bioreactor . The bioreactor pH was maintained at 6.5-7.5 . The temperature was kept constant at 37 +/- 1 degrees C . Raw materials and original wastewater containing penicillin antibiotics were obtained from Fako Pharmaceutical Factory (Fako) in Istanbul, Turkey . Anaerobic sludge used for treatment was obtained from Pakmaya Baker's Yeast Producing Factory (Pakmaya) in Izmit, Turkey and the Fako . A mathematical model based on substrate (total COD) concentration was developed assuming that only three consecutive reactions, namely, hydrolysis, acidogenesis and methanogenesis, are significant . From the experimental data, a model that can be used for COD calculation as a function of time was developed using the first- and the second-order kinetic approaches . Making use of the developed model equation, it was proved that the anaerobic treatment of high strength (COD > 25,000 mg O(2)/liter) antibiotic production wastewater fits the second-order kinetics. J Ind Microbiol Biotechnol, 2004 Nov, 31(10), 469 - 74 Epub 2004 Nov. A simulation study comparing the impact of experimental error on the performance of experimental designs and artificial neural networks used for process screening; Soria MA et al.; Many variables and their interactions can affect a biotechnological process . Testing a large number of variables and all their possible interactions is a cumbersome task and its cost can be prohibitive . Several screening strategies, with a relatively low number of experiments, can be used to find which variables have the largest impact on the process and estimate the magnitude of their effect . One approach for process screening is the use of experimental designs, among which fractional factorial and Plackett-Burman designs are frequent choices . Other screening strategies involve the use of artificial neural networks (ANNs) . The advantage of ANNs is that they have fewer assumptions than experimental designs, but they render black-box models (i.e., little information can be extracted about the process mechanics) . In this paper, we simulate a biotechnological process (fed-batch growth of baker's yeast) to analyze and compare the effect of random experimental errors of different magnitudes and statistical distributions on experimental designs and ANNs . Except for the situation in which the error has a normal distribution and the standard deviation is constant, it was not possible to determine a clear-cut rule for favoring one screening strategy over the other . Instead, we found that the data can be better analyzed using both strategies simultaneously. Bioprocess Biosyst Eng . 2004 Oct 5; {Epub ahead of print} Control of yeast fed-batch process through regulation of extracellular ethanol concentration; Cannizzaro C et al.; At high growth rates, the biomass yield of baker's yeast ( Saccharomyces cerevisiae) decreases due to the production of ethanol . For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, mu, is fixed at a level below the point of ethanol production, i.e., mu(crit) . Optimally, growth should be maintained at mu(crit), but in practice, this is difficult because mu(crit) is dependent upon strain and culture conditions . In this work, growth was maintained at a point just above mu(crit) by regulating ethanol concentration in the bioreactor . The models used for control design are shown, as are the experimental results obtained when this strategy was implemented . This technique should be applicable to all microorganisms that exhibit an "overflow" type metabolism. Clin Exp Allergy, 2004 Oct, 34(10), 1634 - 41 Mould extracts increase the allergic response to ovalbumin in mice; Instanes C et al.; BACKGROUND: Exposure to moulds in indoor air is thought to induce asthma in susceptible persons . Moulds may contain several potent allergens . However, more importantly, moulds may increase the allergic response to other allergens (adjuvant effect) . Previously, we have found that a beta-1,3-glucan from the cell wall of the fungus Sclerotinia sclerotiorum increases the allergic response to the model allergen ovalbumin (OVA) in a mouse model . OBJECTIVE: In the present study, we wanted to confirm the adjuvant effect of another beta-1,3-glucan, MacroGard (MG) from baker's yeast in this model . More importantly, we wished to explore the putative effects of extracts from the moulds Cladosporium herbarum (CH) and Penicillium chrysogenum (PC) using the very same model as used to explore effects of beta-glucans . METHODS: Groups of eight Balb/c mice were injected with OVA alone, OVA+extract or OVA+MG, into one footpad . On day 21, all mice were reinjected with OVA, before exsanguination on day 26 . The levels of OVA-specific IgE, IgG1 and IgG2a in serum were measured by ELISA . RESULTS: Compared with OVA alone, OVA+MG, OVA+CH extract and OVA+PC extract increased OVA-specific IgE and IgG1 levels significantly . For all groups, the levels of IgG2a anti-OVA remained similar to those of the OVA-alone group . CONCLUSIONS: Our results show that extracts from CH and PC, and the beta-1,3/1,6-glucan from baker's yeast have adjuvant effects on the allergic response in mice. Lab Chip, 2004 Oct, 4(5), 464 - 72 Epub 2004 Jul 29. Cell transport via electromigration in polymer-based microfluidic devices; Witek MA et al.; Electrokinetic transport of Escherichia coli and Saccharomyces cerevisiae (baker's yeast) cells was evaluated in microfluidic devices fabricated in pristine and UV-modified poly(methyl methacrylate)(PMMA) and polycarbonate (PC) . Chip-to-chip reproducibility of the cell's apparent mobilities (micro(app)) varied slightly with a RSD of approximately 10% . The highest micro(app) for baker's yeast cells was observed in UV-modified PC with 0.5 mM PBS (pH = 7.4), and the lowest was measured in pristine PMMA with 20 mM PBS (pH = 7.4) . Baker's yeast in all devices migrated toward the cathode because of their smaller electrophoretic mobility compared to the EOF . In 0.5 mM and 1 mM PBS, E . coli cells migrated toward the anode in all cases, opposite to the direction of the EOF due to their larger electrophoretic mobility . E . coli cells in 20 mM PBS migrated toward the cathode, which indicated that the electrophoretic mobility of E . coli cells decreased at higher ionic strengths . Observed differential migrations of E . coli and baker's yeast cells in appropriately prepared polymer microchips were used as the basis for selective introduction into microfluidic devices of only one type of cell . As a working model, experiments were performed with E . coli and RBCs (red blood cells) . RBCs migrated toward the cathode in pristine PMMA with 1 mM and 20 mM PBS (pH = 7.4), opposite to the direction of the E . coli cells . By judicious choice of the buffer concentration in which the cell suspension was prepared and the polymer material, RBCs or E . coli cells were selectively introduced into the microdevice, which was monitored via laser backscatter signals. Biotechnol Bioeng, 2004 Dec 5, 88(5), 567 - 74 High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol; Varga E et al.; In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF) . In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction . In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C . The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry . Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions . This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading . Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained . After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation . The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration . A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer . It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2. J Am Chem Soc, 2004 Oct 13, 126(40), 12827 - 32 Systematic investigation of Saccharomyces cerevisiae enzymes catalyzing carbonyl reductions; Kaluzna IA et al.; Eighteen key reductases from baker's yeast (Saccharomyces cerevisiae) have been overproduced in Escherichia coli as glutathione S-transferase fusion proteins . A representative set of alpha- and beta-keto esters was tested as substrates (11 total) for each purified fusion protein . The stereoselectivities of beta-keto ester reductions depended both on the identity of the enzyme and the substrate structure, and some reductases yielded both L- and D-alcohols with high stereoselectivities . While alpha-keto esters were generally reduced with lower enantioselectivities, it was possible in all but one case to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form . Taken together, the results demonstrate not only that individual yeast reductases can be used to supply important chiral building blocks, but that GST-fusion proteins allow rapid identification of synthetically useful biocatalysts (along with their corresponding genes). Biotechnol Prog, 2004 Sep-Oct, 20(5), 1534 - 42 Physicochemical parameters involved in the interaction of Saccharomyces cerevisiae cells with ion-exchange adsorbents in expanded bed chromatography; Vergnault H et al.; Expanded bed adsorption (EBA) is an interesting primary technology allowing the adsorption of target proteins from unclarified feedstock in order to combine separation, concentration, and purification steps . However, interactions between cells and adsorbent beads during the EBA process can strongly reduce the performance of the separation . So, to minimize these interactions, the mechanisms of cell adsorption on the support were investigated . Adsorption kinetics of the baker's yeast Saccharomyces cerevisiae on the anion exchanger Q Hyper Z were directly performed under real EBA operating conditions, in a lab-scale UpFront 10 column . The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast) . For both types, a complete series of experiments for determining the adsorption profile versus time was performed, varying the superficial velocity or the pH . In parallel, the surface physicochemical properties of the cells (surface charge and electron-donor and electron-acceptor components) and of the support were determined . First of all, whatever the yeast types, the relation between cell adsorption and bed expansion has been highlighted, demonstrating the important role of hydrodynamic . However, for the type II yeast cells, adsorption increased dramatically, compared to the type I, even though it was shown that both types exhibited the same surface charge . In fact, there were strong differences in the Lewis acidic and basic components of the two yeasts . These differences explain the variable affinity toward the support, which was characterized by a