Yeast

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 acid starvation and Gcn4p regulate adhesive growth and FLO11 gene expression in Saccharomyces cerevisiae; Braus GH et al.; In baker's yeast Saccharomyces cerevisiae, cell-cell and cell-surface adhesion are required for haploid invasive growth and diploid pseudohyphal development . These morphogenetic events are induced by starvation for glucose or nitrogen and require the cell surface protein Flo11p . We show that amino acid starvation is a nutritional signal that activates adhesive growth and expression of FLO11 in both haploid and diploid strains in the presence of glucose and ammonium, known suppressors of adhesion . Starvation-induced adhesive growth requires Flo11p and is under control of Gcn2p and Gcn4p, elements of the general amino acid control system . Tpk2p and Flo8p, elements of the cAMP pathway, are also required for activation but not Ste12p and Tec1p, known targets of the mitogen-activated protein kinase cascade . Promoter analysis of FLO11 identifies one upstream activation sequence (UASR) and one repression site (URS) that confer regulation by amino acid starvation . Gcn4p is not required for regulation of the UASR by amino acid starvation, but seems to be indirectly required to overcome the negative effects of the URS on FLO11 transcription . In addition, Gcn4p controls expression of FLO11 by affecting two basal upstream activation sequences (UASB) . In summary, our study suggests that amino acid starvation is a nutritional signal that triggers a Gcn4p-controlled signaling pathway, which relieves repression of FLO11 gene expression and induces adhesive growth.

Farmaco, 2003 Oct, 58(10), 1029 - 32
Chemo-enzymatic synthesis of levodropropizine; Caselli E et al.; Levodropropizine, an antitussive drug, was prepared in high enantiomeric excess in three steps, starting from dichloroacetone (2) . Monosubstitution of 2 with sodium benzoate and subsequent baker's yeast reduction stereoselectively afforded the corresponding chlorohydrin in 73% ee, which was converted to levodropropizine and enantiomerically enriched up to 95% ee by fractional crystallisation.

Cancer Lett, 2003 Aug 20, 198(2), 153 - 60
Protective effects of fungal (1-->3)-beta-D-glucan derivatives against oxidative DNA lesions in V79 hamster lung cells; Slamenova D et al.; beta-Glucans belong to the class of substances known as biological response modifiers with a broad range of activity . We have investigated two types of glucans: (1-->3)-beta-D glucan from the baker's yeast Saccharomyces cerevisiae and beta-glucan-chitin complex from the mycelium of filamentous fungus Aspergillus niger . Since these fibrillar beta-glucans are insoluble in water, their water-soluble derivatives--carboxymethyl glucan (CM-G), sulfoethyl glucan (SE-G), and carboxymethyl chitin-glucan (CM-CG) were prepared and tested . The aim of the present work was to investigate the protective effect of the prepared glucan derivatives against oxidative DNA damage induced by H2O2 and visible light-excited Methylene Blue in V79 hamster lung cells . The level of DNA damage (DNA strand breaks) was measured using the single cell gel electrophoresis, so called comet assay . Our findings demonstrate that all three tested glucans reduce oxidative DNA damage . The ability to reduce genotoxic activity increased in the order: CM-G<SE-G<CM-CG . We suggest that the analyzed glucans exhibit protective effects against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen.

Biotechnol Appl Biochem, 2004 Feb, 39(Pt 1), 89 - 97
Expression of GAL genes in a mutant strain of Saccharomyces cerevisiae lacking GAL80: quantitative model and experimental verification; Verma M et al.; The regulatory network of GAL genes is a model system for the production of foreign proteins . A mathematical model based on steady state was developed for the expression of GAL (galactosidase) genes in a mutant strain of Saccharomyces cerevisiae lacking GAL80 . The transcriptional and translational responses of the GAL switch were predicted at various steady-state glucose concentrations . The model predicted ultrasensitive transcriptional response with a Hill coefficient ( h ) of 1.9 and 3.2 for genes with one and two binding sites respectively . Further, a lesser degree of ultrasensitivity was predicted for translational response with an h value of 1.3 for genes with one binding site and 2.1 for genes with two binding sites . The ultrasensitivity was due to dimerization of regulatory protein Gal4p and co-operative binding of Gal4p to DNA . The steady-state predictions were experimentally verified through measurements of alpha-galactosidase (for one binding site) and beta-galactosidase (for two binding sites) . The steady state model was further extended to represent the dynamic expression profile and the same was verified experimentally . The growth phase and the synthesis of foreign protein could be distinctly separated using a mutant strain of Saccharomyces cerevisiae (baker's yeast).

Anticancer Res, 2003 May-Jun, 23(3B), 2751 - 6
Fungal beta-(1-3)-D-glucan derivatives exhibit high antioxidative and antimutagenic activity in vitro; Krizkova L et al.; The antioxidative activity and antimutagenic effects of the water-soluble beta-(1-3)-D-glucan derivatives from biotechnologically important species, in particular carboxymethyl-glucan (CM-G) and sulfoethyl-glucan (SE-G) both from the baker's yeast Saccharomyces cerevisiae, and carboxymethyl-chitin-glucan (CM-CG) from filamentous fungus Aspergillus niger, were evaluated . The luminol-dependent photochemical method using trolox as a standard showed that CM-CG, SE-G and CM-G possessed high antioxidative properties . CM-CG exhibited the highest antioxidative activity (2.15 +/- 0.14 nmol exhibits the same activity as 1 nmol of trolox), followed by SE-G (2.99 +/- 0.15 nmol) and CM-G (4.59 +/- 0.14 nmol) . These glucans were experimentally confirmed to exhibit different, statistically significant activity in reducing the damage of chloroplast DNA of the flagellate Euglena gracilis induced by ofloxacin and acridine orange . Our findings suggest that the antimutagenic effect of CM-CG, SE-G and CM-G against ofloxacin is based on their antioxidative capability to scavenge reactive oxygen species (p < 0.001) . As far as acridine orange is concerned, the reduction of the chloroplast DNA lesion could be a result of the absorptive capacity of the glucans (p < 0.001) . We found out that the water-soluble beta-(1-3)-D-glucan derivatives possess very high antioxidative activity as well as expressive antimutagenic effects, exerted through different mode of action.

Int J Food Microbiol, 2003 Sep 1, 86(1-2), 79 - 86
Ura- host strains for genetic manipulation and heterologous expression of Torulaspora delbrueckii; Hernandez-Lopez MJ et al.; Recently, the industrial and academic interest in the yeast Torulaspora delbrueckii has increased notably due to its high resistance to several stresses . This characteristic has made of this organism a very attractive model to study the molecular basis of the stress response in yeast . However, very little is known about the physiology and genetics of this yeast, and the tools for its manipulation have not been developed . Here, we have generated Ura(-) strains of the baker's yeast T . delbrueckii IGC5323 by either 5-FOA-aided selection or transformation with a PCR-based disruption cassette, natMX4, which confers nourseothricin resistance . Furthermore, the mutant and disruptant strains were used as recipient of a plasmid containing the xlnB cDNA from Aspergillus nidulans . Our results indicate that Torulaspora transformants produce active recombinant protein at a similar level to that found for Saccharomyces.

Biochem Soc Trans, 2003 Aug, 31(Pt 4), 745 - 7
Introduction to mannan-binding lectin; Kilpatrick DC; Mannan-binding lectin (MBL) was first discovered as a plasma opsonin for baker's yeast and was independently characterized biochemically . It belongs to the small subfamily of collectins: C-type lectins possessing a collagen-like domain . MBL is synthesized by the liver and secreted into the bloodstream . It is believed to be an important component of innate immunity, acting as an ante-antibody and/or as a disease modifier . It is thought to influence disorders as diverse as meningococcal disease, rheumatoid arthritis, cystic fibrosis and recurrent miscarriage . Lack of MBL may be most relevant in the context of a co-existing secondary immune deficiency . Replacement therapy, first carried out 30 years ago with unfractionated plasma, appears promising . The development of a recombinant product should permit the extension of MBL therapy to randomized clinical trials of sufficient size to provide clear evidence about the physiological significance of this intriguing glycoprotein.

Chemosphere, 2003 Sep, 52(10), 1807 - 17
Thermal and spectroscopic studies on sorption of nickel(II) ion on protonated baker's yeast; Padmavathy V et al.; Protonated form (Hy) of yeast was subjected to thermal analysis (TGA and DTG) in the temperature range 60-800 degrees C . Chemically bound water volatilizes around 200 degrees C and the matrix undergoes extensive oxidative decomposition at 450 degrees C, the weight loss reaching 75% at 800 degrees C . The sorption capacity of the matrix for nickel(II) ion increases on heat treatment from 60 to 200 degrees C (from 16.9 to 25.0 mg/g), but was reduced on heating to higher temperatures at an initial nickel(II) ion concentration of 1200 mg/g . The FTIR spectra of Hy and nickel(II) ion saturated yeast, indicated that biosorption occurs on the sugar and nucleic acid regions, possibly involving --COOH and --NH groups.

Anal Bioanal Chem, 2003 Jul, 376(6), 832 - 7 Epub 2003 Jun 17.
Polymeric FAD used as enzyme-friendly mediator in lactate detection; Leonida MD et al.; We present the fabrication and properties of lactate biosensors . The novel feature is the use of polymerized flavin adenine dinucleotide (FAD) as mediator for electron transfer . The biosensors were prepared using lactate dehydrogenase (LDH), lactate oxidase (LOX), or baker's yeast (BY) immobilized at the surface of the electrode . The sensors using purified enzymes showed good sensitivity, linearity, and stability . The sensitivity of the BY electrodes was slightly lower . The advantages of this type of sensors are discussed in connection with potential applications.

Bioresour Technol, 2003 Sep, 89(3), 281 - 7
Kinetics of biosorption of cadmium on Baker's yeast; Vasudevan P et al.; In the present study the kinetics of biosorption of cadmium(II) ions by deactivated protonated yeast converted to sodium form was investigated for different initial concentrations of the metal ion (10-100 ppm) and different sorbent dosages (0.1-2.0 g) at a pH of 6.5 . The adsorption process occurred in four distinct steps and the rates for these steps decreased sequentially . The rate of cadmium uptake in each case was pseudo-second-order with respect to metal ion concentration . The amount sorbed at equilibrium was found to be directly proportional to the initial metal ion concentration divided by the sorbent mass.

J Math Biol, 2003 Jun, 46(6), 479 - 503
Words in DNA sequences: some case studies based on their frequency statistics; Basu S et al.; One of the critical requirements of data analysis involving large DNA sequences is an effective statistical summarization of those sequences . In this article DNA sequences have been analyzed based on word frequencies . Our analysis focuses on the detection of structural signature of a genome reflected in word frequencies and identification of phylogenetic relationships among different species reflected in the variation of word distributions in their DNA sequences . We have carried out a statistical study of the complete genome of baker's yeast, of various ribosomal RNA sequences from different prokaryotic and eukaryotic organisms and of the full genomes of some bacteriophages . Our exploratory analysis amply demonstrates the usefulness of DNA word frequencies in reducing the dimensionality of large sequences while retaining some of the structural information there that can have biological significance . Some conceptual issues that arise in course of our investigation have been addressed . A few interesting problems related to the statistics of DNA words have been pointed out with some indication of their possible solutions . The work has been partially motivated by the fact that sequence alignment and homology techniques that are quite popular for comparing and analyzing relatively smaller DNA sequences of nearly equal sizes are not applicable to data consisting of large sequences with widely varying sizes, which may contain segments with unknown or no biological functions, and consequently their comparison through functional homology is either impossible or extremely difficult.

Clin Exp Immunol, 2003 Jun, 132(3), 473 - 6
Anti-Saccharomyces cerevisiae antibodies (ASCA) and autoimmune liver diseases; Muratori P et al.; Antibodies to the baker's yeast Saccharomyces cerevisiae (ASCA), recently proposed as a serological marker of Crohn's disease, have also been detected in other autoimmune disorders . The aim of this study was to determine prevalence and clinical significance of ASCA in autoimmune liver disease . The presence of IgG and IgA ASCA was evaluated using a commercially available immunoassay in 215 patients with autoimmune liver disease (primary biliary cirrhosis, PBC, 123 cases; autoimmune hepatitis, AIH, 67 cases; primary sclerosing cholangitis, PSC, 25 cases), 48 with inflammatory bowel disease and 19 healthy blood donors . Anti neutrophil cytoplasmic antibodies with the perinuclear pattern (p-ANCA) were assessed by indirect immunofluorescence in PSC patients . The main clinical and biochemical parameters between ASCA-positive and negative patients were analysed and compared . ASCA are predominant in Crohn's disease (70%); among liver patients, PSC and AMA-negative PBC show the highest ASCA prevalence (53% and 44%) . In PBC ASCA correlate with higher levels of circulating IgA (P < 0.05) . In PSC the detection of either ASCA or p-ANCA is neither associated with any clinical or biochemical feature, nor with an underlying inflammatory bowel disease . ASCA can not be considered an additional serological marker of autoimmune liver disease, but the possibility of detecting such a reactivity in autoimmune liver disorders should be considered; their correlation with elevated IgA in PBC suggests that ASCA may be an indirect sign of enhanced mucosal immunity; in PSC patients neither ASCA nor p-ANCA predict the occurrence of a concomitant inflammatory bowel disease.

J Agric Food Chem, 2003 Jun 4, 51(12), 3629 - 35
Generation of thiols by biotransformation of cysteine-aldehyde conjugates with baker's yeast; Huynh-Ba T et al.; Baker's yeast was shown to catalyze the transformation of cysteine-furfural conjugate into 2-furfurylthiol . The biotransformation's yield and kinetics were influenced by the reaction parameters such as pH, incubation mode (aerobic and anaerobic), and substrate concentration . 2-Furfurylthiol was obtained in an optimal 37% yield when cysteine-furfural conjugate at a 20 mM concentration was anaerobically incubated with whole cell baker's yeast at pH 8.0 and 30 degrees C . Similarly to 2-furfurylthiol, 5-methyl-2-furfurylthiol (11%), benzylthiol (8%), 2-thiophenemethanethiol (22%), 3-methyl-2-thiophenemethanethiol (3%), and 2-pyrrolemethanethiol (6%) were obtained from the corresponding cysteine-aldehyde conjugates by incubation with baker's yeast . This work indicates the versatile bioconversion capacity of baker's yeast for the generation of thiols from cysteine-aldehyde conjugates . Thanks to its food-grade character, baker's yeast provides a biochemical tool to produce thiols, which can be used as flavorings in foods and beverages.

Cell Immunol, 2003 Jan, 221(1), 1 - 5
Immunopotentiation of intraepithelial lymphocytes in the intestine by oral administrations of beta-glucan; Tsukada C et al.; Mice were orally administered with beta-glucan, isolated from baker's yeast, daily for one week (25mg/day/mouse) and several immunoparameters in the digestive tract were examined . The most prominent change was an increase in the number of intraepithelial lymphocytes (IEL) in the intestine, although the number of lymphocytes in the liver remained unchanged . The absolute number of both alphabetaT cells and gammadeltaT cells expressing CD8 antigens increased among IEL in the intestine . Primarily, liver lymphocytes showed a spontaneous production of Type 0 cytokine (simultaneous production of IFNgamma and IL-4) while IEL did not produce any cytokines without stimulation . However, mice administered with beta-glucan produced Type 1 cytokine, namely, production of IFNgamma alone . These results suggest that beta-glucan may be an important potentiator for mucosal immunity in the digestive tract.

Curr Genet, 2003 Jul, 43(4), 245 - 54 Epub 2003 May 08.
Evolution, biochemistry and genetics of protein kinase C in fungi; Schmitz HP et al.; From yeast to humans, protein kinase C is an enzyme of central importance in signal transduction processes . In baker's yeast, Saccharomyces cerevisiae, a single gene encoding Pkc1p has been identified . Mutant analyses revealed that Pkc1p function is essential to ensure cellular integrity through regulation of a specific MAP kinase cascade . Due to the involvement of different defective mammalian isozymes in various diseases and the model character of simple eukaryotes, increasing attention has been paid to the structure and regulation of the enzymes of fungal origin . This review summarizes the knowledge gathered so far.

J Nutr, 2003 May, 133(5 Suppl 1), 1579S - 80S
Integrating trace element metabolism from the cell to the whole organism; Thiele DJ; The redox chemistry of copper (Cu) makes this both a powerful enzyme catalyst and a dangerous reactant that generates hydroxyl radical . Although virtually all cells from microbes to mammals must acquire Cu to drive important biochemical reactions, the potential toxicity of Cu demands an exquisite level of vectorial transport and homeostatic control . Our laboratory is interested in how organisms acquire Cu through the action of high-affinity plasma membrane Cu transporters of the copper transport protein (Ctr) class of proteins . We have isolated Ctr Cu transporters from baker's yeast and fission yeast and from flies, mice and mammals . This review will focus on understanding how the Ctr high-affinity Cu transport proteins function, from their biochemical mechanism of action in yeast and cultured metazoan cells to their roles in Cu delivery and mammalian embryonic development.

Free Radic Biol Med, 2003 May 15, 34(10), 1315 - 25
Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae; Chen SR et al.; Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress . When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies . Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes . These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast . Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults . These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death . In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses . Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 853 - 65
Purification of glucose-6-phosphate dehydrogenase from baker's yeast in aqueous two-phase systems with free triazine dyes as affinity ligands; Xu Y et al.; To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced . In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase . The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration . A two-step affinity extraction process was established for the purification of G6PDH from baker's yeast . The total yield of G6PDH was 66.9% and purification factor was 2.35.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 787 - 97
Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems; Oliveira GG et al.; This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker's yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems . First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design . The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase . The purification factor (PurTOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (KProt) was 0.47.

J Agric Food Chem, 2003 May 7, 51(10), 3103 - 7
Chemoenzymatic synthesis of aroma active 5,6-dihydro- and tetrahydropyrazines from aliphatic acyloins produced by baker's yeast; Kurniadi T et al.; Twenty-five acyloins were generated by biotransformation of aliphatic aldehydes and 2-ketocarboxylic acids using whole cells of baker's yeast as catalyst . Six of these acyloins were synthesized and tentatively characterized for the first time . Subsequent chemical reaction with 1,2-propanediamine under mild conditions resulted in the formation of thirteen 5,6-dihydropyrazines and six tetrahydropyrazines . Their odor qualities were evaluated, and their odor thresholds were estimated . Among these pyrazine derivatives, 2-ethyl-3,5-dimethyl-5,6-dihydropyrazine (roasted, nutty, 0.002 ng/L air), 2,3-diethyl-5-methyl-5,6-dihydropyrazine (roasted, 0.004 ng/L air), and 2-ethyl-3,5-dimethyltetrahydropyrazine (bread crustlike, 1.9 ng/L air) were the most intensive-smelling aroma active compounds.

J Environ Sci Health B, 2003 May, 38(3), 243 - 55
Efficient adsorption of the mycotoxins zearalenone and T-2 toxin on a modified yeast glucan; Freimund S et al.; 1,3-Beta-D-glucan derived from baker's yeast was chemically modified in two steps yielding crosslinked carboxymethyl glucan as the sodium salt (2) . After cation exchange with hexadecyltrimethylammonium chloride, a hydrophobic adsorbent (3) was obtained which showed an excellent binding of the estrogenic mycotoxin zearalenone with a maximum adsorption of up to 183 mg/g . Compound 3 additionally showed a relatively high adsorption capacity for the trichothecene T-2 toxin of at least 10 mg/g . Starting from 2, various derivatives were prepared by cation exchange using quaternary ammonium salts bearing substituents besides methyl from four to 18 carbon atoms . The adsorption of T-2 toxin on these derivatives were compared with compound 3 leading to the conclusion that 3 is the best adsorbent of all investigated tetraalkylammonium-modified derivatives of 2.

FEMS Yeast Res, 2002 Aug, 2(3), 251 - 7
Novel insights into the osmotic stress response of yeast; Mager WH et al.; Response to hyperosmolarity in the baker's yeast Saccharomyces cerevisiae has attracted a great deal of attention of molecular and cellular biologists in recent years, from both the fundamental scientific and applied viewpoint . Indeed the underlying molecular mechanisms form a clear demonstration of the intricate interplay of (environmental) signalling events, regulation of gene expression and control of metabolism that is pivotal to any living cell . In this article we briefly review the cellular response to conditions of hyperosmolarity, with focus on the high-osmolarity glycerol mitogen-activated protein kinase pathway as the major signalling route governing cellular adaptations . Special attention will be paid to the recent finding that in the yeast cell also major structural changes occur in order to ensure maintenance of cell integrity . The intriguing role of glycerol in growth of yeast under (osmotic) stress conditions is highlighted.

FEMS Yeast Res, 2002 Dec, 2(4), 481 - 94
The three zinc-containing alcohol dehydrogenases from baker's yeast, Saccharomyces cerevisiae; Leskovac V et al.; This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker's yeast, Saccharomyces cerevisiae . The opening section deals with the substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates . In the following sections, the kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism . The complete primary structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented . All known artificial mutations in the primary structure of the YADH are covered in full and described in detail . Further, the chemical mechanism of action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism . Finally, the bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate specificity and the enantioselectivity of the yeast enzyme.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 665 - 71
Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor; Belo I et al.; Fed-batch is the dominating mode of operation in high-cell-density cultures of Saccharomyces cerevisae in processes such as the production of baker's yeast and recombinant proteins, where the high oxygen demand of these cultures makes its supply an important and difficult task . The aim of this work was to study the use of hyperbaric air for oxygen mass transfer improvement on S . cerevisiae fed-batch cultivation . The effects of increased air pressure up to 1.5 MPa on cell behavior were investigated . The effects of oxygen and carbon dioxide were dissociated from the effects of total pressure by the use of pure oxygen and gas mixtures enriched with CO(2) . Fed-batch experiments were performed in a stirred tank reactor with a 600 mL stainless steel vessel . An exponential feeding profile at dilution rates up to 0.1 h(-)(1) was used in order to ensure a subcritical flux of substrate and, consequently, to prevent ethanol formation due to glucose excess . The ethanol production observed at atmospheric pressure was reduced by the bioreactor pressurization up to 1.0 MPa . The maximum biomass yield, 0.5 g g(-)(1) (cell mass produced per mass of glucose consumed) was attained whenever pressure was increased gradually through time . This demonstrates the adaptive behavior of the cells to the hyperbaric conditions . This work proved that hyperbaric air up to 1.0 MPa (0.2 MPa of oxygen partial pressure) could be applied to S . cerevisiae cultivation under low glucose flux . Above that critical oxygen partial pressure value, i.e., for oxygen pressures of 0.32 and 0.5 MPa, a drastic cell growth inhibition and viability loss were observed . The increase of carbon dioxide partial pressure in the gas mixture up to 48 kPa slightly decreased the overall cell mass yield but had negligible effects on cell viability.

Environ Sci Pollut Res Int, 2003, 10(1), 44 - 8
Minimum specific cost control of technological processes realized in a living objects-containing microenvironment; Amelkin AA et al.; The purpose of the present work is to work out an approach for the development of software and the choice of hardware structures when designing subsystems for automatic control of technological processes realized in living objects containing limited space (microenvironment) . The subsystems for automatic control of the microenvironment (SACME) under development use the Devices for Air Prophylactic Treatment, Aeroionization, and Purification (DAPTAP) as execution units for increasing the level of safety and quality of agricultural raw material and foodstuffs, for reducing the losses of agricultural produce during storage and cultivation, as well as for intensifying the processes of activation of agricultural produce and industrial microorganisms . A set of interconnected SACMEs works within the framework of a general microenvironmental system (MES) . In this research, the population of baker's yeast is chosen as a basic object of control under the industrial fed-batch cultivation in a bubbling bioreactor . This project is an example of a minimum cost automation approach . The microenvironment optimal control problem for baker's yeast cultivation is reduced from a profit maximum to the maximization of overall yield by the reason that the material flow-oriented specific cost correlates closely with the reciprocal value of the overall yield . Implementation of the project partially solves a local sustainability problem and supports a balance of microeconomical, microecological and microsocial systems within a technological subsystem realized in a microenvironment maintaining an optimal value of economical criterion (e.g . minimum material, flow-oriented specific cost) and ensuring: (a) economical growth (profit increase, raw material saving); (b) high security, safety and quality of agricultural raw material during storage process and of food produce during a technological process; elimination of the contact of gaseous harmful substances with a subproduct during various technological stages; (c) improvement of labour conditions for industrial personnel from an ecological point of view (positive effect of air aeroionization and purification on human organism promoting strengthened health and an increase in life duration, pulverent and gaseous chemical and biological impurity removal) . An alternative aspect of a controlled living microenvironment forming is considered.

Ann Nutr Metab, 2003, 47(1), 16 - 21
Relative bioavailability and antioxidant potential of two coenzyme q10 preparations; Kurowska EM et al.; Coenzyme Q10 (CoQ10) is synthesized by the human body and found in certain foods . Daily supplementation of CoQ10 could protect against heart disease but the bioavailability of CoQ10 supplements depends on the formulation taken . We compared the bioavailability and antioxidant properties of two commercial CoQ10 formulations, a commercial grade CoQ10 powder (commercial grade CoQ) and a new BT-CoQ10 BIO-TRANSFORMED (BT-CoQ10) obtained by fermentation of a soy-based, CoQ10-rich media with baker's yeast . Eleven healthy individuals participated in a randomized two-way crossover trial, with a 3-week washout period . Capsules containing 300 mg of either BT-CoQ10 or commercial grade CoQ10 were given daily for 1 week and multiple blood samples were taken for CoQ10, glutathione and glutathione peroxidase (GPx) determination . In 3 subjects, baseline plasma CoQ10 levels were lower prior to BT than prior to commercial grade CoQ treatment . In the remaining participants, ingestion of BT vs . commercial grade CoQ significantly increased maximum plasma CoQ10 concentration (+126%, p = 0.04) and tended to increase CoQ10 area under the curve from 0 to 24 h (+160%, p = 0.07) . One week of treatment with each formulation increased plasma CoQ10 but did not alter plasma glutathione or GPx activity . The enhanced bioavailability of the BT product might be due to its predominantly reduced, hydrophilic membrane-complex form .

Mol Microbiol, 2003 Mar, 47(5), 1185 - 97
Molecular mechanisms of iron uptake in fungi; Kosman DJ; Fungi, like all free-living organisms, are in competition for limiting nutrients . In accumulating iron, fungi are faced also with a trace metal whose aqueous and redox chemistry make it both relatively bio-unavailable and strongly cytotoxic . Successful adaptation to this environmental context has provided fungi with an iron uptake strategy that has three features: it relies on redox cycling to enhance iron bio-availability and reduce iron cytotoxicity; it includes both high- and low-affinity pathways that are mechanistically distinct; and it is autoregulating so as to maintain intracellular iron homeostasis . Using Saccharomyces cerevisiae as a paradigm, this review summarizes current knowledge about the four pathways by which this yeast accumulates iron . These four pathways include: siderophore iron accumulation; high affinity iron uptake via an iron permease; and two lower affinity uptake pathways through relatively non-specific divalent metal ion transporters . All of these four pathways are directly or indirectly dependent on the activity of metalloreductase activity expressed extracellularly on the plasma membrane . A variety of experimental and genomics data indicate that this resourcefulness is shared by many, if not most, fungi . On the other hand, while the autoregulation of iron metabolism in Baker's yeast is well-understood, little is known about the apparent homeostatic mechanisms in these other yeasts and fungi . The integration of these multiple uptake mechanisms and their regulation into over-all iron homeostasis in yeast concludes this brief review.

Nature, 2003 Feb 20, 421(6925), 848 - 52
Yeast genome duplication was followed by asynchronous differentiation of duplicated genes; Langkjaer RB et al.; Gene redundancy has been observed in yeast, plant and human genomes, and is thought to be a consequence of whole-genome duplications . Baker's yeast, Saccharomyces cerevisiae, contains several hundred duplicated genes . Duplication(s) could have occurred before or after a given speciation . To understand the evolution of the yeast genome, we analysed orthologues of some of these genes in several related yeast species . On the basis of the inferred phylogeny of each set of genes, we were able to deduce whether the gene duplicated and/or specialized before or after the divergence of two yeast lineages . Here we show that the gene duplications might have occurred as a single event, and that it probably took place before the Saccharomyces and Kluyveromyces lineages diverged from each other . Further evolution of each duplicated gene pair-such as specialization or differentiation of the two copies, or deletion of a single copy--has taken place independently throughout the evolution of these species.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 54 - 8
{Cloning of trehalose-6-phosphate synthase gene from S . cerevisiae and its plant expression vector construction}; Chen H et al.; Total RNA, mRNA were isolated from baker's yeast S . cerevisiae, the cDNA was prepared with AMV Reverse Transcriptase from the total mRNA . The gene of trehalose-6-phosphate synthase was cloned from the cDNA with PCR amplification . The gene was sequenced and the results showed that the tpsl gene contains 1507 nucleotides and 99.6% identity with S . cerevisiae . The tps1 gene was constructed on the plant expression vector pBin438.

J Agric Food Chem, 2003 Jan 15, 51(2), 483 - 91
New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing; Codon AC et al.; Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C . The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains . Viability of the heteroplasmons was improved shortly after freezing . However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation . This indicated that mitochondria were not stable and were incompatible with the nucleus . The strains tested regained their original resistance to freezing after recovering their own mitochondria . The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively . No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains . Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains . Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals . The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism . Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C . This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs . For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.

Appl Environ Microbiol, 2003 Jan, 69(1), 715 - 8
Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae; Shima J et al.; The effect of intracellular charged amino acids on freeze tolerance in dough was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast . An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.

Appl Environ Microbiol, 2003 Jan, 69(1), 475 - 82
Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis; Meroth CB et al.; Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB) . The sourdoughs were continuously propagated until the composition of the LAB flora remained stable . Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora . Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant . In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L . mindensis, were detected . In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L . crispatus and L . pontis became the predominant species in sourdough B and L . crispatus, L . panis, and L . frumenti became the predominant species in sourdough C . On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L . johnsonii and L . reuteri were found . The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L . fermentum was also detected . Isolates of the species L . sanfranciscensis and L . fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.

J Synchrotron Radiat, 2003 Jan 1, 10(Pt 1), 4 - 8 Epub 2002 Dec 24.
A structural genomics initiative on yeast proteins; Quevillon-Cheruel S et al.; A canonical structural genomics programme is being conducted at the Paris-Sud campus area on baker's yeast proteins . Experimental strategies, first results and identified bottlenecks are presented . The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.

Carbohydr Res, 2003 Jan 2, 338(1), 5 - 9
Synthesis of a mannose heptasaccharide existing in baker's yeast, Saccharomyces cerevisiae X2180-1A wild-type strain; Zeng Y et al.; A mannose heptasaccharide existing in baker's yeast, Saccharomyces cerevisiae X2180-1A wild-type strain, was effectively synthesized as its allyl glycoside via TMSOTf-promoted condensation of a disaccharide donor 13 with a pentasaccharide acceptor 12, followed by deprotection . The pentasaccharide 12 was constructed by coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9) with allyl 6-O-acetyl-3,4-di-O-benzoyl-alpha-D-mannopyranoside (10), followed by deacetylation . The tetrasaccharide 9 was obtained by coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5) with allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (6), followed by deallylation and trichloroacetimidation . The disaccharides 6 and 13 were readily obtained by known methods.

Int Rev Cytol, 2003, 222, 141 - 96
Chromosomes of the budding yeast Saccharomyces cerevisiae; Loidl J; The mitotic chromosomes of the baker's yeast, Saccharomyces cerevisiae, cannot be visualized by standard cytological methods . Only the study of meiotic bivalents and the synaptonemal complex and the visualization of chromosome-sized DNA molecules on pulsed-field gels have provided some insight into chromosome structure and behavior . More recently, advanced techniques such as in situ hybridization, the illumination of chromosomal loci by GFP-tagged DNA-binding proteins, and immunostaining of chromosomal proteins have promoted our knowledge about yeast chromosomes . These novel cytological approaches in combination with the yeast's advanced biochemistry and genetics have produced a great wealth of information on the interplay between molecular and cytological processes and have strengthened the role of yeast as a leading cell biological model organism . Recent cytological studies have revealed much about the chromosomal organization in interphase nuclei and have contributed significantly to our current understanding of chromosome condensation, sister chromatid cohesion, and centromere orientation in mitosis . Moreover, important details about the biochemistry and ultrastructure of meiotic pairing and recombination have been revealed by combined cytological and molecular approaches . This article covers several aspects of yeast chromosome structure, including their organization within interphase nuclei and their behavior during mitosis and meiosis.