strong electron-donor and a weak electron-acceptor component . These observed behaviors agreed with the colloidal theory . This work demonstrates that all kinds of interaction between the cells and the support (electrostatic, Lifshitz-van der Waals, acid/base) have to be taken into account together with hydrodynamic characteristics inside the bed. J Agric Food Chem, 2004 Oct 6, 52(20), 6165 - 9 Biodegradation of 3-chloro-1,2-propanediol with Saccharomyces cerevisiae; Bel-Rhlid R et al.; A novel enzymatic dehalogenating activity of 3-chloro-1,2-propanediol (3-MCPD) with Saccharomyces cerevisiae (baker's yeast) is reported . All bioconversion assays were carried out under aerobic conditions, at 28 degrees C, and the kinetics were monitored . The biodegradation was performed at different pH values (6.2, 7.0, and 8.2), in the presence and absence of glucose, using racemic 3-MCPD at two different concentrations (7.3 micromol/L and 27 mmol/L) . Optimal conversion (68%) of racemic (R,S)-3-MCPD at a concentration of 27 mmol/L was achieved after 48 h of reaction time, at pH 8.2, and in the presence of glucose . At a concentration of 7.3 micromol/L, 73% degradation was observed after 72 h, at pH 8.2 and in the absence of glucose . Under the same experimental conditions, the conversion of pure (S)-3-MCPD (85%) was higher than that of the (R)-enantiomer (60%). Biosci Biotechnol Biochem, 2004 Sep, 68(9), 1888 - 97 Purification and characterization of glutamine synthetase of Pseudomonas taetrolens Y-30: an enzyme usable for production of theanine by coupling with the alcoholic fermentation system of baker's yeast; Yamamoto S et al.; Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed gamma-glutamylethylamide (theanine) from glutamic acid and ethylamine in a mixture containing the alcoholic fermentation system of baker's yeast for ATP-regeneration . Glutamine synthetase (GS), probably responsible for theanine formation, was isolated from the extract of the organism grown on a medium containing 1% methylamine, 1% glycerol, 0.5% yeast extract, and 0.2% polypepton as carbon and nitrogen sources . The molecular mass was estimated to be 660 kDa by gel filtration and 55 kDa by SDS-polyacrylamide gel electrophoresis, suggesting that Ps . taetrolens Y-30 GS consists of 12 identical subunits . The enzyme required Mg2+ or Mn2+ for its activity . Under the standard reaction condition for glutamine formation (pH 8.0 with 30 mM Mg2+), GS showed 7% and 1% reactivity toward methylamine and ethylamine respectively of that to ammonia . Reactivity to the alkylamines varied with optimum pH of the reaction in response to divalent cation in the mixture: pH 11.0 was the optimum for the Mg2+ -dependent reaction with ethylamine, and pH 8.5 was the optimum for the Mn2+ -dependent reaction . In a mixture of an optimum reaction condition with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn2+), reactivity increased up to 7% of the reactivity to ammonia in the standard reaction condition . The isolated GS formed theanine in the mixture with the yeast fermentation system. Bioresour Technol, 2005 Jan, 96(1), 103 - 9 Biosorption of cadmium and lead ions by ethanol treated waste baker's yeast biomass; Goksungur Y et al.; The biosorption of cadmium and lead ions from artificial aqueous solutions using waste baker's yeast biomass was investigated . The yeast cells were treated with caustic, ethanol and heat for increasing their biosorption capacity and the highest metal uptake values (15.63 and 17.49 mg g(-1) for Cd(2+) and Pb(2+), respectively) were obtained by ethanol treated yeast cells . The effect of initial metal concentration and pH on biosorption by ethanol treated yeast was studied . The Langmuir model and Freundlich equation were applied to the experimental data and the Langmuir model was found to be in better correlation with the experimental data . The maximum metal uptake values (qmax, mg g(-1)) were found as 31.75 and 60.24 for Cd(2+) and Pb(2+), respectively . Competitive biosorption experiments were performed with Cd(2+) and Pb(2+) together with Cu(2+) and the competitive biosorption capacities of the yeast biomass for all metal ions were found to be lower than in non-competitive conditions. Plant J, 2004 Oct, 40(1), 120 - 30 AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes; Sauer N et al.; Three members of the Arabidopsis sucrose transporter gene family, AtSUC6-AtSUC8 (At5g43610; At1g66570; At2g14670), share a high degree of sequence homology in their coding regions and even in their introns and in their 5'- and 3'-flanking regions . A fourth sucrose transporter gene, AtSUC9 (At5g06170), which is on the same branch of the AtSUC-phylogenetic tree, shows only slightly less sequence homology . Here we present data demonstrating that two genes from this subgroup, AtSUC6 and AtSUC7, encode aberrant proteins and seem to represent sucrose transporter pseudogenes, whereas AtSUC8 and AtSUC9 encode functional sucrose transporters . These results are based on analyses of splice patterns and polymorphic sites between these genes in different Arabidopsis ecotypes, as well as on functional analyses by cDNA expression in baker's yeast . For one of these genes, AtSUC7 (At1g66570), different, ecotype-specific splice patterns were observed in Wassilewskija (Ws), C24, Columbia wild type (Col-0) and Landsberg erecta (Ler) . No incorrect splicing and no sequence polymorphism were detected in the cDNAs of AtSUC8 and AtSUC9, which encode functional sucrose transporters and are expressed in floral tissue . Finally, promoter-reporter gene plants and T-DNA insertion lines were analyzed for AtSUC8 and AtSUC9. Biochem Biophys Res Commun, 2004 Oct 8, 323(1), 58 - 64 Exchangeable zinc ions transiently accumulate in a vesicular compartment in the yeast Saccharomyces cerevisiae; Devirgiliis C et al.; The baker's yeast Saccharomyces cerevisiae was used as a model to visualize intracellular labile zinc under conditions of nutritional zinc imbalance . Zinc-specific staining was performed in yeast cells using both Zinquin fluorescence and zinc-selenium autometallography . Both techniques resulted in specific labeling of an intracellular vesicular compartment that was present in wild type cells as well as in the vacuolar Zn transporter mutants Deltazrc1 and Deltacot1 . This compartment, that closely resembles mammalian zincosomes, appeared rapidly under conditions of zinc availability and was independent of endocytosis . However, persistence of the zinc loaded vesicles in nutritional zinc deficiency was dependent on the presence of functional Zrc1 and Cot1 vacuolar transporters . Overall our findings indicate that labile zinc in yeast cells enters a dynamic vesicular compartment which could represent an extremely important defence to buffer both zinc excess and deficiency . Mol Cell Biol, 2004 Sep, 24(18), 7998 - 8006 Targeted gene knockout reveals a role in meiotic recombination for ZHP-3, a Zip3-related protein in Caenorhabditis elegans; Jantsch V et al.; The meiotically expressed Zip3 protein is found conserved from Saccharomyces cerevisiae to humans . In baker's yeast, Zip3p has been implicated in synaptonemal complex (SC) formation, while little is known about the protein's function in multicellular organisms . We report here the successful targeted gene disruption of zhp-3 (K02B12.8), the ZIP3 homolog in the nematode Caenorhabditis elegans . Homozygous zhp-3 knockout worms show normal homologue pairing and SC formation . Also, the timing of appearance and the nuclear localization of the recombination protein Rad-51 seem normal in these animals, suggesting proper initiation of meiotic recombination by DNA double-strand breaks . However, the occurrence of univalents during diplotene indicates that C . elegans ZHP-3 protein is essential for reciprocal recombination between homologous chromosomes and thus chiasma formation . In the absence of ZHP-3, reciprocal recombination is abolished and double-strand breaks seem to be repaired via alternative pathways, leading to achiasmatic chromosomes and the occurrence of univalents during meiosis I . Green fluorescent protein-tagged C . elegans ZHP-3 forms lines between synapsed chromosomes and requires the SC for its proper localization. Vopr Pitan, 2004, 73(3), 22 - 5 {Vitamin B1 and B2 ratio as a method of brewer's yeast identification}; Local nanomechanical motion of the cell wall of Saccharomyces cerevisiae; Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E . Young Drive East, Los Angeles, CA 90095, USAWe demonstrate that the cell wall of living Saccharomyces cerevisiae (baker's yeast) exhibits local temperature-dependent nanomechanical motion at characteristic frequencies . The periodic motions in the range of 0.8 to 1.6 kHz with amplitudes of approximately 3 nm were measured using the cantilever of an atomic force microscope (AFM) . Exposure of the cells to a metabolic inhibitor causes the periodic motion to cease . From the strong frequency dependence on temperature, we derive an activation energy of 58 kJ/mol, which is consistent with the cell's metabolism involving molecular motors such as kinesin, dynein, and myosin . The magnitude of the forces observed ( approximately 10 nN) suggests concerted nanomechanical activity is operative in the cell. Am J Physiol Cell Physiol, 2004 Sep, 287(3), C580 - 9 Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases; Ton VK et al.; The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism . It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes . In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca(2+) homeostasis machinery, with particular emphasis on Ca(2+)-transporting ATPases . Putative Ca(2+)-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca(2+) pumps . High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca(2+) transport and trafficking . The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca(2+),Mn(2+)-ATPases (SPCA) . Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump . Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin . Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease. Eukaryot Cell, 2004 Aug, 3(4), 955 - 65 Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins; de Groot PW et al.; Covalently linked cell wall proteins (CWPs) of the dimorphic fungus Candida albicans are implicated in virulence . We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells . Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry {LC/MS/MS}) methods . HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively . In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant beta-1,3-glucanase . Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods . The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins . Nonsecretory proteins were absent in our cell wall preparations . The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins . In addition, Pga24p shows similarity to the flocculins of baker's yeast . (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions . The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown . These results indicate that a substantial number of the covalently linked CWPs of C . albicans are actively involved in cell wall remodeling and expansion and in host-pathogen interactions. Dev Cell, 2004 Aug, 7(2), 150 - 1 Signaling mucins in the (S)limelight; Clevers H; Mucins may be the ugly ducklings of molecular biology . Their large size, repetitive nature, and unglamorous biological activities have not favored their study . However, integral membrane mucins have conserved intracellular C termini that may influence intracellular signaling . In a recent issue of Genes & Development, Cullen et al . show that the C terminus of membrane mucin-like Msb2 activates a CDC42/MAPK cascade to control filamentous growth of baker's yeast. Bioprocess Biosyst Eng, 2004 Dec, 27(1), 9 - 15 Epub 2004 Aug 04. On-line estimation of biomass concentration using a neural network and information about metabolic state; Vanek M et al.; This paper deals with the design of a neural network-based biomass concentration estimation system . This system is enhanced by the incorporation of information about the actual metabolism of the microorganism cultivated, which is taken from an on-line knowledge-based system . Two different design approaches have been investigated using the fed-batch cultivation of baker's yeast as the model process . In the first, metabolic state (MS) data were passed as additional input to the neural network; in the second, these data were used to select a neural network suitable for the specific MS . Two neural network types--feed-forward (Levenberg-Marquardt) and cascade correlation--were applied to this system and tested, and the performances of these neural networks were compared. Biotechnol Lett, 2004 Aug, 26(15), 1237 - 40 Fermentative capacity of baker's yeast exposed to hyperbaric stress; Campelo AF et al.; Baker's yeast suspensions were incubated at different pressures (from 1 bar to 6 bar) and different gases {air, O(2) and a mixture of 8% (v/v) CO(2), 21% O(2) and N(2)} . Raising the air pressure from 1 bar to 6 bar stimulated cell growth but had no effect on leavening ability or viability of the cells . A 50% reduction of the CO(2) produced in dough occurred with 6 bar O(2) which also stopped growth . The fermentative capacity of the cells was stimulated by the cells exposure to increased CO(2) partial pressure up to 0.48 bar. Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1442 - 8 Superior molasses assimilation, stress tolerance, and trehalose accumulation of baker's yeast isolated from dried sweet potatoes (hoshi-imo); Nishida O et al.; Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan . Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker's yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains . Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae . One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128 . Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher . ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs . The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128 . These results suggest that ONY1 has potential commercial use as baker's yeast for frozen dough and high-sugar dough. Anticancer Res, 2004 May-Jun, 24(3a), 1455 - 63 Induction of apoptosis in breast cancer cells by Saccharomyces cerevisiae, the baker's yeast, in vitro; Ghoneum M et al.; The present study was undertaken to evaluate the effect of phagocytosis of killed yeast on the induction of apoptosis in human metastatic breast cancer cells (MCF-7 and ZR-75-1) and non-metastatic breast cancer cells (HCC70) . Heat-killed Saccharomyces cerevisiae, baker's and brewer's yeast, was cultured with cancer cells at a ratio of yeast to cancer cells = 10:1, and the percent apoptotic cancer cells was determined by flow cytometry and cytospin preparation . Upon phagocytosis of yeast, breast cancer cells underwent apoptosis . Induction of apoptosis was time- and dose-dependent . Apoptosis was detected as early as 0.5 h (13%), increased to 19% at 2 h and peaked (38%) at 4 h . Metastatic cancer cells were found to be more susceptible to yeast-induced apoptosis than non-metastatic cells; 629% increase for MCF-7 as compared to cells alone, 258% for ZR-75 cells, while HCC70 cells showed a 178% increase . Phagocytosis is associated with the disruption of mitochondrial membrane potential and activation of initiator and effector caspases 8, 9 and 3 . However, inhibitors of these caspases did not inhibit yeast-induced apoptosis in cancer cells, suggesting that yeast induces apoptosis in breast cancer cells by a mechanism that is independent of caspase activation . This data may have clinical implications. Ann Allergy Asthma Immunol, 2004 Jun, 92(6), 673 - 5 Anaphylaxis to wheat beer; Herzinger T et al.; BACKGROUND: Despite its worldwide and abundant consumption, beer has rarely been found to cause anaphylaxis . Barley malt contained in lager beers seems to be an important elicitor . OBJECTIVE: To report the unusual case of severe anaphylaxis following the ingestion of wheat beer . METHODS: A 59-year-old man experienced angioedema, generalized urticaria, and unconsciousness after ingestion of wheat beer . He tolerated lager beer well . For diagnostic evaluation, skin prick tests, oral challenge tests, and identification of specific IgE antibodies were performed . RESULTS: Skin prick test results with standard series of common aeroallergens and food allergens were negative with the exception of a 1 + reaction to wheat flour . The results of skin prick tests with native materials were positive for 2 brands of wheat beer and wheat malt shred but negative for baker's yeast, hops, and a brand of lager beer . Oral challenges with wheat beer or wheat flour elicited urticaria . By CAP-FEIA, specific IgE antibodies to wheat and barley flour but not to hops or baker's yeast were found in serum . Immunoblot analysis revealed that patient's IgE was bound to a protein of approximately 35 kDa in wheat extract . CONCLUSIONS: This is the first report, to our knowledge, on anaphylaxis to beer attributable to wheat allergy. Neurosci Lett, 2004 Jul 15, 365(1), 19 - 22 Cholinergic modulation of baker's yeast cell phagocytosis by rat astrocytes; Gomez RM et al.; Cholinergic regulation of baker's yeast cell phagocytosis in rat cultured astrocytes was studied . Phagocytic activity was reduced by 1 x 10(-5) M of atropine or pirenzepine, but not by AF-DX116 or 4-DAMP . In addition, carbachol stimulated phagocytosis in a dose-dependent manner . Furthermore, only 1 x 10(-5)M of atropine, pirenzepine and 4-DAMP significantly reduced enhanced activity induced by 1 x 10(-7)M carbachol . It was also observed that L-NMMA, staurosporine, or U-73122, reduced phagocytosis activity while TFP failed to do so . Nitrite levels in astrocyte supernatants increased after baker's yeast cells were incorporated to astrocyte cultures, correlating with enhanced phagocytosis induced by carbachol stimulation, and were reduced by 1 x 10(-5) M of atropine, pirenzepine or aminopiridine, but not by AF-DX116 or 4-DAMP . Enhanced NO production triggered by astrocyte phagocytosis may have pathological consequences. Plant J, 2004 Jul, 39(2), 219 - 36 Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility; Klein M et al.; ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals . Recent evidence shows that this class of transporters may also be involved in many other cellular processes . Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities . This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation . Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane . Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate . In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs . Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants . These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture. Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1185 - 92 Potent hypocholesterolemic activity of the yeast Kluyveromyces marxianus YIT 8292 in rats fed a high cholesterol diet; Yoshida Y et al.; The hypocholesterolemic activities of 81 yeast strains were examined in rats fed a high cholesterol diet (HCD) . Male Wistar rats were fed an HCD or an HCD supplemented with 10% yeast for 7 d . It was found that the hypocholesterolemic activities of the yeasts varied remarkably between strains . Kluyveromyces marxianus YIT 8292 exhibited the most potent hypocholesterolemic activity among the yeasts that were tested . K . marxianus YIT 8292 significantly decreased not only plasma total cholesterol but also liver total cholesterol when administered as a dietary admixture at a concentration of 3% . In contrast, brewer's yeast and baker's yeast, which have been predominantly used for food, did not exhibit hypocholesterolemic activity even when administered at a concentration of 10% . These results suggest that K . marxianus YIT 8292 may be utilized as a novel food material with the ability to contribute to the prevention of hypercholesterolemia. Proteins, 2004 Aug 1, 56(2), 338 - 45 The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes; Kovina MV et al.; New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E . coli show their spectral sensitivity to ThDP binding . Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes . Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect . A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected) . For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring . The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested . From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen . Appl Environ Microbiol, 2004 Jun, 70(6), 3681 - 6 Endogenous xylose pathway in Saccharomyces cerevisiae; Toivari MH et al.; The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism . We found that S . cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters . In nontransformed S . cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source . The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase . An S . cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes . Overexpression of the GRE3 and ScXYL2 genes in the S . cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source. Appl Environ Microbiol, 2004 Jun, 70(6), 3377 - 82 Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions; Tanghe A et al.; Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications . In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions . We found that the difference in the freezing rate is apparently responsible for the difference in the results . We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect . Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested . Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity . In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions . If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly . Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought. Rev Argent Microbiol, 2004 Jan-Mar, 36(1), 41 - 6 {Increase of rising activity of commercial yeasts by application of stress conditions during their propagation}; Galvagno MA et al.; Rising activity determined as CO2 production of two commercial strains of Saccharomyces cerevisiae could be increased mainly in sweet bread doughs by introducing a "starvation/pulse feeding" schedule of sugar cane molasses during a fed-batch propagation . Such increase was strain dependent . Except for the trehalose intracellular level, other traits related to the yeast industrial performance were unaffected . Applicability of method for baker's yeast industrial production is discussed. Biotechnol Bioeng, 2004 Jun 30, 86(7), 795 - 800 In situ product removal using a crystallization loop in asymmetric reduction of 4-oxoisophorone by Saccharomyces cerevisiae; Buque-Taboada EM et al.; In situ product crystallization was investigated for solid product crystals that were obtained during fermentation . The model reaction was the asymmetric reduction of 4-oxoisophorone (OIP) using baker's yeast (S . cerevisiae) as a biocatalyst . The target product was 6R-dihydro-oxoisophorone (DOIP), also known as levodione, a key intermediate in carotenoid synthesis . DOIP was degraded by baker's yeast mainly to (4S,6R)-actinol, an unwanted byproduct in the process . Actinol formation reached up to 12.5% of the initial amount of OIP in the reactor during a batch process . However, better results were obtained when the dissolved DOIP concentration was controlled using an integrated fermentation-crystallization process because: (a) actinol formation was reduced to 4%; and (b) DOIP crystal formation in the reactor was avoided . DOIP productivity was improved by 50% and its selectivity was raised from 87% to 96% relative to the batch process . In the integrated process, most of the product was recovered as pure crystals; this may already minimize, if not eliminate, the need for organic solvents in the final purification steps . An almost sixfold reduction in biocatalyst consumption per kilogram product was achieved, which also can contribute to the minimization of waste streams . Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 1 - 34 Structure, function, and mechanism of ribonucleotide reductases; Kolberg M et al.; Ribonucleotide reductase (RNR) is the enzyme responsible for the conversion of ribonucleotides to 2'-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA . In the recent years, many new crystal structures have been obtained of the protein subunits of all three classes of RNR . This review will focus upon recent structural and spectroscopic studies, which have offered deeper insight to the mechanistic properties as well as evolutionary relationship and diversity among the different classes of RNR . Although the three different classes of RNR enzymes depend on different metal cofactors for the catalytic activity, all three classes have a conserved cysteine residue at the active site located on the tip of a protein loop in the centre of an alpha/beta-barrel structural motif . This cysteine residue is believed to be converted into a thiyl radical that initiates the substrate turnover in all three classes of RNR . The functional and structural similarities suggest that the present-day RNRs have all evolved from a common ancestral reductase . Nevertheless, the different cofactors found in the three classes of RNR make the RNR proteins into interesting model systems for quite diverse protein families, such as diiron-oxygen proteins, cobalamin-dependent proteins, and SAM-dependent iron-sulfur proteins . There are also significant variations within each of the three classes of RNR . With new structures available of the R2 protein of class I RNR, we have made a comparison of the diiron centres in R2 from mouse and Escherichia coli . The R2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms, and new radical forms are different from non-radical forms . In mouse R2, the binding of iron(II) or cobalt(II) to the four metal sites shows high cooperativity . A unique situation is found in RNR from baker's yeast, which is made up of heterodimers, in contrast to homodimers, which is the normal case for class I RNR . Since the reduction of ribonucleotides is the rate-limiting step of DNA synthesis, RNR is an important target for cell growth control, and the recent finding of a p53-induced isoform of the R2 protein in mammalian cells has increased the interest for the role of RNR during the different phases of the cell cycle. Anal Biochem, 2004 Jun 15, 329(2), 307 - 15 Importance of product/reactant equilibration in the kinetics of the phosphoglucose isomerization reaction by differential stopped flow microcalorimetry; Stodeman M et al.; The kinetics for the isomerization of fructose-6-phosphate to glucose-6-phosphate (F6P --> G6P) by baker's yeast phosphoglucose isomerase (PGI) with regard to k(cat) and K(m) were determined from analysis of differential stopped flow microcalorimeter measurements using the integrated form of the Michaelis-Menten rate equation . Values for K(m) (F6P --> G6P) that were determined at pH 8.0 and ionic strength 0.1M at 293.4, 298.4, 303.4, and 311.5K exhibited a linear dependence on the substrate concentration at each temperature because of the substrate-product equilibrium . The minimum values for K(m) ranged from 2.62+/-0.55 mM at 293.4K to 7.8+/-4.8mM at 311.5K and were the same as the minimum values for the reverse reaction (G6P --> F6P) at 293.4 K and 298.4 K . Minimum values for k(cat) increased with temperature, from 2.78+/-0.34s(-1) at 293.4K to 11.4+/-1.0s(-1) at 311.5K, and for the reverse reaction, G6P --> F6P, from 0.852+/-0.086 s(-1) at 293.4K to 1.46+/-0.06s(-1) at 298.4K . The enzyme efficiency at 311.5K is close to the collision rate for a diffusion-controlled process in solution . The {F6P}/{G6P} equilibrium constants were determined from comparison of the values of k(cat) in both directions and were 0.307+/-0.053 at 293.4K and 0.395+/-0.033 at 298.4K . The heats of reaction in the F6P --> G6P direction increased from -8.96+/-0.26 kJmol(-1) at 311.5K to -8.27+/-0.40 kJmol(-1) at 293.4K, a value in fair agreement with 7.01+/-0.32 kJmol(-1) in the opposite G6P --> F6P direction. J Econ Entomol, 2004 Apr, 97(2), 330 - 9 Disruption of host location of western corn rootworm larvae (Coleoptera: Chrysomelidae) with carbon dioxide; Bernklau EJ et al.; Elevated concentrations of carbon dioxide (CO2) prevented neonate larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, from locating the roots of growing corn in behavioral bioassays conducted in soil tubs . When CO2 was pumped into one end of a soil tub, significantly more larvae were recovered from soil at the treated end than from soil around a growing corn plant at the opposite end of the tub . In controls with ambient air pumped into one end of a soil tub, significantly more larvae were recovered from the soil around the corn plant than from soil on the treated side . Larvae were unable to locate the roots of corn seedlings when CO2-generating materials were mixed into the soil . CO2-concentrations in soil were measured by mass spectrometry with selected ion monitoring at m/z 44 . Granules composed of baker's yeast, yeast nutrients, and an organic substrate were prepared as a CO2 source and were tested in larger soil tub bioassays . Significantly fewer larvae were recovered from corn roots in the soil tubs with yeast granules than from corn roots in control soil tubs . The CO2-generating granules produced soil CO2 concentrations between 15.8 and 18.5 mmol/mol (compared with 1.7-2.6 mmol/mol in control tubs), and this was sufficient to prevent larvae from locating corn roots . In field trials, organic and inorganic CO2- generating treatments resulted in root ratings that were significantly lower than for the control plants. Lab Chip, 2003 Aug, 3(3), 212 - 6 Epub 2003 Jul 24. Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem; Perch-Nielsen IR et al.; Diagnostic PCR has been used to analyse a wide range of biological materials . Conventional PCR consists of several steps such as sample preparation, template purification, and PCR amplification . PCR is often inhibited by contamination of DNA templates . To increase the sensitivity of the PCR, the removal of PCR inhibitors in sample preparation steps is essential and several methods have been published . The methods are either chemical or based on filtering . Conventional ways of filtering include mechanical filters or washing e.g . by centrifugation . Another way of filtering is the use of electric fields . It has been shown that a cell will experience a force when an inhomogeneous electric field is applied . The effect is called dielectrophoresis (DEP) . The resulting force depends on the difference between the internal properties of the cell and the surrounding fluid . DEP has been applied to manipulate cells in many microstructures . In this study, we used DEP as a selective filter for holding cells in a microsystem while the PCR inhibitors were flushed out of the system . Haemoglobin and heparin - natural components of blood - were selected as PCR inhibitors, since the inhibitory effects of these components to PCR have been well documented . The usefulness of DEP in a microsystem to withhold baker's yeast (Saccharomyces cerevisiae) cells while the PCR inhibitors haemoglobin and heparin are removed will be presented and factors that influence the effect of DEP in the microsystem will be discussed . This is the first time dielectrophoresis has been used as a selective filter for removing PCR inhibitors in a microsystem. Mol Cell, 2004 Apr 23, 14(2), 247 - 58 Vpu-mediated degradation of CD4 reconstituted in yeast reveals mechanistic differences to cellular ER-associated protein degradation; Meusser B et al.; In HIV infected cells, the plasma membrane protein CD4 is removed from the secretory pathway by proteasomal digestion . This crucial step of viral infection occurs at the endoplasmic reticulum and is triggered by the HIV encoded protein Vpu . Here we show that this process can be recapitulated in baker's yeast . The analysis in the yeast system revealed that Vpu-induced breakdown of CD4 occurs independently of the cellular ER-associated protein degradation system . Moreover, our system allows direct comparison between Vpu-mediated turnover and cellular ER-associated protein degradation of CD4 . This analysis suggests fundamental mechanistic differences between both pathways: Vpu-induced turnover strictly relies on ubiquitination of CD4 at cytosolic lysine residues prior to export of the substrate from the membrane . In contrast, the cellular ER-associated protein degradation pathway can transport ER-lumenal parts of CD4 into the cytoplasm before ubiquitination and extraction of the membrane anchor. BMC Evol Biol . 