Bioorg Chem, 2002 Oct, 30(5), 350 - 5
Effect of different strains of yeast on stereocontrolled reduction of 5-acetylisoxazolines; Tripathi MK et al.; The stereocontrolled reduction of 3-aryl-5-acetylisoxazolines (1) to the corresponding alcohols (2 and 3) in the presence of four different yeast strains, recognized as Baker's yeast (commercial), Candida krusei (ATCC 14243), Pichia farinosa (NRRL Y110) and Sacchromyces sp . (soil isolate) have been attempted . The C . krusei was found to be diastereoselective for the (R)-1 while the Sacchromyces sp . led to complete reduction to yield the RS- and SS-alcohol in 1:1 ratio at 10 g/L scale.

Biochem J, 2003 Mar 15, 370(Pt 3), 785 - 92
Thermodynamic characterization of yeast triosephosphate isomerase refolding: insights into the interplay between function and stability as reasons for the oligomeric nature of the enzyme; Najera H et al.; The reasons underlying the oligomeric nature of some proteins such as triosephosphate isomerase (TIM) are unclear . It has been proposed that this enzyme is an oligomer, mainly because of its stability rather than for functional reasons . To address this issue, the reversible denaturation and renaturation of the homodimeric TIM from baker's yeast ( Saccharomyces cerevisiae ) induced by guanidinium chloride and urea have been characterized by spectroscopic, functional and hydrodynamic techniques . The unfolding and refolding of this enzyme are not coincident after 'conventional' equilibrium times . Unfolding experiments did not reach equilibrium, owing to a very slow dissociation and/or unfolding process . By contrast, equilibrium was reached in the refolding direction . The simplest equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer . The Gibbs energy changes for monomer folding (delta G (0)(fold) = -16.6+/-0.7 kJ x mol(-1)) and monomer association (delta G (0)(assoc) = -70.3+/-1.1 kJ x mol(-1)) were calculated from data obtained in the two denaturants . From an analysis of the present data and data from the literature on the stability of TIM from different species and for other beta/alpha barrels, and model simulations on the effect of stability in the catalytic activity of the enzyme, it is concluded that the low stability of the monomers is neither the only, nor the main, cause for the dimeric nature of TIM . There is interplay between function and stability.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1414 - 22
Design of the pH profile for asymmetric bioreduction of ethyl 4-chloro-3-oxobutyrate on the basis of a data-driven method; Chen J et al.; The goal of this paper was to design the optimal time-varying operating pH profile in the asymmetric reduction of ethyl 4-chloro-3-oxobutyrate by baker's yeast . Ethyl (S)-4-chloro-3-hydroxybutyrate was produced to reach two important quality indices: reaction yield and product optical purity . The method integrated an orthogonal function approximation and an orthogonal array . The technique used a set of orthonormal functions as the basis for representing the possible profile . The optimal profile could be obtained if the orthogonal coefficients were properly adjusted . The orthogonal array was used to design and analyze the effect of each orthogonal coefficient in order to reach the optimal objective (quality) function . The performance based on the proposed strategy was significantly improved by over 10% compared with the traditional fixed pH or uncontrolled pH values during the reaction . The proposed method can be applied to the required dynamic profile in the bioreactor process to effectively improve the product quality, given good design directions and the advantage of the traditional statistical approach.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1287 - 91
Application of fungi as biocatalysts for the reduction of diethyl 1-oxoalkylphosphonates in anhydrous hexane; Brzezinska-Rodak M et al.; Five different species of microorganisms, namely, Rhodotorula rubra, Rhodotorula glutinis, Cladosporium sp., Verticillium sp., and baker's yeast, turned out to be useful biocatalysts for enantioselective reduction of a variety of diethyl 1-oxoalkylphosphonates . To suppress substrate decomposition, bioreductions were carried out under anhydrous conditions, using lyophilized cells immobilized on Celite R 630 . The influence of reaction conditions such as biotransformation time and chemical additives on the yield of the reaction is discussed.

Exp Anim, 2002 Oct, 51(5), 517 - 9
Changes in activities of enzymes in erythrocytes from ddY mice supplemented with dietary selenium; Arai T et al.; Plasma and erythrocyte enzyme activities were measured in ddY mice supplemented with dietary selenium (Se) from baker's yeast (Saccharomyces serevisiae) or selenious acid at 0.3 ppm Se content . Glutathione peroxidase (GSHpx) activities increased significantly in erythrocytes from mice supplemented with dietary Se . It was concluded that addition of dietary Se as food additive is very effective for activation of GSHpx in mice.

Appl Environ Microbiol, 2002 Dec, 68(12), 6059 - 69
The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation; De Vuyst L et al.; Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast . Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria . This consortium seems to be unique for the Greek traditional wheat sourdoughs studied . Strains of the species W . cibaria have not been isolated from sourdoughs previously . No Lactobacillus pontis or Lactobacillus panis strains were found . An L . brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species . In addition, fermentation capabilities associated with the LAB detected have been studied . During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol . Mannitol was produced from fructose that served as an additional electron acceptor . In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L . sanfranciscensis . Two of the L . paralimentarius isolates tested did not ferment maltose; all strains were homofermentative . In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly . No maltose phosphorylase activity was found either . These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.

Appl Environ Microbiol, 2002 Dec, 68(12), 5981 - 9
Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains; Tanghe A et al.; Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae . Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2 . Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance . These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast . This appears to be the first clear physiological function identified for microbial aquaporins . We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage . Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles . Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene . Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.

J Am Soc Mass Spectrom, 2002 Nov, 13(11), 1282 - 91
Application of ESI-FAIMS-MS to the analysis of tryptic peptides; Barnett DA et al.; High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates gas-phase analyte ions from chemical background, offering substantial improvements in the detection of peptides from complex protein digests . For a digest of enolase 1 (baker's yeast), the focusing and separation offered by FAIMS produced an average intensity gain of 3.5 for the tryptic ions and reductions in background intensity of 5- to 10-fold when compared with ESI-MS . The increased signal-to-background in the ESI-FAIMS-MS experiment resulted in a greater number of identifiable peptides and therefore greater sequence coverage . Compensation voltage (CV) maps for a total of 282 tryptic peptides from thirteen proteins, generated according to charge-state, mass-to-charge ratios, and chain length, show that a majority of tryptic peptides can be detected by operating FAIMS at a few discrete values of CV rather than scanning CV across a wide range . The ability to reduce scanning requirements has potential benefits for coupling FAIMS with LC-MS . In select cases, FAIMS can be used to eliminate isobaric MS overlap between tryptic peptides; however, the primary advantage of FAIMS in an LC-FAIMS-MS analysis is foreseen to be the attenuation of chemical background noise rather than the separation of individual peptides . Using FAIMS to reduce mass spectral noise will offer improved detection of peptides from low abundance proteins in complex biological samples.

FEBS Lett, 2002 Nov 20, 531(3), 453 - 8
Heterologous expression of dihydroflavonol 4-reductases from various plants; Martens S et al.; Dihydroflavonol 4-reductases (DFR) catalyze the stereospecific reduction of dihydroflavonols to the respective flavan 3,4-diols (leucoanthocyanidins) and might also be involved in the reduction of flavanones to flavan-4-ols, which are important intermediates in the 3-deoxyflavonoid pathway . Several cDNA clones encoding DFR have been isolated from different plant species . Despite the important function of these enzymes in the flavonoid pathway, attempts at heterologous expression of cDNA clones in Escherichia coli have failed so far . Here, three well known heterologous expression systems for plant-derived genes were tested to obtain the functional protein of DFR from Gerbera hybrids . Successful synthesis of an active DFR enzyme was achieved in eukaryotic cells, using either baker's yeast (Saccharomyces cerevisiae) or tobacco protoplasts (Nicotiana tabacum), transformed with expression vectors containing the open reading frame of Gerbera DFR . These expression systems provide useful and powerful tools for rapid biochemical characterization, in particular the substrate specificity, of the increasing number of cloned DFR sequences . Furthermore, this tool allows the stereospecific synthesis of (14)C-labeled leucoanthocyanidins in high quality and quantity, which is a prerequisite for detailed biochemical investigation of the less understood enzymatic reactions located downstream of DFR in anthocyanin, catechin and proanthocyanidin biosynthesis.

Biol Pharm Bull, 2002 Nov, 25(11), 1506 - 8
Production of interleukin-1 activity of Kupffer cells from mice treated with the acidic mannan fraction of baker's yeast; Okawa Y et al.; We investigated the production of interleukin-1 (IL-1) activity by Kupffer cells (KC) from mice treated with a neutral mannan fraction (WNM) or an acidic mannan fraction (WAM025) from baker's yeast (Saccharomyces cerevisiae) in vivo and in vitro . The mice administered WAM025 showed an increase in the number of KC and the IL-1 production compared with mice administered WNM . In an in vitro stimulation assay using KC from a normal mouse, it was also found that WAM025 displayed an increase in IL-1 production . Diisopropyl fluorophosphate completely inhibited the production of IL-1 by KC from the mice administered WAM025.

J Org Chem, 2002 Nov 1, 67(22), 7872 - 5
Chemoenzymatic synthesis of phosphocarnitine enantiomers; Mikolajczyk M et al.; Racemic phosphocarnitine 3 has been synthesized starting from diethyl 3-chloro-2-oxopropanephosphonate 4 in three steps involving reduction of 4 to the corresponding 2-hydroxyphosphonate 5, conversion of the latter to phosphonic acid 6, and final reaction with trimethylamine, affording the trimethylammonium salt of 3 . Baker's yeast reduction of 4 and enzymatic kinetic resolution of (+/-)-5 afforded the enantiomerically pure precursors of phosphocarnitine, (R)-(+)-5 and (S)-(-)-5, which were converted to (S)-(-)- and (R)-(+)-phosphocarnitine 3, respectively.

J Biomol NMR, 2002 Aug, 23(4), 323 - 4
1H, 15N and 13C resonance assignments of yeast Saccharomyces cerevisiae calmodulin in the Ca2+-free state; Ishida H et al.; Calmodulin (CaM) is a small Ca2+-binding protein, which has been found in all of eucaryotic cells examined . CaMs isolated from various species have highly conserved amino acid sequence (more than 90% identical), and show the same biological functions . CaM isolated from the baker's yeast (Saccharomyces cerevisiae) (yCaM), however, shares only 60% identity in the amino acid sequence with CaM from vertebrate, and shows quite distinct conformational and biochemical properties compared with those of CaM from other species . The conformational details of yCaM, however, have not been revealed yet . We achieved the chemical shift assignments of yCaM (146 amino acids) in the apo-state using uniformly 15N- and 13C-labeled protein . Consequently, the resonances of 95% atoms in the backbone amides were successfully assigned.

Lipids, 2002 Aug, 37(8), 733 - 40
Identification and expression of mammalian long-chain PUFA elongation enzymes; Leonard AE et al.; In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a delta6-desaturation step followed by retroconversion (Sprecher, H., Biochim . Biophys . Acta 1486, 219-231, 2000) . We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA . The database search led to the isolation of two cDNA clones from human and mouse . These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5 . The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide . Expression of these open reading frames in baker's yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20- and 22-carbon long-chain PUFA, as determined by the conversion of 20:4n-6 to 22:4n-6, 22:4n-6 to 24:4n-6, 20:5n-3 to 22:5n-3, and 22:5n-3 to 24:5n-3 . The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20- and C22-carbon) n-6 and n-3 PUFA substrates by the significant increase in the levels of 24:4n-6 and 24:5n-3, respectively . This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 1116 - 25
Feedback stabilization of fed-batch bioreactors: non-monotonic growth kinetics; Smets IY et al.; This paper deals with the design of a feedback controller for fed-batch microbial conversion processes that forces the substrate concentration C(S) to a desired setpoint, starting from an arbitrary (initial) substrate concentration when non-monotonic growth kinetics apply . This problem is representative for a lot of industrial fermentation processes, with the baker's yeast fermentation as a well-known example . It is assumed that the specific growth rate mu is function of the substrate concentration only . A first approach exploits the availability of on-line measurements of both the substrate and biomass concentration . A second approach is merely based on on-line measurements of the biomass concentration, which provide an estimate for the specific growth rate . After a reformulation of the substrate concentration setpoint into a specific growth rate setpoint, it is demonstrated that the fed-batch process can still be stabilized around any desired operating point along the non-monotonic kinetics.

Dig Dis Sci, 2002 Sep, 47(9), 2079 - 81
Seroreactivities against Saccharomyces cerevisiae and Mycobacterium avium subsp . paratuberculosis p35 and p36 antigens in Crohn's disease patients; Shafran I et al.; Crohn's disease (CD) is an idiopathic chronic inflammatory bowel disease (IBD) . Accurate diagnosis of the disease is of great clinical importance to assess its prognosis and success of therapy . Recent studies have validated and confirmed the potential utility of anti-Saccharomyces cerevisiae (baker's yeast; ASCA) IgG/IgA antibodies and anti-M . avium ss . paratuberculosis p35/p36 antibodies, separately, as serological markers to identify patients with CD . The efficacy of these markers was evaluated in the same patients with Crohn's disease . The anti-ASCA IgA/IgG and the anti-M . avium ss . paratuberculosis p35/p36 antibodies were positive in 60% (36/60) and 86.7% (52/60) of CD patients, respectively . When all the serologic markers were considered, the sensitivity in detecting CD was increased to 95.0% (57/60); 21 of 24 ASCA-negative patients were p35/p36-positive and five of eight of p35/p36-negative patients were ASCA-positive . This investigation further establishes the utility of p35 and p36 recombinant clones for the diagnosis of CD, and reveals the complimentary role of ASCA and p35 and p36 for effective detection of CD . Larger studies are needed to investigate the combined use of these serologic markers for the diagnosis of CD.

Appl Environ Microbiol, 2002 Oct, 68(10), 4780 - 7
Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs; Teunissen A et al.; The routine production and storage of frozen doughs are still problematic . Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation . As a result, the yeast loses gassing power significantly during storage of frozen doughs . We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles . Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor . Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions . AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs . Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar . AT25 had only half of the DNA content of S47, and its cell size was much smaller . Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47 . When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production . Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.

J Ind Microbiol Biotechnol, 2002 Sep, 29(3), 124 - 8
Ethanol production from corn cob hydrolysates by Escherichia coli KO11; de Carvalho Lima KG et al.; Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11 . When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar . When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h) . Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h . Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula . A simulation of an industrial process integrating pentose fermentation by E . coli and hexose fermentation by yeast was carried out . At the first step, E . coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h . Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth . After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l . This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration.

Mol Biol Rep, 2002, 29(1-2), 255 - 7
Loss of fermentative capacity in baker's yeast can partly be explained by reduced glucose uptake capacity; Rossell S et al.; Initial attempts to increase fermentative capacity of baker's yeast focussed on the overproduction of single enzymes, which proved to be insufficient . Nowadays many components of the system are monitored simultaneously in a search for a correlation with fermentative capacity . However, this strategy has not yet proven fruitful either . Here we investigate an element previously neglected, the glucose transporter, and find that a loss of glucose transport capacity correlates with a decrease of fermentative capacity during nutrient starvation . However the correlation is not unique, suggesting that the loss of fermentative capacity cannot be attributed to an inactivation of glucose transport alone . Our results suggest the necessity to use a detailed kinetic model as an underlying working hypothesis and to use Metabolic Control Analysis to examine the pathway's control properties.

Yeast, 2002 Sep 30, 19(13), 1171 - 82
Cloning and analysis of CoEXG1, a secreted 1,3-beta-glucanase of the yeast biocontrol agent Candida oleophila; Segal E et al.; Lytic enzymes may have a role in the biological control of fungi . The yeast biocontrol agent, Candida oleophila, is an excellent subject to research this matter . In the present study, CoEXG1, which encodes for a secreted 1,3-beta-glucanase, is the first gene to be cloned from C . oleophila . It was isolated from a partial genomic library and analysed . Its open reading frame and putative promoter were expressed in baker's yeast, Saccharomyces cerevisiae . The reading frame, expressed under the inducible GAL1 promoter, caused an increased secretion of beta-glucanase, and the putative promoter region activated the lacZ reporter gene, to which it was fused . Sequencing analysis revealed that CoEXG1 carries the signature pattern of the 5 glycohydrolases family and has a putative secretion leader, as well as a high degree of identity to yeast 1,3-beta-glucanases . The GenBank Accession No . of CoEXG1 is AF393806 .

Biochem Pharmacol, 2002 Oct 15, 64(8), 1279 - 92
Novel competitive irreversible inhibitors of aldehyde dehydrogenase (ALDH1): restoration of chemosensitivity of L1210 cells overexpressing ALDH1 and induction of apoptosis in BAF(3) cells overexpressing bcl(2); Quash G et al.; 4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new amino-substituted derivatives of alpha, beta acetylenic thiolester compounds as inhibitors of aldehyde dehydrogenase 1, (ALDH1) . Of these compounds, the dimethyl derivative (DIMATE) was a competitive irreversible inhibitor (K(i) approximately 280 microM) of baker's yeast ALDH1 in vitro showing 80% inhibition at 400 microM when preincubated with the enzyme for 30min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 microM even after 60min preincubation . ATE inhibited ALDH1 activity in ALDH1-transfected L1210 T cells resistant to hydroperoxycyclophosphamide (HCPA) and inhibited growth synergistically in the presence of HCPA . In non-transfected L1210 counterparts ATE did not potentiate growth inhibition by HCPA . DIMATE was a 30-100-fold more effective growth inhibitor than ATE . Endogenous ALDH1 activities of BAF(3) cells over-expressing different levels of bcl(2) (0-100%) were similar (16-20mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 microM . Up to 4 microM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 microM DIMATE, DNA-fragmentation increased concomitantly with ALDH1 inhibition . No DNA-fragmentation was observed with ALDH1 irreversible inhibitors devoid of a thiolester group or with thiolesters which were not inhibitors of ALDH1 . It was seen only with competitive irreversible inhibitors having the methanethiol and enzyme-inhibitory moieties . The methanethiol putatively released from DIMATE by ALDH1 esterase activity plays a role, albeit undefined, in lowering intramitochondrial glutathione levels which decreased by 47% as DNA-fragmentation increased.

Electrophoresis, 2002 Jul, 23(13), 2048 - 56
Monitoring the migration behavior of living microorganisms in capillary electrophoresis using laser-induced fluorescence detection with a charge-coupled device imaging system; Girod M et al.; Remarkably high apparent peak efficiencies (10(6)-10(9) theoretical plates per meter) in capillary electrophoresis (CE) could be achieved in the separation of two different kinds of bacteria and Baker's yeast using poly(ethylene oxide) as a necessary buffer additive . In these applications no deliberate stacking procedure was implemented . Seemingly, the investigated organisms in this study behave differently than molecules under an applied electric field . For molecules, these extremely high efficiencies are very unusual . Using a 488 nm argon-ion laser coupled to a charge-coupled device (CCD) camera it was possible to monitor the migration behavior of stained microorganisms over a length of 10 cm . This part simulates the very beginning of the CE run . In specific cases 60-70% of the monitored detection window could be filled with analyte without significant loss in peak efficiency . For a mixture of two different microorganisms the occurring separation process could be followed in detail . The effect of buffer concentration, polymer type, polymer molecular weight, polymer concentration, pH, and the effect of injection time was investigated . The expansion of fast and reproducible CE separations to other unicellular organisms may become a powerful tool in microbiological science and technology.

Water Sci Technol, 2002, 45(12), 189 - 95
Ammonia recovery from high strength agro industry effluents; Altinbas M et al.; The aim of the study was to investigate ammonia recovery from high strength agro industry effluents involving significant amounts of ammonia, by applying magnesium ammonium phosphate (MAP) precipitation technology . Two types of industrial effluents have been tested in the study . The first plant was an opium alkaloid processing industry and the second one was a baker's yeast industry . High chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN) and unacceptable dark brown color characterized effluents from both industries . Effluents from the biologically treated opium alkaloid and baker's yeast industries were both applied at the stoichiometric ratio (Mg:NH4:PO4 = 1:1:1) and above the stoichiometric ratio (Mg:NH4:PO4 = 1.1:1:1.1) to MAP precipitation . NH4 removals of 61-80% were achieved at the pH of 9.2 at the stoichiometric ratio, whereas 83% NH4 removal was obtained at the pH of 9.2 above the stoichiometric ratio . Experimental studies performed on both anaerobically and/or aerobically treated baker's yeast and opium alkaloid industry effluents have clearly indicated that MAP precipitation was an appropriate treatment option for NH4 removal or struvite recovery from high ammonia content agro industry effluents . Additional ammonia recovery studies were conducted on ozonated and Fenton's oxidation applied effluents and these have also indicated that the amounts of struvite and the quality of MAP precipitate was increased significantly . In this framework, MAP sludge recovered from combined biological and Fenton's oxidation treatment effluents were considered as a more valuable slow release fertilizer for agricultural use.

Biochemistry (Mosc), 2002 Jun, 67(6), 667 - 71
Isolation and properties of noncovalent complex of transketolase with RNA; Solovjeva ON; A method for isolation of homogenous transketolase from baker's yeast using immunoaffinity chromatography was significantly simplified . It was demonstrated that transketolase could be isolated from fresh yeast in the form of a complex with a high molecular weight RNA . Storage of yeast led to the dissociation of the complex to a low molecular weight complex and then to the free enzyme . Conditions were chosen for complex dissociation and free enzyme isolation . In comparison to the free enzyme, the specific activities of the high and low molecular weight complexes were decreased 20-25- and 3-5.5-fold, respectively . The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate (donor substrate) did not change for the low molecular weight complex, while the time of binding to calcium increased . The latter was necessary for the complete manifestation of the enzymatic activity . Changes in the circular dichroism spectrum between 300 and 360 nm after the addition of thiamine diphosphate, which characterize the formation of the catalytically active holoenzyme, were significantly lower for the low molecular weight complex than for the free enzyme.

Anal Biochem, 2002 Jul 15, 306(2), 197 - 203
LC/MS analysis of NAD biosynthesis using stable isotope pyridine precursors; Evans J et al.; A liquid chromatographic-electrospray ionization ion trap mass spectrometry (LC/MS) method has been developed to measure the biosynthetic incorporation of specific precursors into NAD . The stable isotope-labeled precursors tryptophan, quinolinic acid, nicotinic acid, and nicotinamide were added to the media of human liver tumor cells (SK-HEP) grown in culture . The cells were harvested, the NAD was extracted, and the ratio of labeled to unlabeled NAD was measured using the newly developed LC/MS assay . The quantity of NAD formed from each precursor relative to an internal standard (fully labeled 13C, 15N-labeled NAD prepared from baker's yeast) was measured . The detection limit (signal-to-noise ratio 5:1) of the LC/MS method was 37 fmol (25 pg) of NAD and was linear from 20.0 ng to 25 pg . All reported NAD levels were normalized relative to cellular protein measurements . At 50 microM precursor concentrations, nicotinamide was the dominant precursor and NAD levels in the cell rose well above normal levels . Other precursors were minimally incorporated . The same methods were applied to NAD biosynthesized by macrophages derived from peripheral blood monocytes . However, the NAD concentration in macrophages was about 5% of that in SK-HEP cells and the incorporation of stable isotope-labeled substrates remained below measurable levels.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 310 - 7 Epub 2002 May 01.
Fed-batch cultivation of baker's yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen; Jorgensen H et al.; An industrial strain of Saccharomyces cerevisiae (DGI 342) was cultivated in fed-batch cultivations at a specific growth rate of 0.2 h(-1) . The yeast was then exposed to carbon or nitrogen starvation for up to 8 h, to study the effect of starvation on fermentative capacity and content of protein, trehalose and glycogen . Nitrogen starvation triggered the accumulation of trehalose and glycogen . After 8 h of starvation, the content of trehalose and glycogen was increased 4-fold and 2-fold, respectively . Carbon starvation resulted in a partial conversion of glycogen into trehalose . The trehalose content increased from 45 to 64 mg (g dry-weight)(-1), whereas the glycogen content in the same period was reduced from 55 to 5 mg (g dry-weight)(-1) . Glycogen was consumed faster than trehalose during storage of the starved yeast for 1 month . Nitrogen starvation resulted in a decrease in the protein content of the yeast cells, and the fermentative capacity per gram dry-weight decreased by 40% . The protein content in the carbon-starved yeast increased as a result of starvation due to the fact that the content of glycogen was reduced . The fermentative capacity per gram dry-weight was, however, unaltered.

Appl Biochem Biotechnol, 2002 Jun, 101(3), 239 - 49
pH-sensitive chitosan films for baker's yeast immobilization; Oztop HN et al.; Dried baker's yeast cells were immobilized on a chitosan film, which is a natural polymer . Prepared chitosan films were treated with glutaraldehyde to facilitate the immobilization of the cells . The effects of the amount of glutaraldehyde, incubation time, pH, and temperature on immobilization were investigated . The amount of glutaraldehyde was chosen to be 0.01% (weight) . The highest amount of yeast immobilization was obtained with 5 h incubation . It was determined that optimum temperature for immobilization is 25 degrees C, and the optimum pH for immobilization is 6 . Immobilized cells were allowed to stand for 3 d in distilled water and buffer solution (pH 6) to investigate the desorption, but no desorption was found . The maximum immobilization capacities were found to be 90 microg protein cm(-2) film in optimum conditions.

Appl Biochem Biotechnol, 2002 Jun, 101(3), 211 - 27
Scale-up of microbubble dispersion generator for aerobic fermentation; Hensirisak P et al.; A laboratory-scale microbubble dispersion (MBD) generator was shown to improve oxygen transfer to aerobic microorganisms when coupled to the conventional air-sparger . However, the process was not demonstrated on a large scale to prove its practical application . We investigated the scale-up of a spinning-disk MBD generator for the aerobic fermentation of Saccharomyces cerevisiae (baker's yeast) . A 1-L spinning-disk MBD generator was used to supply air for 1- and 50-L working volume fermentation of baker's yeast . For the two levels investigated, the MBD generator maintained an adequate supply of surfactant-stabilized air microbubbles to the microorganisms at a relatively low agitation rate (150 rpm) . There was a significant improvement in oxygen transfer to the microorganism relative to the conventional sparger . The volumetric mass transfer coefficient, kLa, for the MBD system at 150 rpm was 765 h(-1) compared to 937 h(-1) for the conventional sparger at 500 rpm . It is plausible to surmise that fermentation using larger working volumes may further improve the kLa values and the dissolved oxygen (DO) levels because of longer hold-up times and, consequently, improve cell growth . There was no statistically significant difference between the cell mass yield on substrate (0.43 g/g) under the MBD regime at an agitation rate of 150 rpm and that achieved for the conventional air-sparged system (0.53 g/g) at an agitation rate of 500 rpm . The total power consumption per unit volume of broth in the 50-L conventional air-sparged system was threefold that for the MBD unit for a similar product yield . Practical application of the MBD technology can be expected to reduce power consumption and therefore operating costs for aerobic fermentation.

Food Chem Toxicol, 2002 Jul, 40(7), 965 - 78
Sequence analysis and resistance to pepsin hydrolysis as part of an assessment of the potential allergenicity of ice structuring protein type III HPLC 12; Baderschneider B et al.; The recently published WHO/FAO guidelines on the assessment of allergenicity of novel food proteins provide a strategy with which to approach the determination of the potential of novel proteins in foods to be allergens . Key to this strategy are the assessment of sequence similarity to known allergens and the assessment of the resistance to pepsin hydrolysis . Ice structuring proteins (also commonly referred to as anti-freeze or thermal hysteresis proteins) are a group of naturally occurring proteins that bind to ice and structure ice crystal formation . The amino acid sequence of the ice structuring protein (ISP) type III HPLC 12 (ISP type III) was compared in silico with the sequences of known allergens . Secondly, the resistance to pepsin hydrolysis of ISP type III and its glycoconjugates (produced in recombinant baker's yeast) was assessed . The results indicate that ISP type III has no sequence similarity with known allergenic proteins . Both ISP type III and ISP type III glycoconjugates contained within the fermentation product were hydrolysed readily by pepsin (50% loss in <10 min at pH 1.5) to give peptide fragments that were too small to be allergenic or to trigger cross-linking to IgE . In an accompanying study, we demonstrated that IgE from fish-allergic individuals did not bind ISP Type III . Therefore, in accordance with the WHO/FAO strategy, the assessment of ISP type III and ISP type III glycoconjugates by sequence analysis together with lack of resistance to pepsin hydrolysis and the absence of IgE binding supports the conclusion that both are unlikely to present a potential sensitisation hazard.

Appl Environ Microbiol, 2002 Jun, 68(6), 3024 - 30
Gene expression analysis of cold and freeze stress in Baker's yeast; Rodriguez-Vargas S et al.; We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions . We found both up- and down-regulation of genes, although repression was more common . We identified and sequenced several cold-induced genes exhibiting the largest differences . We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport . These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism . We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress . The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain . In contrast, both genes were repressed in frozen cells of the sensitive strain . Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance . Growth of wild-type cells at 10 degrees C was not affected by high ERG10 expression . However, YEpERG10 transformant cells exhibited increased freezing tolerance . Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity . These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 265 - 72
Effect of pH on the stability of hexokinase and glucose 6-phosphate dehydrogenase; Souza MA et al.; Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods . The stability of the enzymes can be affected by several variables, pH being one of them . The effect of pH on the stability of HK and G6PDH was evaluated in this work . Baker's yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors . The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5) . The residual activity of HK and G6PDH, expressed as micromol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4 degrees C . It was observed that up to 4 h both enzymes were stable in all buffers used . However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.

Bioresour Technol, 2002 May, 82(3), 285 - 9
Biosorption of monovalent and divalent ions on baker's yeast; Vasudevan P et al.; Biosorption of monovalent ions Na+ and K+, by deactivated protonated yeast (Saccharomyces cerevisiae) at controlled pH, was compared with biosorption of divalent ions Ca2+ and Mg2+ to help to understand the underlying bindingmechanisms . The adsorption for monovalent ions was accompanied by H+ release . Divalent ions were sorbed by proton displacement, and also an additional mode not accompanied by release of H+ . The sorption uptake of both monovalent and divalent metal ions increased with pH in the range 3-7 peaking at 6.75 . Equilibrium sorption isotherms at pH = 6.75 showed that the totalmaximum biosorptive capacity for metal ions decreased in the following order: Ca > Mg > Na > or = K.

FEMS Immunol Med Microbiol, 2002 Mar 25, 33(1), 41 - 5
beta-1,3-Glucan reduces growth of Mycobacterium tuberculosis in macrophage cultures; Hetland G et al.; The effect of beta-1,3-D-glucans SSG, from Sclerotinia sclerotiorum, or soluble (sMG) or particulate (pMG) MacroGard from baker's yeast on growth of Mycobacterium tuberculosis H37Rv in cultures of peritoneal macrophages from BALB/c mice was examined . After 24 h intracellular bacteria from lysed macrophages were cultured and the number of cfu counted . SSG given with challenge, but not 24 h after, reduced the number of M . tuberculosis cfu significantly . pMG, but not sMG, given with challenge had an even stronger inhibitory effect, which was enhanced after serum opsonization of the particles . The effect of serum-treated pMG was abrogated by addition of a monoclonal antibody to CD11b . The results indicate that beta-glucans inhibit growth of M . tuberculosis in host cells in vitro, probably due to cellular stimulation and/or competitive inhibition of uptake of bacteria via CR3 (CD11b/18).