2004 Mar 08;4(1):9. Upstream plasticity and downstream robustness in evolution of molecular networks; Maslov S et al.; BACKGROUND: Gene duplication followed by the functional divergence of the resulting pair of paralogous proteins is a major force shaping molecular networks in living organisms . Recent species-wide data for protein-protein interactions and transcriptional regulations allow us to assess the effect of gene duplication on robustness and plasticity of these molecular networks . RESULTS: We demonstrate that the transcriptional regulation of duplicated genes in baker's yeast Saccharomyces cerevisiae diverges fast so that on average they lose 3% of common transcription factors for every 1% divergence of their amino acid sequences . The set of protein-protein interaction partners of their protein products changes at a slower rate exhibiting a broad plateau for amino acid sequence similarity above 70% . The stability of functional roles of duplicated genes at such relatively low sequence similarity is further corroborated by their ability to substitute for each other in single gene knockout experiments in yeast and RNAi experiments in a nematode worm Caenorhabditis elegans . We also quantified the divergence rate of physical interaction neighborhoods of paralogous proteins in a bacterium Helicobacter pylori and a fly Drosophila melanogaster . However, in the absence of system-wide data on transcription factors' binding in these organisms we could not compare this rate to that of transcriptional regulation of duplicated genes . CONCLUSIONS: For all molecular networks studied in this work we found that even the most distantly related paralogous proteins with amino acid sequence identities around 20% on average have more similar positions within a network than a randomly selected pair of proteins . For yeast we also found that the upstream regulation of genes evolves more rapidly than downstream functions of their protein products . This is in accordance with a view which puts regulatory changes as one of the main driving forces of the evolution . In this context a very important open question is to what extent our results obtained for homologous genes within a single species (paralogs) carries over to homologous proteins in different species (orthologs). Biotechnol Prog, 2004 Mar-Apr, 20(2), 403 - 11 Understanding and improving NADPH-dependent reactions by nongrowing Escherichia coli cells; Walton AZ et al.; We have shown that whole Escherichia coli cells overexpressing NADPH-dependent cyclohexanone monooxygenase carry out a model Baeyer-Villiger oxidation with high volumetric productivity (0.79 g epsilon-caprolactone/L.h ) under nongrowing conditions (Walton, A . Z.; Stewart, J . D . Biotechnol . Prog . 2002, 18, 262-268) . This is approximately 20-fold higher than the space-time yield for reactions that used growing cells of the same strain . Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively . Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E . coli cells could support even more efficient NADPH-dependent bioconversions if a more suitable enzyme-substrate pair were identified . This was demonstrated by reducing ethyl acetoacetate with whole cells of an E . coli strain that overexpressed an NADPH-dependent, short-chain dehydrogenase from baker's yeast (Saccharomyces cerevisiae) . Under glucose-fed, nongrowing conditions, this reduction proceeded with a space-time yield of 2.0 g/L.h and a final product titer of 15.8 g/L using a biocatalyst:substrate ratio (g/g) of only 0.37 . These values are significantly higher than those obtained previously . Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 +/- 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions . This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway. Appl Biochem Biotechnol, 2004 Spring, 113-116, 509 - 23 Optimization of steam pretreatment of corn stover to enhance enzymatic digestibility; Varga E et al.; Among the available agricultural byproducts, corn stover, with its yearly production of 10 million t (dry basis), is the most abundant promising raw material for fuel ethanol production in Hungary . In the United States, more than 216 million t of corn stover is produced annually, of which a portion also could possibly be collected for conversion to ethanol . However, a network of lignin and hemicellulose protects cellulose, which is the major source of fermentable sugars in corn stover (approx 40% of the dry matter {DM}) . Steam pretreatment removes the major part of the hemicellulose from the solid material and makes the cellulose more susceptible to enzymatic digestion . We studied 12 different combinations of reaction temperature, time, and pH during steam pretreatment . The best conditions (200 degrees C, 5 min, 2% H2SO4) increased the enzymatic conversion (from cellulose to glucose) of corn stover more then four times, compared to untreated material . However, steam pretreatment at 190 degrees C for 5 min with 2% sulfuric acid resulted in the highest overall yield of sugars, 56.1 g from 100 g of untreated material (DM), corresponding to 73% of the theoretical . The liquor following steam explosion was fermented using Saccharomyces cerevisiae to investigate the inhibitory effect of the pretreatment . The achieved ethanol yield was slightly higher than that obtained with a reference sugar solution . This demonstrates that baker's yeast could adapt to the pretreated liquor and ferment the glucose to ethanol efficiently. J Chromatogr A, 2004 Mar 12, 1029(1-2), 103 - 12 Designed chimaeric galactosyl-mimodye ligands for the purification of Pseudomonas fluorescens beta-galactose dehydrogenase; Mazitsos CF et al.; Two chimaeric galactosyl-mimodye ligands were designed and applied to the purification of Pseudomonas fluorescens galactose dehydrogenase (GaDH) . The chimaeric affinity ligands comprised a triazine ring on which were anchored: (i) an anthraquinone moiety that pseudomimics the adenine part of NAD+, (ii) a galactosyl-mimetic moiety (D-galactosamine for ligand BM1 or shikimate for ligand BM2), bearing an aliphatic 'linker', that mimics the natural substrate galactose, and (iii) a long hydrophilic 'spacer' . The mimodye-ligands were immobilised to 1,1-carbonyldiimidazole-activated agarose chromatography support, via the spacer's terminal amino-group, to produce the respective mimodye adsorbents . Both immobilized mimodyes successfully bound P . fluorescens GaDH but failed to bind the enzyme from rabbit muscle . Adsorbent BM1 bound GaDH from green peas and Baker's yeast, but adsorbent BM2 failed to do so . The mimodye-ligand comprising D(+)-galactosamine (BM1), compared to BM2, exhibited higher purifying ability and enzyme recovery for P . fluorescens GaDH . The dissociation constants (KD) of BM1 and BM2 for P . fluorescens GaDH were determined by analytical affinity chromatography to be 5.9 microM and 15.4 microM, respectively . The binding capacities of adsorbents BM1 and BM2 were 18 U/mg adsorbent and 6 U/mg adsorbent, respectively . Adsorbents BM1 and BM2 were integrated in two different protocols for the purification P . fluorescens GaDH . Both protocols comprised as a common first step DEAE anion-exchange chromatography, with a second step of affinity chromatography on BM1 or BM2, respectively . The purified GaDH obtained from the protocols using BM1 and BM2 showed specific activities equal to 1077 and 854 U/mg, respectively . The former is the highest reported so far and the enzyme appeared as a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Inorg Chem, 2004 Mar 22, 43(6), 1957 - 63 The reaction of dimethyltin(IV) dichloride with thiamine diphosphate (H2TDP): synthesis and structure of {SnMe2(HTDP)(H2O)}Cl.H2O, and possibility of a hitherto unsuspected role of the metal cofactor in the mechanism of vitamin-B1-dependent enzymes; Casas JS et al.; The complex {SnMe(2)(HTDP)(H(2)O)}Cl.H(2)O, synthesized by reaction between dimethyltin(IV) dichloride and thiamine diphosphate hydrochloride (H(3)TDPCl) in water, was characterized by X-ray diffractometry and IR and Raman spectroscopy in the solid state, and by electrospray mass spectrometry (ESMS) and NMR spectroscopy ((1)H, (13)C, (31)P, (119)Sn and inverse-detection (1)H,(15)N HMBC) in aqueous solution . In the solid state the HTDP(-) anion chelates the metal via one oxygen atom of each phosphate group {Sn-O = 2.062(3), 2.292(3) A}, and another oxygen atom belonging to the terminal phosphate links the SnMe(2)(2+) cations into chains . The tin atom has distorted octahedral coordination involving the trans methyl groups, the above-mentioned diphosphate oxygen atoms, and the oxygen atom of the coordinated water molecule . The thiamine moiety has F conformation . NMR studies suggest that the interaction between the organometallic cation and the HTDP(-) ligand persists in D(2)O solution, which is in keeping with the ESMS spectrum showing a peak corresponding to {SnMe(2)(HTDP)} . Both in the solid state and in solution, the acidic HTDP(-) proton in the complex is located on the N(1') atom of the pyrimidine ring . The enzymatic behavior of native pyruvate decarboxylase (EC 4.1.1.1, PDC), obtained from baker's yeast, was compared in a coupled assay with that shown by the "SnMe(2)-holoenzyme" created by incubation of apoPDC with {SnMe(2)(HTDP)(H(2)O)}Cl.H(2)O . The SnMe(2)-holoenzyme exhibited about 34% of the activity of the native enzyme (with a Michaelis-Menten constant of 2.7 microM, as against 6.4 microM for native PDC), so confirming the very low specificity of PDC regarding the identity of its metal ion cofactor . In view of the observed protonation of N(1'), it is suggested that the role of divalent cations in the mechanism of thiamine-diphosphate-dependent enzymes may be not only to anchor the cofactor in its binding site but also to shift the acidic proton of HTDP(-) from the diphosphate group to N(1'); protonation of N(1') is widely believed to be important for enzyme function, but the origin of the proton has never been clarified. Mol Genet Genomics, 2004 May, 271(4), 387 - 93 Epub 2004 Mar 11. Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts; Gojkovic Z et al.; The ability to propagate under anaerobic conditions is an essential and unique trait of brewer's or baker's yeast ( Saccharomyces cervisiae) . To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species . While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen . From the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis . Here we show that S . kluyveri, which separated from the S . cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and mitochondrial DHODases . We show that these two S . kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen . Only the cytoplasmic DHODase promotes growth in the absence of oxygen . Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired a bacterial gene for DHODase, which subsequently allowed cell growth gradually to become independent of oxygen. Mol Biol Evol, 2004 Jun, 21(6), 1081 - 4 Epub 2004 Mar 10. NUMTs in sequenced eukaryotic genomes; Richly E et al.; Mitochondrial DNA sequences are frequently transferred to the nucleus giving rise to the so-called nuclear mitochondrial DNA (NUMT) . Analysis of 13 eukaryotic species with sequenced mitochondrial and nuclear genomes reveals a large interspecific variation of NUMT number and size . Copy number ranges from none or few copies in Anopheles, Caenorhabditis, Plasmodium, Drosophila, and Fugu to more than 500 in human, rice, and Arabidopsis . The average size is between 62 (baker's yeast) and 647 bps (Neurospora), respectively . A correlation between the abundance of NUMTs and the size of the nuclear or the mitochondrial genomes, or of the nuclear gene density, is not evident . Other factors, such as the number and/or stability of mitochondria in the germline, or species-specific mechanisms controlling accumulation/loss of nuclear DNA, might be responsible for the interspecific diversity in NUMT accumulation. Biochemistry, 2004 Mar 16, 43(10), 2926 - 34 Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor; Roux C et al.; The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs . Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity . Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets . Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design . To date, inhibition and crystallographic studies of all PMIs are still largely unexplored . As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively . We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P . 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes . We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI . All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity. Cancer Epidemiol Biomarkers Prev, 2004 Feb, 13(2), 299 - 303 Effects of a high-selenium yeast supplement on celecoxib plasma levels: a randomized phase II trial; Frank DH et al.; A combination of celecoxib and selenium was used in a randomized double-blind Phase II trial as a preliminary study to a multicenter Phase III colorectal cancer chemoprevention trial using these two agents together . The purpose of this trial was to determine whether high-selenium baker's yeast {(Saccharomyces cerevisiae) 200 microg once daily} in combination with celecoxib (400 mg once daily) altered the steady-state plasma concentration of celecoxib or produced clinically significant toxicities . Seventy-three healthy subjects (ages 40-75 years) were recruited to the 6-week study from the general local population and were randomized to either the celecoxib plus selenized baker's yeast group or the celecoxib plus placebo group after a 2-week run in period of celecoxib only . Blood samples were taken at baseline (to document that there was no evidence of celecoxib intake), after the 2-week run-in period on celecoxib to verify steady-state blood levels of this agent, and at end of study (4 weeks postrandomization) . Toxicities were monitored at 2 weeks after initiation of celecoxib, at 4 weeks after initiation, and at the end of the study . Blood level concentrations of celecoxib did not differ between the two groups as determined by high-performance liquid chromatography analysis nor were there significant differences in blood chemistry values between the two groups . Subjects' self-report of general physical toxicities was uncommon and limited to National Cancer Institute toxicity grade 2 or less; however, 2 female participants (3%) were removed from the study medications because of grade 2 edema and significant weight gain after 2 and 2.5 weeks of celecoxib administration . In conclusion, high-selenium yeast and celecoxib can be taken at the described doses with minimum short-term negative effects . In future Phase III chemoprevention trials of celecoxib, weight gain should be carefully monitored, and participants should be made aware of this potential side effect before study entry. Methods Mol Med, 2004, 94, 191 - 5 Production of antigens in Chlamydomonas reinhardtii: green microalgae as a novel source of recombinant proteins; Fuhrmann M; Recombinant small-scale proteins are produced in a number of systems, from bacteria like Escherichia coli, through lower eukaryotes like baker's yeast, up to mammalian cell cultures . However, the need for safe and cheap sources of large amounts of recombinant proteins for different purposes, including material sciences, diagnostics, and, of course, medical therapy, has forced the development of alternative production systems . Green microalgae are cheap and easily grown and offer a high protein content, which would seem to make them ideal hosts for the large-scale sustainable production of recombinant proteins in the future . In selected species, recombinant DNA can be introduced into the genomes of the nucleus, the chloroplast, and even the mitochondria, and thus the system offers both prokaryotic (chloroplast, mitochondria) and eukaryotic translation systems for a tailored expression of virtually any protein. Appl Environ Microbiol, 2004 Feb, 70(2), 1088 - 96 Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients; Di Cagno R et al.; This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients . Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients . This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE) . A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli . After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally . Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin . The minimal agglutinating activity was ca . 250 times higher than that of doughs chemically acidified or started with baker's yeast . Two types of bread, containing ca . 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients . Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread . When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values . The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread . These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans. Biotechnol Prog, 2004 Jan-Feb, 20(1), 128 - 33 Ability of different biomaterials to enantioselectively catalyze oxidation and reduction reactions; Nagaoka H; We studied the ability of different biomaterials to enantioselectively catalyze oxidation or reduction reactions with the help of substrate rac-1-m or p-ArCH(OH)Me and the 1-o-ArC(O)Me derivatives . Apoenzyme (NAD(P)(+)-dependent secondary alcohol dehydrogenase(NAD(P)-E)) and cofactor (NAD(P)(+)) were activated by preincubating immobilized aqueous plant leaf (e.g., young wheat leaves), cereal tissue (wheat bran), vegetable (e.g., carrot), and seaweed (e.g., wakame seaweed) solutions, and the NAD(P)-E oxidized only (R)-isomers highly enantioselectively . Thus, greater than 99% ee(s) of (S)-isomers (1m-5m and 1p-5p) can be obtained from corresponding rac-1-m or p-ArCH(OH)Me . Further, immobilized chlorella cells and immobilized baker's yeast can reduce highly stereoselectively; greater than 99% ee(s) of (S)-isomers (1o-5o) can be obtained from corresponding 1-o-ArC(O)Me . Specific use of each isomer ((S)-6 and (R)-6) with greater than 99% ee(s) of racemic-1-2-NpCH(OH)Me becomes possible through selective use of NAD(P)-E eluted from artemisia vulgaris indica leaves and young wheat leaves . We suggest that the pH of the reaction media can determine not only the direction of NAD(P)-E, toward enantioselectively catalyzed oxidation (pH > 7.0) or reduction reaction (pH < 7.0), but also the regioselective reactivity of NAD(P)-E to the substrate o- (pH < 7.0), m-, and p-substituted groups (pH > 7.0) . Thus, in comparison to current biocatalysts, several biomaterials can serve as asymmetric reagent bases, providing easily obtained, low-cost natural catalysts with stereoselectivity, regioselectivity, and substrate specificity that work under mild conditions for asymmetric synthesis of organic compounds. Water Res, 2004 Jan, 38(2), 385 - 92 The effect of levitated water on fermentation kinetics; Sapunov VN et al.; The rate of anaerobic glucose fermentation by baker's yeast is found to be altered when tap water is replaced with "levitated" (i.e., hydrodynamically processed) water . To analyze the effect in more detail, we developed a fermentation kinetics model that differentiates between (i) nutrient transport into the cell, (ii) the "catabolic" and (iii) the "anabolic" reactions . As a result, the levitated water affects specifically the glucose uptake kinetics, whereas the other kinetic parameters remain unchanged . Remarkably, the sign of the effect changes with the water used to prepare the culture . When levitated water is used for both the culture preparation and the fermentation, the rate constant of glucose transport is increased by (67+/-25)%, relative to ordinary tap-water . When the culture is prepared in ordinary water and only the fermentation is performed in levitated water, the rate constant of glucose transport decreased by (50+/-12)% . Three-week old levitated water has no discernable effect any more. Food Chem Toxicol, 2004 Feb, 42(2), 321 - 33 Safety evaluation of ice-structuring protein (ISP) type III HPLC 12 preparation . Lack of genotoxicity and subchronic toxicity; Hall-Manning T et al.; Ice-structuring proteins (ISPs) naturally occur in a range of species (including edible