Int Rev Cytol, 2002, 215, 149 - 87
Osmotic adaptation in yeast--control of the yeast osmolyte system; Hohmann S; The yeast Saccharomyces cerevisiae (baker's yeast or budding yeast) is an excellent eukaryotic model system for cellular biology with a well-explored, completely sequenced genome . Yeast cells possess robust systems for osmotic adaptation . Central to the response to high osmolarity is the HOG pathway, one of the best-explored MAP kinase pathways . This pathway controls via different transcription factors the expression of more than 150 genes . In addition, osmotic responses are also controlled by protein kinase A via a general stress response pathway and by presently unknown signaling systems . The HOG pathway partially controls expression of genes encoding enzymes in glycerol production . Glycerol is the main yeast osmolyte, and its production is essential for growth in a high osmolarity medium . Upon hypo-osmotic shock, yeast cells transiently stimulate another MAP kinase pathway, the so-called PKC pathway, which appears to orchestrate the assembly of the cell surface and the cell wall . In addition, yeast cells show signs of a regulated volume decrease by rapidly exporting glycerol through Fps1p . This unusual MIP channel is gated by osmotic changes and thereby plays a key role in controlling the intracellular osmolyte content . Yeast cells also possess two aquaporins, Aqy1p and Aqy2p . The production of both proteins is strictly regulated, suggesting that these water channels play very specific roles in yeast physiology . Aqy1p appears to be developmentally regulated . Given the strong yeast research community and the excellent tools of genetics and functional genomics available, we expect yeast to be the best-explored cellular organism for several years ahead, and osmotic responses are a focus of interest for numerous yeast researchers.

J Biol Chem, 2002 Jun 14, 277(24), 21666 - 74 Epub 2002 Apr 19.
In vitro evolution of recognition specificity mediated by SH3 domains reveals target recognition rules; Panni S et al.; We have designed a repertoire of 10(7) different SH3 domains by grafting the residues that are represented in the binding surfaces of natural SH3 domains onto the scaffold of the human Abl-SH3 domain . This phage-displayed library was screened by affinity selection for SH3 domains that bind to the synthetic peptides, APTYPPPLPP and LSSRPLPTLPSP, which are peptide ligands for the human Abl or Src SH3 domains, respectively . By characterizing the isolates, we have observed that as few as two or three amino acid substitutions lead to dramatic changes in recognition specificity . We propose that the ability to shift recognition specificity with a small number of amino acid replacements is an important evolutionary characteristic of protein binding modules . Furthermore, we have used the information obtained by these in vitro evolution experiments to generate a scoring matrix that evaluates the probability that any SH3 domain binds to the peptide ligands for the Abl and Src SH3 domains . A table of predictions for the 28 SH3 domains of baker's yeast is presented.

Mol Microbiol, 2002 Feb, 43(4), 835 - 42
Protein phosphatase 2A on track for nutrient-induced signalling in yeast; Zabrocki P et al.; Early studies identified two bona fide protein phosphatase 2A (PP2A)-encoding genes in Saccharomyces cerevisiae, designated PPH21 and PPH22 . In addition, three PP2A-related phosphatases, encoded by PPH3, SIT4 and PPG1, have been identified . All share as much as 86% sequence similarity at the amino acid level . This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4 regulation will also be discussed . Whereas a role for PP2A in yeast morphology and cell cycle has been readily recognized, uncovering its function in yeast signal transduction is a more recent breakthrough . Via their interaction with phosphorylated Tap42, PP2A and Sit4 play a pivotal role in target of rapamycin (TOR) signalling . PPH22 overexpression mimics overactive cAMP-PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets . The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling . We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient-induced signalling in baker's yeast.

J Agric Food Chem, 2002 Apr 10, 50(8), 2350 - 5
Generation of roasted notes based on 2-acetyl-2-thiazoline and its precursor, 2-(1-hydroxyethyl)-4,5-dihydrothiazole, by combined bio and thermal approaches; Bel Rhlid R et al.; Roasted notes contribute to the flavor of thermally processed foods such as meat and bread . 2-Acetyl-2-thiazoline is one of the key volatile compounds responsible for the roasted and popcorn-like aroma character . We report here on the biogeneration of flavoring preparations with intense roasted notes, which are characterized by a high content of 2-acetyl-2-thiazoline . These flavoring preparations were obtained by fermentation of cysteamine, ethyl-L-lactate, and D-glucose with baker's yeast . The precursor of 2-acetyl-2-thiazoline, 2-(1-hydroxyethyl)-4,5-dihydrothiazole, was prepared under mild conditions by microbial reduction of the carbonyl group of 2-acetyl-2-thiazoline using baker's yeast as biocatalyst . The addition of 2-(1-hydroxyethyl)-4,5-dihydrothiazole as aroma precursor to pizza dough resulted in an increase of the roasted note.

Biochemistry, 2002 Apr 2, 41(13), 4264 - 72
Crystallographic study of the recombinant flavin-binding domain of Baker's yeast flavocytochrome b(2): comparison with the intact wild-type enzyme; Cunane LM et al.; Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c . The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction . To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K . Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis . The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder . The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding . While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c . This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.

Folia Microbiol (Praha), 2001, 46(5), 391 - 6
Subcellular shifts of trimeric G-proteins following activation of baker's yeast by glucose; Kotyk A et al.; Addition of glucose to a resting cell suspension of the yeast Saccharomyces cerevisiae was accompanied by marked shifts of the G alpha-protein subunits from the plasma membrane to the cell interior . This process was rapid with half-times between < 10 and 20 s . The decrease of the plasma membrane pool of the Gi alpha/Go alpha- and Gq alpha/Gl 1 alpha-protein subunits correlated with an increase in acid-sensitive forms of these proteins which was recovered in the mitochondrial and/or lysosomal membrane fraction . In contrast to cells from higher organisms glucose-stimulated yeast exhibits an extremely rapid type of the redistribution (internalization) . The question remains open as to the functional significance of the internalized forms of the G-proteins as these remain sequestered from the plasma membrane well after glucose has been consumed.

IUBMB Life, 2001 Dec, 52(6), 285 - 9
Novel approaches to tackling malarial drug resistance using yeast; Sibley CH et al.; Yeasts have a justified reputation as one of the world's most versatile organisms . Baker's yeast continues to live up to this recognition by joining the war against malaria . Yeast can now be used to study antifolate drug resistance patterns that depend on the dihydrofolate reductase enzyme (DHFR) from the malaria parasite.

EMBO J, 1983, 2(9), 1571 - 5
Analysis of sequences conferring autonomous replication in baker's yeast; Kearsey S; A method is presented for rapid sequencing and mapping of elements which support autonomous replication in yeast . The strategy relies on a novel phage M13 vector which allows detection of ARS (autonomously replicating sequence) function in cloned fragments . Deletion mapping of an ARS element linked to the HO gene of Saccharomyces cerevisiae has identified a 57-bp region 3' to the gene, which is essential for autonomous replication . This region shows sequence homology to other ARS elements.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 210 - 6
The development of low temperature inactive (Lti) baker's yeast; Gysler C et al.; The construction of a novel baker's yeast variety via traditional genetic techniques is described . The phenotype was designated "Lti" ("Low temperature inactive") . Lti mutations with the desired characteristics within a genetically well-defined haploid laboratory strain of Saccharomyces cerevisiae were isolated, and two different approaches were taken to obtain baker's yeast strains, which exhibit reduced fermenting activity at refrigeration temperatures . In a first approach, a chosen Lti strain carrying mutation lti9 was combined with other laboratory strains carrying defined MAL alleles . In a second approach, the same lti mutation was introduced in the genetic background of polyploid commercial baker's yeast strains that harbor important "industrial" properties . Lti strains arising from both approaches were characterized with specifically developed screening procedures . Strains of the "academic" Lti strain family displayed between 85% and 92% of the biomass yield of a commercial reference strain, whereas strains of the "industrial" Lti strain family showed a variation between 60% and 115% . Lti strains from both families varied strongly among each other in their activity in model doughs: at 8 degrees C they displayed activities between 5% and 30%, and at 30 degrees C between 40% and 113% of a commercial reference baker's yeast strain.

Yakugaku Zasshi, 2002 Jan, 122(1), 71 - 88
{Development of new methods in asymmetric reactions and their applications}; Node M; Several novel methods using chiral reagents and biocatalysts for asymmetric reactions are described . Among those reactions, asymmetric reduction via a novel tandem Michael addition/Meerwein-Ponndorf-Verley reduction of acyclic alpha,beta-unsaturated ketones using a chiral mercapto alcohol, asymmetric synthesis of allene-1,3-dicarboxylate via crystallization induced asymmetric transformation, and improved asymmetric nitroolefination of lactones and lactames at alpha-carbon using new chiral reagents were developed . In the reactions using biocatalysts, asymmetric dealkoxycarbonylation of bicyclic beta-keto diesters having sigma-symmetry with lipase or esterase to give optically active beta-keto esters, the asymmetric reduction of bicyclic 1,3-diketones having sigma-symmetry with Baker's yeast to give optically active keto alcohols, and the asymmetric aldol reaction of glycine with threonine aldolase were also developed . The above mentioned products were effectively utilized as chiral building blocks for the asymmetric synthesis of natural products and drugs.

Water Res, 2002 Feb, 36(3), 609 - 16
Decolorization of wastewater of a Baker's yeast plant by membrane processes; Mutl SH et al.; The aim of this study is to develop a membrane-based treatment scheme to remove colorants from the effluent of a baker's yeast plant . For this purpose microfiltration (MF), ultrafiltration (UF) and nanofiltraton (NF) membranes with differing molecular weight cut-offs (MWCOs) were tested . To evaluate the effectiveness of membrane processes in treating the waste stream, optical density (OD), COD, color measurements together with permeation fluxes were used . Effects of pretreatment methods (coagulation and coarse filtration) and feed composition on OD, color, COD were studied . In addition, gel filtration analysis was employed to characterize feed and permeate streams in terms of MW distribution of organics that are present . Maximum rejections obtained were 94%, 89% and 72% for OD, color and COD, respectively, when 0.8 microm microfiltration membrane and 400 Da NF membrane were used in series . It was also observed that addition of intermediate UF steps did not increase overall rejections and final permeate flux of NF membrane . Based on these observations, an efficient scheme was offered.

Rev Biol Trop, 2001 Mar, 49(1), 77 - 84
Effect of three food types on the population growth of Brachionus calyciflorus and Brachionus patulus (Rotifera: Brachionidae); Sarma SS et al.; We compared the population growth of B . calyciflorus and B . patulus using the green alga Chlorella vulgaris, baker's yeast Saccharomyces cerevisiae or their mixture in equal proportions as food . Food was offered once every 24 h in two concentrations (low: 1 x 10(6) and high: 3 x 10(6) cells ml-1) separately for each species . The experiments were terminated after 15 days . In general, at any food type or concentration, B . patulus reached a higher population density . A diet of Chlorella alone supported a higher population growth of both rotifer species than yeast alone . B . calyciflorus and B . patulus achieved highest population densities (103 +/- 8 ind . ml-1 and 296 +/- 20 ind . ml-1, respectively) on a diet of Chlorella at 3 x 10(6) cells ml-1 . When cultured using the mixture of Chlorella and yeast, the maximal population densities of B . calyciflorus were lower than those grown on Chlorella . Under similar conditions, the maximal abundance values of B . patulus were comparable in both food types . Regardless of food type and density the rate of population increase per day (r) for B . calyciflorus varied from 0.13 +/- 0.03 to 0.63 +/- 0.04 . These values for B . patulus ranged from 0.19 +/- 0.01 to 0.37 +/- 0.01 . The results indicated that even though Chlorella was a superior food for the tested rotifers, yeast can be effectively used at low concentrations to supplement algal requirements in rotifer culture systems.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 111 - 24
Fermentative capacity after cold storage of baker's yeast is dependent on the initial physiological state but not correlated to the levels of glycolytic enzymes; Nilsson A et al.; Growth and starvation of baker's yeast was monitored by on-line microcalorimetry and cells originating from four different physiological states were stored at low temperature (4 degrees C) for up to 26 days . The different physiological states were designated F (respiro-Fermentative phase of growth), R (initial Respiratory phase of growth), -N (non-growing state because of Nitrogen depletion), and -NC (non-growing state because of both Nitrogen and Carbon depletion) . The cells were tested before and after cold storage for their fermentative capacity, and characterised by 2D gel analysis (and subsequent quantitative silver staining and image analysis with software PDQUEST) for their levels of six enzymes of the glycolytic pathway (hexokinase 2 (Hxk2p), fructose bisphosphate aldolase (Fba1p), glyceraldehyde-3-phosphate dehydrogenase (Tdh3p), enolase A (Enolp), enolase B (Eno2p), and triose phosphate isomerase (Tpi1p)) and two enzymes of the fermentative branch (pyruvate decarboxylase (Pdc1p) and alcohol dehydrogenase (Adh1p)) . The enzymes Hxk2p, Tdh3p, Eno2p, Pdc1p and Adh1p were down-regulated by 25-80% during the transition between the F and R states . During the transition to non-growing states (-N and -NC states), the levels of Hxk2p, Tdh3p and Eno2p were further reduced . However, after cold storage, the glycolytic and fermentative enzymes of the different physiological states were expressed to the same extent . In contrast, the fermentative capacity differed between the states; the R-state cells were superior compared to cells from the other states tested and preserved more than 50% of their initial fermentative capacity (6 mmol ethanol per gram dry weight and hour) . Our data therefore clearly demonstrate that persistence of fermentative capacity during total starvation at low temperature after as long as 1 month is strongly dependent on the physiological state from which the cells originate . However, the level of expression of the glycolytic enzymes could not explain the difference in fermentative capacity of the different physiological states after cold storage.

Adv Biochem Eng Biotechnol, 2002, 75, 51 - 80
Three-phase oxygen absorption and its effect on fermentation; Nagy E; The absorption rate of oxygen in the presence of a second, dispersed, organic phase can be significantly increased due to the higher solubility and diffusivity of oxygen in the organic phase . The oxygen supply of micro-organisms, which is very often a limiting factor during fermentation, can be improved, and the critical level of oxygen in the fermentation broth can be avoided by using dispersed organic phase . This paper reviews the models of the enhanced absorption rates and their integration into mass balance equations of fermentation . Several calculations were carried out to illustrate the effect of the dispersed organic phase and kinetic parameters on the absorption rates and on fermentation with double-substrate-limitation kinetics applying batch and continuous operation modes . Using software taking from the literature, the effect of the organic phase on the baker's yeast production is also presented in fed-batch mode.

Lipids, 2001 Oct, 36(10), 1099 - 103
Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase; Chuang LT et al.; Conjugated linoleic acid (CLA; 18:2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; delta9,12-18:2) . Using a growing baker's yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 microM) on elongation of LA (25 microM) to eicosadienoic acid (EDA; delta11,14-20:2) . Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA . The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium . In the meantime, only these two isomers, when added at 50 microM to the media, were elongated to conjugated FDA (c11,t13- and t12,c14-20:2) by the same recombinant elongase at the rate of 28 and 24%, respectively . The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase . Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on delta6-desaturation as well as elongation of LA which results in a decrease in long-chain n-6 fatty acids and consequently the eicosanoid synthesis.

Chemistry, 2001 Nov 5, 7(21), 4562 - 71
Biocatalytic reduction of beta,delta-diketo esters: a highly stereoselective approach to all four stereoisomers of a chlorinated beta,delta-dihydroxy hexanoate; Wolberg M et al.; A stereoselective chemoenzymatic synthesis of all four stereoisomers of tert-butyl 6-chloro-3,5-dihydroxy-hexanoate (6a) is presented . The key step of the sequence is a highly regio- and enantioselective single-site reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (1a) by two enantiocomplementary biocatalysts . Alcohol dehydrogenase from Lactobacillus brevis (recLBADH) afforded a 72% yield of enantiopure tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate {(S)-2a} . The enantiomer (R)-2a was prepared with 90-94% ee by Baker's yeast reduction in a biphasic system (50% yield) . Both biotransformations were performed on a gram scale . The beta-keto group of the enantiomeric delta-hydroxy-beta-keto esters 2a thus obtained was reduced by syn- and anti-selective borohydride reductions . Permutation of the reduction methods yielded all four stereoisomers of the crystalline target compound 6a (> or = 99.3% ee, dr > or = 205:1), which is a versatile 1,3-diol building block . recLBADH accepts a variety of beta,delta-diketo esters as was determined in a photometric assay . tert-Butyl 3,5-dioxo-hexanoate (1b) and tert-butyl 3,5-dioxo-heptanoate (1c) were reduced on a preparative scale as well to afford the corresponding delta-hydroxy-beta-keto esters (R)-2b and (R)-2c with 99.4% ee and 98.1% ee, respectively.

Chirality, 2001, 13(10), 694 - 8
Determination of the absolute configuration of bicyclo{3.3.1}nonane-2,7-dione by circular dichroism spectroscopy and chemical correlation; Butkus E et al.; A study of the enantiomers of bicyclo{3.3.1}nonane-2,7-dione, a chiral molecule containing two carbonyl chromophores, was performed . Enantiomers of this structure were obtained by HPLC resolution and the (+)-(1R,5S)-enantiomer by enantiospecific synthesis from(+)-(1S,5S)-bicyclo{3.3.1}nonane-2,6-dione . The title structure is an interesting molecule to demonstrate the validity of the octant rule . The location of the major chair-chair conformer into octants placing each chromophore into the origin of the octants led to the opposite configuration assignments . In order to prove unequivocally absolute configuration, enantiospecific synthesis of the title compound was carried out . The kinetic resolution of racemic bicyclo{3.3.1}nonane-2,6-dione using baker's yeast afforded (+)-(1S,5S)-2,6-dione . Employing a reaction sequence analogous to one developed earlier by us with racemic substrates led to carbonyl group shift giving enantiomerically pure (+)-(1R,5S)-bicyclo{3.3.1}nonane-2,7-dione . The absolute configuration of the investigated compound was established by combined use of the octant rule and chemical correlation .

Appl Biochem Biotechnol, 2001 Sep, 95(3), 209 - 20
Characterization of yeast strains for wine production: effect of fermentation variables on quality of wine produced; Ndip RN et al.; Sixteen yeast strains isolated from grapefruit (Citrus paradis), orange (Citrus sinensis) and pineapple (Ananas comosus) were characterized using standard microbiological procedures . The species were identified as Saccharomyces uvarum, S . cerevisiae, S . carlbergensis, and S . ellipsoideus . Their abilities for wine production were tested by using sugar and ethanol tolerance tests . The best biochemically active strain, S . ellipsoideus, was used along with commercially available baker's yeast (S . cerevisiae) to produce wine from grapefruit, orange, and pineapple juices . After fermentation for 14 d with S . cerevisiae and 21 d with S . ellipsoideus, wines produced were compared with Baron de Valls (standard) . The highest (10.47% {v/v}) and lowest (7.68% {v/v}) alcohol concentrations with corresponding residual sugar concentrations of 1.88% (w/v) and 7.7% (w/v) were produced from orange after fermentation with S . cerevisiae and S . ellipsoideus, respectively . S . ellipsoideus was found to be the best yeast strain producing wine with the highest acceptable score of 7.41 from orange . The study revealed the possibility of producing wine from our locally available fruits using simple, cheap, and adaptable technology with biochemically characterized yeast strains.

Biol Chem, 2001 Oct, 382(10), 1431 - 8
A eubacterial origin for the human tRNA nucleotidyltransferase?
Reichert AS, Thurlow DL, Morl M.
tRNA CCA-termini are generated and maintained by tRNA nucleotidyltransferases . Together with poly(A) polymerases and other enzymes they belong to the nucleotidyltransferase superfamily . However, sequence alignments within this family do not allow to distinguish between CCA-adding enzymes and poly(A) polymerases . Furthermore, due to the lack of sequence information about animal CCA-adding enzymes, identification of corresponding animal genes was not possible so far . Therefore, we looked for the human homolog using the baker's yeast tRNA nucleotidyltransferase as a query sequence in a BLAST search . This revealed that the human gene transcript CGI-47 (#AF151805) deposited in GenBank is likely to encode such an enzyme . To identify the nature of this protein, the cDNA of the transcript was cloned and the recombinant protein biochemically characterized, indicating that CGI-47 encodes a bona fide CCA-adding enzyme and not a poly(A) polymerase . This confirmed animal CCA-adding enzyme allowed us to identify putative homologs from other animals . Calculation of a neighbor-joining tree, using an alignment of several CCA-adding enzymes, revealed that the animal enzymes resemble more eubacterial ones than eukaryotic plant and fungal tRNA nucleotidyltransferases, suggesting that the animal nuclear cca genes might have been derived from the endosymbiotic progenitor of mitochondria and are therefore of eubacterial origin.

Appl Environ Microbiol, 2001 Dec, 67(12), 5420 - 4
Enzyme-linked immunosorbent assay specific for (1-->6) branched, (1-->3)-beta-D-glucan detection in environmental samples; Milton DK et al.; (1-->3)-beta-D-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments . A specific enzyme immunoassay was developed to quantify (1-->6) branched, (1-->3)-beta-D-glucans in environmental samples . The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1-->3)-beta-D-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall beta-D-glucans as a detector reagent . The assay was highly specific for (1-->6) branched, (1-->3)-beta-D-glucans (such as that from Saccharomyces cerevisiae) and did not show any response at 200 ng/ml to curdlan, laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and endotoxins . The detection level was 0.8 ng/ml for baker's yeast glucan and Betafectin . A coefficient of variation of 7.8% was obtained for (1-->3)-beta-D-glucans in house dust samples . Metal working fluids spiked with (1-->3)-beta-D-glucans inhibited the glucan assay . Because the assay is specific for (1-->6) branched, (1-->3)-beta-D-glucans and is sensitive and reproducible, it will be useful for the investigation of health effects from exposure to this class of biologically active molecules.

J Agric Food Chem, 2001 Nov, 49(11), 5265 - 9
Determination of cellular carbohydrates in peanut fungal pathogens and baker's yeast by capillary electrophoresis and electrochromatography; Zhang M et al.; In this work, the quantitation of cellular carbohydrates, namely chitin and glucan, in peanut fungal pathogens and baker's yeast was carried out by capillary electrophoresis (CE) and capillary electrochromatography (CEC) . The chitin and glucan of the fungi were hydrolyzed by the enzymes chitinase and glucanase, respectively, to their corresponding sugar monomers N-acetylglucosamine (GlcNAc) and glucose (Glc) . These two monosaccharides were then tagged with 6-aminoquinoline (6-AQ) to allow their separation and detection in CE and CEC . The 6-AQ derivatives of GlcNAc and Glc formed the basis for the determination by CE and CEC of chitin and glucan in peanut fungi and baker's yeast . Several parameters affecting the separation of the 6-AQ derivatives of GlcNAc and Glc, including the separation voltage and the composition of the running electrolyte, were investigated . Under the optimized separation conditions, the contents of cellular carbohydrates including N-acetylglucosamine, chitin, glucose, and glucan in some fungi, such as Sclerotinia minor, Sclerotium rolfsii, and baker's yeast, were successfully determined . The method described here allowed the assessment of genetic differences in Sclerotium rolfsii isolates from various locations.

Infect Immun, 2001 Dec, 69(12), 7559 - 64
Role of glucan and surface protein BAD1 in complement activation by Blastomyces dermatitidis yeast; Zhang MX et al.; Our previous studies showed that Blastomyces dermatitidis yeast activates the human complement system, leading to deposition of opsonic complement fragments onto the yeast surface . This report examines the influence of altered surface expression of glucan or BAD1 protein (formerly WI-1) on the yeast's ability to activate and bind C3 . Compared to the wild type, a glucan-deficient mutant yeast delayed initiation of C3 deposition and reduced C3-binding capacity by 50% . Linkage of baker's-yeast beta-glucan to the glucan-deficient yeast restored initial C3 deposition kinetics to the wild-type level and partially restored C3-binding capacity, suggesting that beta-glucan is an initiator of complement activation and a C3 acceptor . The role of BAD1 in B . dermatitidis yeast-complement interaction was also assessed . BAD1 knockout yeast initiated faster C3 deposition and increased C3-binding capacity compared to the wild-type yeast or a BAD1-reconstituted yeast, suggesting either a lack of an intrinsic ability in BAD1 or an inhibitory role of BAD1 in complement activation and binding . However, both complement activation and the capacity for C3 binding by the wild-type yeast were enhanced in normal human serum supplemented with an anti-BAD1 monoclonal antibody (MAb) or in immune sera from blastomycosis patients . Microscopic analysis revealed that more initial C3-binding sites were formed on yeast in the presence of both naturally occurring complement initiators and exogenous anti-BAD1 MAb, suggesting that anti-BAD1 antibody enhanced the ability of B . dermatitidis yeast to interact with the host complement system . Thus, glucan and BAD1 have distinctly different regulatory effects on complement activation by B . dermatitidis.

Mol Biol Cell, 2001 Nov, 12(11), 3644 - 57
Novel stress-responsive genes EMG1 and NOP14 encode conserved, interacting proteins required for 40S ribosome biogenesis; Liu PC et al.; Under stressful conditions organisms adjust the synthesis, processing, and trafficking of molecules to allow survival from and recovery after stress . In baker's yeast Saccharomyces cerevisiae, the cellular production of ribosomes is tightly matched with environmental conditions and nutrient availability through coordinate transcriptional regulation of genes involved in ribosome biogenesis . On the basis of stress-responsive gene expression and functional studies, we have identified a novel, evolutionarily conserved gene, EMG1, that has similar stress-responsive gene expression patterns as ribosomal protein genes and is required for the biogenesis of the 40S ribosomal subunit . The Emg1 protein is distributed throughout the cell; however, its nuclear localization depends on physical interaction with a newly characterized nucleolar protein, Nop14 . Yeast depleted of Nop14 or harboring a temperature-sensitive allele of emg1 have selectively reduced levels of the 20S pre-rRNA and mature18S rRNA and diminished cellular levels of the 40S ribosomal subunit . Neither Emg1 nor Nop14 contain any characterized functional motifs; however, isolation and functional analyses of mammalian orthologues of Emg1 and Nop14 suggest that these proteins are functionally conserved among eukaryotes . We conclude that Emg1 and Nop14 are novel proteins whose interaction is required for the maturation of the 18S rRNA and for 40S ribosome production.

J Org Chem, 1999 Sep 3, 64(18), 6603 - 6608
Baker's Yeast-Mediated Reductions of alpha-Keto Esters and an alpha-Keto-beta-Lactam . Two Routes to the Paclitaxel Side Chain; Kayser MM et al.; Baker's yeast (Saccharomyces cerevisiae) has been used to reduce a series of alkyl esters derived from pyruvate and benzoylformate . Both the yield and enantioselectivities of these reductions were maximized when methyl esters were used, and the (R)-alcohols were isolated in all instances . Yeast-mediated ester hydrolysis was a significant side reaction for products derived from long-chain alcohols . In the case of ethyl benzoylformate, the addition of methyl vinyl ketone increased the enantioselectivity of the reduction . These reductions were applied to two syntheses of the paclitaxel C(13) side chain {(2R,3S)-N-benzoyl-3-phenylisoserine} . In the first, a racemic alpha-keto-beta-azido ester was reduced by whole cells of Baker's yeast to afford a diastereomeric mixture in which the desired product predominated and could be isolated chromatographically . In the second, an easily synthesized alpha-keto-beta-lactam was reduced by yeast cells to afford the desired cis isomer as well as the undesired trans diastereomer . Substituting a yeast strain deficient in fatty acid synthase in this reduction suppressed formation of the trans diastereomer . These results suggest that a single enzyme is responsible for both the D- and L-cis-alcohols resulting from reduction of the alpha-keto-beta-lactam . All of the yeast strains used in this project are available commercially, and these biocatalytic reductions require only common laboratory equipment.

Yeast, 2001 Sep 30, 18(13), 1257 - 67
Expression and activity of the Hxt7 high-affinity hexose transporter of Saccharomyces cerevisiae; Ye L et al.; High-affinity hexose transport is required for efficient utilization of low hexose concentrations by the baker's yeast Saccharomyces cerevisiae . These low concentrations occur during the late exponential phase of batch growth on hexoses, during hexose-limited chemostat or fed-batch culture, or during growth on sugars such as sucrose and raffinose that are hydrolysed to hexoses outside the cell . The expression of the Hxt7 high-affinity glucose transporter of S . cerevisiae was examined during batch growth on glucose medium in a wild-type strain and a strain expressing only HXT7 (i.e . with null mutations in HXT1-HXT6) . In the wild-type strain, HXT7 transcription was repressed at high glucose and was detected when the glucose in the culture approached depletion . In the HXT7-only strain, transcription of HXT7 was constitutive throughout the glucose growth phase and was increased further at low glucose concentrations . After glucose depletion, the levels of HXT7 mRNA declined rapidly in both strains . In contrast, the Hxt7 protein was relatively stable after glucose depletion . By monitoring the subcellular localization of an Hxt7::GFP fusion protein it was observed that Hxt7 was localized in the plasma membrane, even when expressed at high glucose concentrations in the HXT7-only strain . After glucose depletion Hxt7 was gradually endocytosed and targeted to the vacuole for degradation . The Hxt7::GFP fusion protein was a fully functional hexose transporter with a catalytic centre activity of approximately 200/sec . It is concluded that repression of HXT7 and degradation of Hxt7 at high glucose concentrations is dependent on a high glucose transport capacity .

Eur J Biochem, 2001 Sep, 268(18), 4918 - 27
The catalytic role of tyrosine 254 in flavocytochrome b2 (L-lactate dehydrogenase from baker's yeast) . Comparison between the Y254F and Y254L mutant proteins; Gondry M et al.; Flavocytochrome b2 catalyses the oxidation of L-lactate to pyruvate in yeast mitochondrial intermembrane space . Its flavoprotein domain is a member of a family of FMN-dependent 2-hydroxy-acid-oxidizing enzymes . Numerous solution studies suggest that the first step of the reaction consists of proton abstraction from lactate C2, leading to a carbanion that subsequently yields electrons to FMN . The crystal structure suggests that the enzyme base is His373, and that Tyr254 may be hydrogen bonded to the substrate hydroxyl . Studies carried out with the Y254F mutant {Dubois, J., Chapman, S.K., Mathews, F.S., Reid, G.A . & Lederer, F . (1990) Biochemistry 29, 6393-6400} showed that Tyr254 does not act as a base but stabilizes the transition state . As the mutation did not induce any change in substrate affinity, the question of the existence of the hydrogen bond in the Michaelis complex remained open . Similar results with glycolate oxidase, mutated at the same position, led to the suggestion that these enzymes actually operate via a hydride transfer mechanism {Macheroux, P., Kieweg, V., Massey, V., Soderlind, E., Stenberg, K . & Lindqvist, Y . (1993) Eur . J . Biochem . 213, 1047-1054} . In the present work, we have re-investigated the matter by analysing the properties of a Y254L mutant flavocytochrome b2, as well as the behaviour of the Y254F enzyme with two substrates other than lactate, and a series of inhibitors . The Y254L protein is less efficient with L-lactate than the wild-type enzyme by a factor of 500, but the substrate affinity is unchanged . In contrast, L-phenyllactate and mandelate, poor substrates (the latter acting more as an inhibitor), exhibit an increased affinity . In addition, the Y254L mutant enzyme is more efficient with phenyllactate than lactate as a substrate . In order to rationalize these observations, we have modelled phenyllactate and mandelate in the active site, using previously described modelling experiments with lactate as a starting point . The results indicate that mandelate cannot bind in an orientation allowing proton abstraction by His373, due to steric interference by the side chains of Ala198 and Leu230 . It might possibly adopt a binding mode as proposed previously for lactate, which leads to a hydride transfer and with which the 198 and 230 side chains do not interfere . However, other researchers {Sinclair, R., Reid, G.A . & Chapman, S.K . (1998) Biochem . J . 333, 117-120} showed that A198G, L230A and A198G/L230A mutant enzymes exhibit a strongly improved mandelate dehydrogenase activity . These results indicate that relief of the steric crowding facilitates catalysis by enabling a better mandelate orientation at the active site, suggesting that its productive binding mode is similar to that proposed for lactate in the carbanion mechanism . The modelling studies therefore support the hypothesis of a carbanion mechanism for all substrates . In addition, we present the effect of the two mutations at position 254 on the binding of a number of competitive inhibitors (such as sulfite, D-lactate, propionate) and of inhibitors that are known to bind at the active site both when the flavin is oxidized and when it is in the semiquinone state (propionate, oxalate and L-lactate at high concentrations) . Unexpectedly, the results indicate that the integrity of Tyr254 is necessary for the binding of these inhibitors at the semiquinone stage.