plants and fish) that need to protect themselves against freeze damage . ISPs have potential applications in a number of areas including cryopreservation and frozen foods manufacture . However, these materials are not currently generally available for commercial use . ISP type III HPLC 12 is of particular interest and although it is likely to be consumed naturally, its toxicological safety has not previously been assessed . This paper presents data from a set of in vitro and in vivo genotoxicity assays (bacterial mutation, chromosome aberration, mammalian cell gene mutation and rat bone marrow micronucleus) and a 3-month repeat-dose gavage study in the rat using high levels of ISP type III HPLC 12 preparation produced by recombinant baker's yeast . No evidence was seen of a genotoxic potential (using levels accepted as limit concentrations for the assays used) or notable subchronic toxicity following oral administration for 3 months in the rat at up to 580 mg ISP type III HPLC 12/kg/day, the highest dose tested (which was considered to be a NOAEL). Appl Microbiol Biotechnol, 2004 Jun, 64(5), 686 - 90 Epub 2003 Dec 10. Behaviour of dehydrated baker's yeast during reduction reactions in a biphasic medium; Cappaert L et al.; The behaviour of dry baker's yeast ( Saccharomyces cerevisiae type II, Sigma) used as biocatalyst without preliminary growth for the synthesis of 2-heptanol from 2-heptanone in a biphasic system is presented . Cells undergo intracellular trehalose consumption with a stoichiometric ethanol production during the first 15 h of the process . This metabolism is then replaced by acetate accumulation . These reactions are disconnected from the biocatalytic reaction and do not provide reduced cofactors . 2-Heptanone is metabolised by two pathways . The first leads to 2-heptanol (molar yield close to 55%, enantioselectivity higher than 99%, with a slight decrease at the end of the process) and the second corresponds to material incorporation into the biomass . This latter phenomenon is assumed to provide the biocatalyst with the reduced cofactors needed for the reduction process . Overall, the process yielded ca . 1.4 g/l 2-heptanol in 50 h reaction, which is close to that observed with fresh cells previously grown for 15 h. Eukaryot Cell, 2003 Dec, 2(6), 1200 - 10 The yeast protein kinase C cell integrity pathway mediates tolerance to the antifungal drug caspofungin through activation of Slt2p mitogen-activated protein kinase signaling; Reinoso-Martin C et al.; The echinocandin caspofungin is a new antifungal drug that blocks cell wall synthesis through inhibition of beta-(1-3)-glucan synthesis . Saccharomyces cerevisiae cells are able to tolerate rather high caspofungin concentrations, displaying high viability at low caspofungin doses . To identify yeast genes implicated in caspofungin tolerance, we performed a genome-wide microarray analysis . Strikingly, caspofungin treatment rapidly induces a set of genes from the protein kinase C (PKC) cell integrity signaling pathway, as well as those required for cell wall maintenance and architecture . The mitogen-activated protein kinase Slt2p is rapidly activated by phosphorylation, triggering signaling through the PKC pathway . Cells lacking genes such as SLT2, BCK1, and PKC1, as well as the caspofungin target gene, FKS1, display pronounced hypersensitivity, demonstrating that the PKC pathway is required for caspofungin tolerance . Notably, the cell surface integrity sensor Wsc1p, but not the sensors Wsc2-4p and Mid2p, is required for sensing caspofungin perturbations . The expression modulation of PKC target genes requires the transcription factor Rlm1p, which controls expression of several cell wall synthesis and maintenance genes . Thus, caspofungin-induced cell wall damage requires Wsc1p as a dedicated sensor to launch a protective response through the activated salvage pathway for de novo cell wall synthesis . Our results establish caspofungin as a specific activator of Slt2p stress signaling in baker's yeast. J Biol Chem, 2004 Feb 27, 279(9), 8116 - 25 Epub 2003 Dec 03. A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha; Stasyk OV et al.; Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression . We identified an H . polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue . Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells . Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein . In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells . A mutant deleted for the GCR1 gene was additionally deficient in fructose repression . Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H . polymorpha . Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H . polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor . Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport. Appl Environ Microbiol, 2003 Dec, 69(12), 7453 - 61 Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis; Meroth CB et al.; Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast . The doughs were continuously propagated until the composition of the microbiota remained stable . A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota . The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum . In sourdough A (traditional process with rye flour), C . humilis dominated under the prevailing fermentation conditions . In rye flour sourdoughs B and C, fermented at 30 and 40 degrees C, respectively, S . cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit . In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant . Isolates identified as C . humilis and S . cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively . The yeast species isolated from the sourdoughs were also detected by PCR-DGGE . However, in the gel, additional bands were visible . Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S . uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S . cerevisiae . The last four species were also detected in sourdoughs A, B, and C. FEMS Yeast Res, 2003 Dec, 4(3), 329 - 38 Construction of a Trp- commercial baker's yeast strain by using food-safe-grade dominant drug resistance cassettes; Estruch F et al.; We have designed a food-safe-grade module for gene disruptions in commercial baker's yeast strains, which contains the G418 resistance cassette, KanMX4, flanked by direct repeats from the MEL1 gene of Saccharomyces cerevisiae . This module was used to obtain a Trp(-) auxotrophic mutant of the polyploid HY strain by successive targeting to the TRP1 locus and later in vivo excision of the kan(r) marker . Southern blot analysis indicated that HY contains five copies of the TRP1 gene . However, after four disruption rounds, a strain named HYtrpM(4), unable to grow in the absence of tryptophan, was selected . Southern and Northern analysis of HYtrpM(4) cells showed that a remaining functional wild-type copy was still present, suggesting that the level of phosphoribosylanthranylate isomerase activity, resulting from a single copy of TRP1, is too low to sustain growth . Accordingly, a high reversion frequency of the Trp(-) phenotype, through gene conversion, was found in cells of the mutant strain . Nevertheless, this was not a drawback for its use as a recipient strain of heterologous genes . Indeed, YEpACT-X24 transformants were stable after 25 generations and expressed and secreted high levels of active recombinant xylanase . These data indicate that the new Trp(-) strain can be used to generate a stable recombinant yeast that fulfils all the requirements and market criteria for commercial utilisation. Nutr Health, 2003, 17(2), 131 - 8 Effect of fungi fermentation on organoleptic properties, energy content and in-vitro multienzyme digestibility of cassava products (flour & gari); Akindahunsi AA et al.; The present study sought to investigate the effect of fungi fermentation on the energy content, sensory quality and the digestibility (in vitro) of cassava products (flour and gari) . The fungi fermented cassava products (gari and flour) were produced, by fermenting cassava mash with pure strains of some common saprophytes, namely, Aspergillus flavus, Aspergillus niger, Rhizopus oryzae and Saccharomyces spp (Baker's yeast and palm wine yeast) for 72 hrs before processing into cassava flour and gari, the forms in which cassava is popularly consumed in Nigeria . Parameters determined include energy (Bomb calorimetry), digestibility (in vitro) and sensory quality by trained taste panel . The results of the study indicated that fungi fermentation of the cassava mash significantly (P < 0.05) increased the acceptability of the colour, texture, aroma and taste of the "gari", with that of Rhizopus oryzae fermentation having the highest general acceptability . Furthermore, the results also indicated that fungi fermentation of cassava mash significantly increased (P < 0.05) the in vitro multienzyme protein digestibility of the cassava products . In view of this, fungi fermentation could be used to improve the sensory quality and protein digestibility of cassava products without any significant (P > 0.05) effect on the energy-giving role of cassava products. J Biol Chem, 2004 Feb 13, 279(7), 5169 - 76 Epub 2003 Nov 10. Activation of the Saccharomyces cerevisiae heat shock transcription factor under glucose starvation conditions by Snf1 protein kinase; Hahn JS et al.; Heat shock transcription factor (HSF) is an evolutionarily conserved protein that mediates eukaryotic transcriptional responses to stress . Although the mammalian stress-responsive HSF1 isoform is activated in response to a wide array of seemingly unrelated stresses, including heat shock, pharmacological agents, infection and inflammation, little is known about the precise mechanisms or pathways by which this factor is activated by many stressors . The baker's yeast Saccharomyces cerevisiae encodes a single HSF protein that responds to heat stress and glucose starvation and provides a simple model system to investigate how a single HSF is activated by multiple stresses . Although induction of the HSF target gene CUP1 by glucose starvation is dependent on the Snf1 kinase, HSF-dependent heat shock induction of CUP1 is Snf1-independent . Approximately 165 in vivo targets for HSF have been identified in S . cerevisiae using chromatin immunoprecipitation combined with DNA microarrays . Interestingly, approximately 30% of the HSF direct target genes are also induced by the diauxic shift, in which glucose levels begin to be depleted . We demonstrate that HSF and Snf1 kinase interact in vivo and that HSF is a direct substrate for phosphorylation by Snf1 kinase in vitro . Furthermore, glucose starvation-dependent, but not heat shock-dependent HSF phosphorylation, and enhanced chromosomal HSF DNA binding to low affinity target promoters such as SSA3 and HSP30, occurred in a Snf1-dependent manner . Consistent with a more global role for HSF and Snf1 in activating gene expression in response to changes in glucose availability, expression of a subset of HSF targets by glucose starvation was dependent on Snf1 and the HSF carboxyl-terminal activation domain. Rapid Commun Mass Spectrom, 2003, 17(21), 2430 - 8 Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection; Morozov VN et al.; Analysis of interactive networks between proteins and other molecular constituents is of paramount importance to delineate complex cellular processes . In order to facilitate this process, new technologies that allow rapid, high-throughput parallel screening, as well as identification of constituents, are necessary . A particularly powerful combination in this regard could be the use of multiprotein microarrays coupled with mass spectrometry (MS) . In the initial step of the method development we applied MS to single-protein microarrays . We demonstrated that even a simplified version of the method allows rapid parallel label-free assay of specific protein interactions with multiple metabolites derived from complex artificial and natural mixtures . The microarrays fabricated by the electrospray deposition technique and cross-linked in glutaraldehyde vapor were brought into contact with droplets of solution containing either a natural extract of baker's yeast cells or an artificial cocktail of metabolites . After washing, the microarrays were placed into 75% methanol to denature proteins and release specifically bound metabolites . The eluates were then analyzed by electrospray ionization mass spectrometry (ESI-MS) to simultaneously detect all the metabolites bound . Such a procedure applied to ten different proteins demonstrated that 50-400 ng of cross-linked protein is enough to obtain ion intensities from metabolites that are well distinguishable above noise . The compatibility of microplates and different microarray designs with MS detection is discussed . Appl Microbiol Biotechnol, 2004 Feb, 63(6), 635 - 46 Epub 2003 Oct 28. Production of lipid compounds in the yeast Saccharomyces cerevisiae; Veen M et al.; This review describes progress using the yeast Saccharomyces cerevisiae as a model organism for the fast and efficient analysis of genes and enzyme activities involved in the lipid biosynthetic pathways of several donor organisms . Furthermore, we assess the impact of baker's yeast on the production of novel, high-value lipid compounds . Yeast can be genetically modified to produce selected substances in relatively high amounts . A major advantage in choosing yeast as an object for metabolic engineering is the fact that the lipid pathways in this organism have been described in detail and are well characterized . We focus on the de novo production of three major families of lipid products . These are: (1) sterols, providing some previously known and some novel applications as examples of the lipid pathway enhancement that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega-3 and omega-6 fatty acids which do not occur naturally in yeast . We utilize the current knowledge and point out perspectives and problems for future biotechnological applications in the field of lipid compounds. Exp Gerontol, 2003 Oct, 38(10), 1051 - 63 A high-throughput screening system for genes extending life-span; Chen C et al.; We developed a high-throughput functional genomic screening system that allows identification of genes prolonging life-span in the baker's yeast Saccharomyces cerevisiae . The method is based on isolating yeast mother cells with extended number of cell divisions as indicated by the increased number of bud scars on their surface . Fluorescently labelled Wheat Germ Agglutinin was used for specific staining of chitin, a major component of bud scars . Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of 12 yeast transformants with a potentially prolonged life-span . The transgene in one of the lines was identified as ferritin light chain (FTL) and studied in more detail . Yeast cells containing FTL showed an enhanced iron and H(2)O(2) resistance, a reduced cell death rate and an increased number of cell divisions . Overexpression of FTL in the nematode Caenorhabditis elegans resulted in a life-span increase of 8% confirming our yeast observations in a multicellular organism . Our data demonstrate that this method permits a fast screening of libraries for hunting genes involved in ageing processes. Z Naturforsch {C}, 2003 Sep-Oct, 58(9-10), 691 - 6 Synthesis of montroumarin; Saeed A; A simple stereoselective synthesis of montroumarin {(3S)-6,8-dihydroxy-3-phenyl-3,4-dihydroisocoumarin} isolated from Montrouziera sphaeroidea has been achieved . Condensation of benzoyl chloride with 3,5-dimethoxyhomophthalic acid afforded 6,8-dimethoxy-3-phenylisocoumarin (3) which on sequential saponification and esterification yielded the keto ester 5 . Enantioselective reduction of the latter with baker's yeast directly furnished the (3S)-6,8-dimethoxy-3-phenyl-3,4-dihydroisocoumarin (6) in good enantioselectivity which on demethylation provided montroumarin . All of the synthesized compounds were examined in vitro for antifungal activity. Biotechnol Adv, 1987, 5(2), 271 - 97 Influence of modifying agents on enzyme inactivation studies . An analysis using a series mechanism and a form of the Hill-type equations; Sadana A et al.; A series deactivation model is utilized to theoretically examine the influence of different modifying agents on enzyme deactivation kinetics . A form of the Hill-type equation is used to describe the effect of the modifying agents on the model parameters . Modification-induced inactivation equations are presented for the acetylation and succinylation of E . Coli asparaginase, for the site-specific reagent and substrate modification of flavocytochrome b(2) from Baker's yeast, and for the guanidinium chloride inactivation of cathepsin D . The analysis of more data for these and other enzymes would help further substantiate the technique presented and enhance the applicability of the model. Antonie Van Leeuwenhoek, 2003, 84(2), 125 - 34 Osmotolerance and leavening ability in sweet and frozen sweet dough . Comparative analysis between Torulaspora delbrueckii and Saccharomyces cerevisiae baker's yeast strains; Hernandez-Lopez MJ et al.; The response of Saccharomyces cerevisiae and freeze-tolerant Torulaspora delbrueckii strains to osmotic stress and their CO2 production capacity in sweet and frozen-sweet dough has been examined . T . delbrueckii strains, IGC5321 and IGC5323 showed higher leavening ability than Saccharomyces, specially after exposure to hyperosmotic stress of bread dough containing 20% sucrose and 2% salt added . In addition, Torulaspora and especially T . delbrueckii IGC5321 exhibited no loss of CO2 production capacity during freeze-thaw stress . Overall, these results appeared to indicate that Torulaspora cells are more tolerant than Saccharomyces to osmotic stress of bread dough . This trait correlated with a low invertase activity, a slow rate of trehalose mobilisation and the ability to respond rapidly to osmotic stress . Growth behaviour on high osmotic synthetic media was also examined . Cells of the IGC5321 strain showed intrinsic osmotolerance and ion toxicity resistance . However, T . delbrueckii IGC5323 exhibited a clear phenotype of osmosensitivity . Hence, this characteristic may not be essential or the only determinant for leavening ability in salted high-sugar dough. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(10), 2229 - 40 Advanced oxidation of biologically pretreated Baker's Yeast Industry effluents for high recalcitrant COD and color removal; Altinbas M et al.; The aim of this study was to investigate the effectiveness of chemical oxidation by applying ozonation, ozonation with hydrogen peroxide and Fenton's processes for decolorization and residual COD removal of biologically pretreated baker's yeast industry (BYI) effluents . Baker's yeast industry effluents characterizing with high COD, TKN, dark color, and non-biodegradable organic pollutants . The batch tests were performed to determine the optimum operating conditions including pH, O3, H2O2, and FeSO4 dosages, molar ratio of Fe2+/H2O2 and reaction time . It was noticed that H2O2 significantly reduced the reaction times for the same ozone dosages: however, COD and color removals were not remarkable . In the Fenton's oxidation studies, the removal efficiencies of COD and color for 30 min reaction time for three different types of BYI effluents were found about 86 and 92%, respectively . Experimental results of the presented study have clearly indicated that the Fenton's oxidation technology is capable to fate almost all parts of the organics which consist of both soluble initial and microbial inert fractions of COD for baker's yeast effluents . Effluents from the Fenton's oxidation process can satisfy effluent standards for COD and color in general. J Biol Inorg Chem, 2003 Nov, 8(8), 803 - 9 Epub 2003 Sep 27. The many highways for intracellular trafficking of metals; Luk E et al.; Metal ions such as copper and manganese represent a unique problem to living cells in that these ions are not only essential co-factors for metalloproteins, but are also potentially toxic . To aid in the homeostatic balance of essential but toxic metals, cells have evolved with a complex network of metal trafficking pathways . The object of such pathways is two-fold: to prevent accumulation of the metal in the freely reactive form (metal detoxification pathways) and to ensure proper delivery of the ion to target metalloproteins (metal utilization pathways) . Much of what we currently know regarding these complex pathways of metal trafficking has emerged from molecular genetic studies in baker's yeast, Saccharomyces cerevisiae . In this review, we shall briefly highlight the current understanding of factors that function in the trafficking and handling of copper, including copper detoxification factors, copper transporters and copper chaperones . In addition, very recent findings on the players involved in manganese trafficking will be presented . The goal is to provide a paradigm for the intracellular handling of metals that may be applied in a more general sense to metals that serve essential functions in biology. Mol Biol Cell, 2003 Oct, 14(10), 4272 - 84 Epub 2003 Jun 27. Amino a