Rapid Commun Mass Spectrom, 2001, 15(18), 1685 - 92
Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis; Poutanen M et al.; Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence . This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae . This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) . The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities . Mass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification . LC/MS/MS was found to be more sensitive and to provide more accurate data, and was very useful when analyzing small amounts of sample . Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation .

Lett Appl Microbiol, 2001 Sep, 33(3), 211 - 5
Effect of selected natural antimicrobials on Baker's yeast activity; Pattison TL et al.; AIMS: To evaluate the responses of Baker's yeast (Saccharomyces cerevisiae) activity to the natural antimicrobials acetic acid, calcium lactate, a lactate-containing cocktail and lactic acid compared to calcium propionate . METHODS AND RESULTS: A dough fermentometer test was used to measure Baker's yeast activity in the presence of these natural antimicrobials and calcium propionate . Yeast activity generally decreased as a function of increasing antimicrobial concentrations, but the lactate-containing cocktail showed no relationship between concentration and yeast activity reduction . At in situ concentrations, calcium propionate resulted in the highest yeast activity reduction, followed by calcium lactate, acetic acid, the lactate-containing cocktail and lactic acid in decreasing order . CONCLUSION: Based on yeast activity reduction, all natural antimicrobials tested showed potential as possible replacements for calcium propionate . SIGNIFICANCE AND IMPACT OF THE STUDY: This has practical implications since calcium propionate inhibits Baker's yeast activity and attracts negative consumer perceptions as a chemical bread preservative.

Biotechnol Bioeng, 2001 Oct 5, 75(1), 29 - 38
Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast; Chin-Joe I et al.; Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols . In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae) . A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction . The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product . Excess glucose as the electron donor, i.e . a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization . Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor . Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate . In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement .

Trends Parasitol, 2001 Sep, 17(9), 407 - 9
Trapping parasite secretory proteins in baker's yeast; Nene V et al.; Because the function of signal sequences has been conserved during evolution it has been possible to develop both bioinformatics resources to identify them and techniques to clone genes that encode secretory proteins . The latter entail insertion of heterologous signals upstream of signal peptide deleted reporter genes . We discuss the advantages of using Saccharomyces cerevisiae for signal sequence trap technology . The yeast protein-translocation system appears to be less discriminating than that of higher eukaryotes - for example, a Theileria parva cysteine protease gene containing a recessed, nonclassical signal allows access to the secretory pathway--and yeast technology could have general application in studying elements of parasite protein trafficking.

Microbiol Mol Biol Rev, 2001 Sep, 65(3), 404 - 21, table of contents
Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase; Helmstaedt K et al.; Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins . In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer . Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors . Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell . The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.

Appl Environ Microbiol, 2001 Sep, 67(9), 4346 - 8
Generation of a novel Saccharomyces cerevisiae strain that exhibits strong maltose utilization and hyperosmotic resistance using nonrecombinant techniques; Higgins VJ et al.; A yeast strain capable of leavening both unsugared and sweet bread dough efficiently would reduce the necessity of carrying out the expensive procedure of producing multiple baker's yeast strains . But issues involving the use of genetically modified foods have rendered the use of recombinant techniques for developing yeast strains controversial . Therefore, we used strong selection and screening systems in conjunction with traditional mass mating techniques to develop a strain of Saccharomyces cerevisiae that efficiently leavens both types of dough.

Appl Environ Microbiol, 2001 Sep, 67(9), 4279 - 85
Improved properties of baker's yeast mutants resistant to 2-deoxy-D-glucose; Rincon AM et al.; We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-D-glucose and had improved fermentative capacity on sweet doughs . Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media . Two of the mutants also had high levels of phosphatase active on 2-deoxy-D-glucose-6-phosphate . Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose . However, the three mutants were slower when fermenting plain doughs . Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.

Vopr Pitan, 2001, 70(3), 22 - 4
{Use of organic forms of selenium in nutrition of patients with gastrointestinal diseases}; Shakhovskaia AK et al.; Selenium level was determined in sera of 27 patients including 15 patients with chronic gastrointestinal diseases and 12 patients with intestinal malabsorption after some kinds of stomach or small intestine resection . Deficiency of this trace element was found in 5 patients and suboptimal concentration in 4 patients in the first group and in 3 and 5 patients of second group respectively . Supplementation of patients with 45 mg of selenium organic form based on autolysis extract of selenious baker's yeast resulted in increase of selenium level in 23 (85%) patients including all of them with selenium deficiency or suboptimal level . It was concluded about high bioavailability of selenium in supplement studied . Perturbed intestinal absorption after surgical resection of stomach or small intestine may be considered as an indication for prophylactic taking of organic selenium form preparation.

J Biol Chem, 2001 Sep 28, 276(39), 36419 - 24 Epub 2001 Jul 26.
Diazaborine treatment of Baker's yeast results in stabilization of aberrant mRNAs; Jungwirth H et al.; Upon Northern blotting, Saccharomyces cerevisiae that was treated with diazaborine showed aberrant mRNAs that were extended at the 3'-end and terminated at secondary processing sites . These bands were also detected in untreated Deltaupf1, Deltaxrn1, and rat7-1 mutants . This finding demonstrates that the aberrant mRNAs also occur in untreated strains in small quantities and can reach the cytoplasm, where they are normally degraded by Xrn1p . Diazaborine treatment stabilizes these mRNAs . The detection of the aberrant bands in the untreated rat7-1 strain indicates that Rat7 is involved in quality control of RNA . The aberrant mRNAs were not detected after diazaborine treatment of a DRG1-1 mutant . Drg1p, a member of the family of AAA (ATPases associated with a variety of cellular activities) proteins, which are thought to represent specific chaperones, may be involved in the process of unfolding the mRNA-ribonucleoprotein complex or in the recognition of aberrant mRNA molecules in the cytoplasm.

Planta, 2001 Jun, 213(2), 214 - 22
The lipopolysaccharides of the phytopathogen Xanthomonas campestris pv . campestris induce an oxidative burst reaction in cell cultures of Nicotiana tabacum; Meyer A et al.; The lipopolysaccharides (LPSXcc) of the phytopathogenic bacteria Xanthomonas campestris pv . campestris (X.c.c.) were purified from an exopolysaccharide-deficient mutant strain . The isolated LPSxcc induced an oxidative burst reaction in cell-suspension cultures of the non-host plant tobacco (Nicotiana tabacum L.) SRI . The oxidative burst elicited by LPSXcc differed from that induced by yeast elicitor (YE), a cell wall preparation of baker's yeast . The LPSXcc-induced oxidative burst was characterised by a slow increase in H2O2 production and an extended decline . Both the LPSXcc-and YE-induced oxidative bursts were completely blocked by the NAD(P)H-oxidase inhibitor diphenylene-iodonium . When LPSXcc and YE were applied in combination, a synergistic effect and the establishment of refractory states in the generation of H2O2 were observed . The amount of cytosolic calcium was measured in transgenic tobacco cell cultures carrying the apoaequorin gene by coelenterazine-derived chemiluminescence . Whereas YE induced a calcium peak within 1 min after application, LPSXcc induced a long-term calcium signal without transients . To our knowledge this is the first report on the elicitation of an oxidative burst in plant cell cultures by isolated LPS of a phytopathogenic bacterium.

J Am Chem Soc, 2001 Feb 28, 123(8), 1547 - 55
Highly stereoselective reagents for beta-keto ester reductions by genetic engineering of baker's yeast; Rodriguez S et al.; While whole cells of baker's yeast (Saccharomyces cerevisiae) are a convenient biocatalytic reducing agent for a wide variety of carbonyl compounds, mixtures of stereoisomeric alcohols are often observed since the organism contains a large number of reductase enzymes with overlapping substrate specificities but differing stereoselectivities . We sought to improve the performance of baker's yeast for beta-keto ester reductions by using recombinant DNA techniques to alter the levels of three enzymes known to play important roles in these reactions (fatty acid synthase, Fasp; aldo-keto reductase, Ypr1p; alpha-acetoxy ketone reductase, Gre2p) . A complete set of "first-generation" yeast strains that either lack or overexpress each of these three enzymes was created and tested for improvements in stereoselective reductions of a series of beta-keto esters . On the basis of these results, multiply modified ("second-generation") strains were created that combined gene knockout and overexpression in single strains . In some cases, these additional modifications further improved the stereoselectivities of beta-keto ester reductions, thereby making several beta-hydroxy ester building blocks readily available by reactions that can be performed by nonspecialists . This work also revealed that additional yeast proteins participate in reducing beta-keto esters, and further progress using this strategy will require either additional genetic manipulations or the expression of yeast reductases in hosts that lack enzymes with overlapping substrate specificity.

J Chromatogr A, 2001 Jun 22, 920(1-2), 299 - 308
Separation and identification of enzymatic sucrose hydrolysis products by high-performance anion-exchange chromatography with pulsed amperometric detection; Farine S et al.; An accurate carbohydrate analysis method, namely high-performance anion-exchange chromatography with pulsed amperometric detection was successfully applied to the study of sucrose hydrolysis under enzymatic (baker's yeast invertase) conditions . The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of D-glucose, D-fructose and five intermediate fructans using a CarboPac PA-100 (Dionex) analytical anion-exchange column . Highly reproducible results were obtained . The unknown fructans were collected from a semi-preparative CarboPac PA-100 (Dionex) column, neutralized and then desalted on a column containing mixed bed resin AG 501-X8 (D) before identification of the chemical structure . This procedure permitted us to obtain about 20 microg of pure product which is not enough for NMR analysis . Detailed GC-MS analytical data of the methylated compounds indicated that these oligosaccharides were beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (1-kestose), beta-D-Fru-(2 --> 6)-alpha-D-glucopyranoside (6-beta fructofuranosylglucose), beta-D-Fru-(2 --> 1)-beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose) coeluating with a disaccharide.

Water Sci Technol, 2001, 43(11), 233 - 41
Application of membrane and ozonation technologies to remove color from agro-industry effluents; Koyuncu I et al.; The results of membrane and ozonation experiments carried out on various agro-industry effluents including fermentation (baker's yeast), corrugated board, opium alkaloid and textile dying industries are presented . The experiments were performed using lab-scale membrane and ozonation reactors . Color removals were in the range of 80 to 99% for the membrane treatment studies . Ozonation experiments have shown that color removals in the range of 83 to 98% are possible for the investigated wastewaters . Final color levels were lower than 100 Pt-Co unit, which is quite acceptable aesthetically . The relative unit treatment costs of ozonation were about two times higher than membrane systems especially for very strong colored effluents including fermentation and opium alkaloid industries . The study has demonstrated that both membrane and ozonation technologies are viable options for color removal.

Comp Biochem Physiol B Biochem Mol Biol, 2001 Jul, 129(4), 831 - 5
Probing the mechanism of a cyanobacterial Delta9 fatty acid desaturase from Spirulina platensis C1 (Arthrospira sp . PCC 9438); Meesapyodsuk D et al.; The initial and rate determining step in the mechanism of fatty acid desaturases has been proposed to be breakage of one of the C&z.sbnd;H bonds at the site of the incipient double bond . This has been investigated and supported for a number of eukaryotic fatty acid desaturases through the use of kinetic isotope effect experiments with deuterated substrates . In order to probe the reaction catalyzed by the cyanobacterial Delta9 desaturase and compare it to the eukaryotic desaturases, the desC gene of Spirulina platensis, strain C1 (Arthrospira sp . PCC 9438) was expressed in a desaturase mutant of baker's yeast . Kinetic isotope effects were performed by culturing yeast transformants with deuterated thia-substituted stearic acids . A large kinetic isotope effect was found for the 9 position, in qualitative agreement with results from eukaryotic desaturases.

Genetika, 2001 Apr, 37(4), 570 - 3
{Lysine overproduction mutations in the yeast Saccharomyces cerevisiae and its transfection into industrial Yeast strains }; Stepanova VP et al.; Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature . A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants . The intracellular content of lysine was also increased by 30% . The genetic nature of lysine overproduction was studied in this strain . An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1R) and the other is involved in the regulation of lysine production (PRL) . Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome . Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses . The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.

Appl Microbiol Biotechnol, 2001 May, 55(4), 476 - 9
Preparation of an organic solvent-tolerant strain from baker's yeast; Kawamoto T et al.; By using immobilized baker's yeast repeatedly in isooctane with occasional reactivation by cultivation, we succeeded in the preparation of an organic solvent-tolerant strain, named KK21, which could grow in the presence of isooctane . This is the first report on an organic solvent-tolerant strain from baker's yeast . Strain KK21 showed high tolerance to organic solvents and maintained a high and stable activity on continuous reduction of n-butyl 3-oxobutanoate in an isooctane-medium two-phase system . Although the morphology of strain KK21 was the same as that of baker's yeast, the saturated fatty acid occupancy (SFA occupancy), which is defined as the percentage of saturated fatty acids in the total fatty acids of phospholipids, of strain KK21 was significantly higher than that of parental baker's yeast when strain KK21 was grown in the presence of isooctane, suggesting that a decrease in fluidity of the cell membrane might play an important role in the tolerance to organic solvents.

Biosci Biotechnol Biochem, 2001 Apr, 65(4), 810 - 6
Identification of 2,3-dihydro-gamma-ionylideneethanol in Cercospora cruenta; Yamamoto H et al.; During our scrutiny of GC-EI-MS date for C15 alcohols as putative intermediates on the ABA biosynthetic pathway in Cercospora cruenta, a trace amount of 5-{2',2'-dimethyl-6'-methylene-1'-cyclohexyl}-3-methyl-4-penten-1-ol (2,3-dihydro-gamma-ionylideneethanol) was identified . Feeding experiments indicated that this compound was not an intermediate to ABA, but a catabolite that originated from gamma-ionylideneacetaldehyde . The stereochemistry of 2,3-dihydro-gamma-ionylideneethanol was deduced to be (3R,1'S) from a comparison with an authentic specimen prepared via baker's yeast asymmetric reduction.

J Agric Food Chem, 2001 May, 49(5), 2228 - 33
Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of baker's yeast; Greiner R et al.; During food processing such as baking, phytate is dephosphorylated to produce degradation products, such as myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates . Certain myo-inositol phosphates have been proposed to have positive effects on human health . The position of the phosphate groups on the myo-inositol ring is thereby of great significance for their physiological functions . Using a combination of high-performance ion chromatography analysis and kinetic studies the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme from baker's yeast (Saccharomyces cerevisiae) was established . The data demonstrate that the phytate-degrading enzyme from baker's yeast dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), D-Ins(1,2)P(2), to finally Ins(2)P (notation 3/4/5/6/1) . Knowledge of the absolute stereochemical specificity of the baker's yeast phytase allows use of the enzyme to produce defined myo-inositol phosphates for kinetic and physiological studies.

Posit Health News, 1998 Fall, (No 17), 22 - 3
Beta glucan vs bacterial infections - continuous daily use of yeast-derived beta glucan is questioned; Phototactic behavior of Daphnia and the continuous monitoring of water quality: interference of fish kairomones and food quality; Laboratory of Aquatic Ecology, K.U . Leuven, Ch . de Beriotstraat 32, B-3000 Leuven, BelgiumWe carried out a laboratory study to evaluate the sensitivity of phototactic behavior of Daphnia magna to sublethal concentrations of pentachlorophenol (PCP) and copper . More specifically, we determined whether the sensitivity of a D . magna clone to those pollutants is influenced by food quality and the presence of fish kairomones . Test animals were fed either unicellular green algae (Scenedesmus acutus) or fresh baker's yeast (Saccharomyces cerevisiae) and were cultured in the presence or absence of fish kairomones . Four concentrations of PCP (0.4, 0.8, 1.2, and 1.6 mg/L PCP) and one concentration of copper (0.02 mg/L Cu2+) in International Standards Organisation (ISO, Geneva, Switzerland) standard medium were applied in the experiments . Animals were exposed for 3 h to the pollutants prior to the experiments . In the absence of fish kairomones, a strong negative relationship between the phototactic index and nominal sublethal concentrations of PCP was found in animals fed either algae or yeast . The sensitivity of the Daphnia clone to sublethal concentrations of PCP was, however, less clear cut in animals fed yeast than in animals fed algae . The detection limit was 0.4 mg/L PCP with algae as food but was as high as 1.2 mg/L PCP when yeast was used as food . The ability to detect sublethal concentrations of copper and PCP using phototactic behavior was lost when the clones were cultured in the presence of fish kairomones . At a concentration of 0.02 mg/L Cu2+ and in the absence of fish kairomones, the D . magna clone tested became significantly less positively phototactic than in the control treatment regardless of the quality of the food used to culture the test animals . These results suggest that assays using the phototactic behavior of Daphnia to monitor water quality should use genetically stable (clonal) material, positively phototactic, and insensitive to the presence of fish kairomones.

Braz J Med Biol Res, 2001 May, 34(5), 645 - 51
Effect of dietary (n-3) highly unsaturated fatty acids on growth and survival of fat snook (Centropomus parallelus, Pisces: Centropomidae) larvae during first feeding; Seiffert ME et al.; The effect of rotifers, Brachionus rotundiformis (S-type), fed three different diets: A (rotifer fed Nannochloropsis oculata), B (rotifer fed N . oculata and baker's yeast, 1:1), and C (rotifer fed N . oculata and baker's yeast, 1:1, and enriched with Selcoregister mark or target), was evaluated based on the survival, growth and swim bladder inflation rate of fat snook larvae . Rotifers of treatment A had higher levels (4.58 mg/g dry weight) of eicosapentaenoic acid (EPA) than B (1.81 mg/g dry weight), and similar levels (0.04 and 0.06 mg/g dry weight, respectively) of docosahexaenoic acid (DHA) . Rotifers of treatment C had the highest levels of EPA (13.2 mg/g dry weight) and DHA (6.08 mg/g dry weight) . Fat snook eggs were obtained by spawning induction with human chorionic gonadotropin . Thirty hours after hatching, 30 larvae/liter were stocked in black cylindric-conical tanks (36-liter capacity) . After 14 days of culture, there were no significant differences among treatments . Mean standard length was 3.13 mm for treatment A, 3.17 mm for B, and 3.39 mm for C . Mean survival rates were very low (2.7% for treatment A, 2.3% for B, and 1.8% for C) . Swim bladder inflation rates were 34.7% for treatment A, 27.1% for B, and 11.9% for C . The lack of differences in growth and survival among treatments showed that the improvement of the dietary value of rotifer may not have been sufficient to solve the problem of larval rearing . Some other factor, probably pertaining to the quality of the larvae, may have negatively influenced survival.

J Agric Food Chem, 2001 Apr, 49(4), 1928 - 33
Yeasts used as fining treatment to correct browning in white wines; Bonilla F et al.; White wine was subjected to several fining treatments using baker's yeast at concentrations of 0.5, 1, 2, 3, 4, and 5 g/L . At all these concentration levels, the yeasts decreased the color of the wine in different degrees . The wine samples treated with the higher yeast concentration were subjected to analysis of phenolic compounds by HPLC and found to exhibit significantly decreased contents of vanillic, syringic and c-coutaric acids, and procyanidins B2 and B4, and colored compounds eluted at high retention times . The efficiency of the yeast-based fining treatment (1 g/L) was compared with traditional treatments such as those involving the use of activated charcoal or PVPP, which were employed at the usual concentrations in Sherry winemaking . This yeast treatment was found to provide results similar to those of the activated charcoal treatment in terms of A(420) . Likewise, significant differences in the degree of retention of various phenols were observed among the three treatments compared . Finally, the wine samples obtained with the different treatments were subjected to a sensory panel . All the wines were found to exhibit improved color, aroma, and flavor with respect to the untreated samples, although the treatment using yeast at 1 g/L provided the best results in terms of aroma.

J Agric Food Chem, 2001 Apr, 49(4), 1861 - 6
Improved organoleptic and nutritive properties of bakery products supplemented with amino acid overproducing Saccharomyces cerevisiae yeasts; Rincon AM et al.; Spontaneous yeast mutants isolated in continuous culture as resistant to toxic amino acid analogues, able to increase up to 40 times their free amino acid pool of Thr, up to 160 times their pool of Met, or up to 20 times their pool of Lys, were characterized with regard to properties of industrial interest . Growth rate, mu (h(-1)), and biomass yield, Y (g/L), of the amino acid overproducing mutants (AA(S)) were in many cases similar to those of the wild type, whereas their free amino acid content was substantially increased in laboratory and industrial media (molasses) . Doughs fermented with 3% baker's yeast and 0.5% AA(S) mutants produced bakery products that displayed texture similar to those fermented with 3.5% baker's yeast, but the former had a considerable improvement of their taste and aroma . On the other hand, bread content of the essential amino acids Lys, Met, and Thr provided by yeast was also increased.

Fish Shellfish Immunol, 2001 Jan, 11(1), 91 - 100
Phagocytosis of Loma salmonae (Microsporidia) spores in Atlantic salmon (Salmo salar), a resistant host, and chinook salmon (Oncorhynchus tshawytscha), a susceptible host; Shaw RW et al.; The in vitro phagocytosis of Loma salmonae spores by macrophages of Atlantic salmon and two strains of chinook salmon were investigated . Opsonisation of L . salmonae with plasma factors increased uptake by head kidney macrophages . Macrophages of Atlantic salmon, which are resistant to the parasite, had a significantly higher phagocytic index (PI) than those of chinook salmon, a susceptible species . This may indicate a possible mechanism contributing to resistance in Atlantic salmon or that L . salmonae is able to evade or suppress initial binding by macrophages of chinook . Non-specific binding or lectinophagocytosis was also suggested by significantly higher PI of spores from EDTA treated plasma when compared with no plasma or heat treated plasma . In comparison, uptake of Baker's yeast Saccharomyces cerevisiae by phagocytes was not significantly different between fish species and strains for all treatments.

Lipids, 2001 Feb, 36(2), 139 - 43
Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system; Chuang LT et al.; Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis . This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids . Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase . A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase . This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s) . No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase . The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers . CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo . Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase . Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

Biotechnol Bioeng, 2001 Apr 20, 73(2), 164 - 8
Gas phase biotransformation reaction catalyzed by baker's yeast; Maugard T et al.; The gas phase continuous production of acetaldehyde from ethanol and hexanol from hexanal using dried baker's yeast was studied as an alternative approach to conventional processes . The effects of water activity, activity of substrates, and amount of yeast on the performance of the continuous bioreactor were investigated . The extent of yeast hydration and ethanol activity are the most important factors affecting yeast activity and stability .

Appl Microbiol Biotechnol, 2001 Jan, 55(1), 55 - 60
Application of high performance anion exchange chromatography to study invertase-catalysed hydrolysis of sucrose and formation of intermediate fructan products; Farine S et al.; Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru) . All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment . The unknown products were subsequently identified by linkage analysis as beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)- alpha-D-glucopyranoside (1-kestose), beta-D-Fru- (2 --> 6)-alpha-D-glucopyranoside (6-beta-fructofuranosylglucose), beta-D-Fru-(2 -->1) -beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide . The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder . Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised . This observation has not yet been reported, as far as we know.

J Protein Chem, 2000 Aug, 19(6), 535 - 42
Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR; Satterlee JD et al.; The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, {N82A}CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy . Proton NMR studies of {N82A}CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H2O vs . D2O) of the buffer solution . No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.

Med Sci Monit, 2001 Jan-Feb, 7(1), 121 - 4
Skin prick test response to enzyme enolase of the baker's yeast (Saccharomyces cerevisiae) in diagnosis of respiratory allergy; Nittner-Marszalska M et al.; BACKGROUND: The aim of the study is to prove that Saccharomyces cerevisiae enolase, the major allergen of the baker's yeast, induces allergic immediate response in patients with inhalant allergy sensitized to Candida albicans extract . MATERIAL AND METHODS: The study was performed in three groups of patients: I . 20 atopic patients with respiratory allergy sensitized to Candida albicans and inhalant allergens (mite, feather, pollens) II . 30 patients with respiratory allergy, positive skin tests to inhalant allergens but negative skin tests to Candida albicans and other fungi; III . 20 nonatopic, healthy individuals . Skin prick test of purified enolase from Saccharomyces cerevisiae (bakers yeast) at concentration 1 and 10 mg/ml was performed in all groups . The results were documented planimetrically . RESULTS: 95% of patients sensitized to Candida albicans extract showed positive skin reactions to Saccharomyces cerevisiae enolase, 10% of patients of group II and none of the patients of the control group had positive skin responses to enolase . The mean wheal size (mm2) in skin prick test to Candida albicans, Saccharomyces cerevisiae enolase at concentration 10 mg/ml was x = 15.17 +/- 11.08, 15.76 +/- 19.67 and at concentration 1 mg/ml 10.02 +/- 10.49, respectively . CONCLUSIONS: 1 . Saccharomyces cerevisiae enolase induces an immediate allergic reaction in skin in subjects with respiratory allergy and positive skin prick test results to Candida albicans and other fungi . 2 . Enolase can be an important allergenic component of the Candida albicans extract.

Biochem Soc Trans, 2000 Dec, 28(6), 751 - 2
Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms; Leipelt M et al.; Cerebrosides are typical membrane lipids of many organisms . They occur in plants, fungi, animals, humans and some prokaryotes . Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells . However, very little is known about the roles played by these lipids in plants and fungi . To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans . Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast . They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides . The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage . We also generated a ceramide glucosyltransferase-knock-out strain of C . albicans which was devoid of cerebrosides . The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media . In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.

Biochem Soc Trans, 2000 Dec, 28(6), 632 - 5
Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases; Meesapyodsuk D et al.; In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae) . Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast . The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids . The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases . The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments . Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes . These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus . These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.

Eur J Biochem, 2001 Feb, 268(3), 645 - 55
Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases . Apparent correlation of pepstatin-insensitivity and pressure response; Fujiwara S et al.; The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated . Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp . 101 (pseudomonapepsin; PCP) and Xanthomonas sp . T-22 (xanthomonapepsin; XCP)) . The specificity constant {k(cat)/K(m(app))} of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures . The calculated apparent activation volume (Delta V((k(cat)/(K(m)) was about 1, 3, 13, and 14 mL.mol(-1) for PCP, XCP, pepsin, and proteinase A, respectively . The hydrolysis of acid-denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis . In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid-denatured myoglobin at higher pressures . The residual activities of these enzymes after the incubation at high pressures implied a pressure-induced stabilization towards autolysis . The changes in the fourth derivative near-UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa . Upon an increase in pressure, the peaks from PCP and XCP red-shifted slightly, whereas pepsin and proteinase A blue-shifted substantially, thus indicating a more polar environment . The intrinsic fluorescence also decreased upon increasing pressure . However, the change for XCP was rather small, but the change for the other three was very large . The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure . An analysis by the center of spectra mass (CSM) gave the Delta G and Delta V of transition as 9.8 kJ x mol(-1) and -24 mL x mol(-1) (pepsin) and 11.7 kJ x mol(-1) and -43 mL x mol(-1) (proteinase A), respectively, by assuming a simple two-state transition . The circular dichroism (CD) showed relatively small changes after 1-h incubations at 400 MPa, indicating that the secondary structures were largely maintained.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 159 - 62
Leavening ability of baker's yeast exposed to hyperosmotic media; Hirasawa R et al.; To develop a simple and rapid method for enhancing the leavening ability of baker's yeast, we examined the fermentation ability of baker's yeast exposed to hyperosmotic media . When baker's yeast cells were incubated at 25 degrees C for 1 h in a hyperosmotic medium containing 0.5% yeast extract, 0.5% peptone and 20% sucrose, the cells showed a higher fermentation ability in the subsequent fermentation test than those untreated . The increased ratios were from 40 to 60% depending on the strains used . Glucose and fructose showed a similar effect to that of sucrose, but sorbitol was less effective . A high correlation between the intracellular glycerol content and fermentation ability after the osmotic treatment suggested that glycerol accumulated during the hyperosmotic treatment was used in the subsequent fermentation as a substrate, lessened the lag time, and consequently enhanced the fermentation ability . Various baker's yeasts also showed a high leavening ability in dough after the hyperosmotic treatment.

J Agric Food Chem, 1998 Jan 19, 46(1), 248 - 254
Natural Abundance (2)H Nuclear Magnetic Resonance Study of the Origin of Raspberry Ketone; Fronza G et al.; The site-specific natural abundance deuterium distribution of raspberry ketone 3 obtained through a variety of methods has been determined through (2)H NMR spectroscopy . This technique provided a means of distinguishing between "natural" raspberry ketones biogenerated from 4-hydroxybenzalacetone (2), obtained from 4-hydroxybenzaldehyde of extractive botanical origin and acetone produced by sugar fermentation by reduction using baker's yeast and other microorganisms, and other raspberry ketone samples obtained in different "non-natural" ways . Of natural origin is also a commercial sample obtained in an unspecified manner . A mechanistic interpretation has been proposed to explain the difference of site-specific deuterium content between the examined samples.

FEBS Lett, 2000 Dec 22, 487(1), 71 - 5
Genomic exploration of the hemiascomycetous yeasts: 12 . Kluyveromyces marxianus var . marxianus; Llorente B et al.; As part of the comparative genomics project 'GENOLEVURES', we studied the Kluyveromyces marxianus var . marxianus strain CBS712 using a partial random sequencing strategy . With a 0.2 x genome equivalent coverage, we identified ca . 1300 novel genes encoding proteins, some containing spliceosomal introns with consensus splice sites identical to those of Saccharomyces cerevisiae, 28 tRNA genes, the whole rDNA repeat, and retrotransposons of the Ty1/2 family of S . cerevisiae with diverged Long Terminal Repeats . Functional classification of the K . marxianus genes, as well as the analysis of the paralogous gene families revealed few differences with respect to S . cerevisiae . Only 42 K . marxianus identified genes are without detectable homolog in the baker's yeast . However, we identified several genetic rearrangements between these two yeast species.

Waste Manag, 2001, 21(1), 99 - 103
A mathematical model for the anaerobic treatment of Baker's yeast effluents; Deveci N et al.; Baker's yeast fermentation produces high strength wastewaters containing high concentrations of organic materials that cannot easily be degraded by biological processes . A two-stage (anaerobic-aerobic) treatment system has been used in order to treat this effluent efficiently . Most of the COD reduction takes place during the anaerobic degradation . The objective of this study was to generate a simple mathematical model for the anaerobic stage . The model, which is based on the elimination of COD, assumes that only three consecutive reactions namely hydrolysis, acidogenesis and methanogenesis are significant . In the laboratory-scale experiments, feed strength was increased from 3600 to 14,000 mg O2 l-1 in a batch reactor . Three reaction rate constants were found as 0.08, 0.004, 0.06 h-1 by analyzing data from the laboratory experiments . The model was tested and found to be congruent with the daily operation data, which were collected from the Pakmaya Baker's Yeast Kocaeli Factory Plant . This plant contains three Upflow Anaerobic Sludge Blanket reactors, which are run in series . The rate constants of the hydrolysis and methanogenesis were similar to the batch experiment case.

J Mol Evol, 2001 Jan, 52(1), 2 - 5
Intron length and codon usage; Vinogradov AE; The correlation was shown between the length of introns and the codon usage of the coding sequences of the corresponding genes, which in some cases can be related to the level of gene expression . The link is positive in the unicellular organisms, i.e., genes with the longer introns show the higher bias of codon usage . It is most pronounced in baker's yeast, where it is definitely related to the level of gene expression--genes with the higher level of expression have the longer introns . The correlation is inverted in multicellular organisms as compared to unicellular ones . Some organisms, however, do not show the link . The presence or absence of the link does not seem to be related to the GC percent of the coding sequences.

Plant J, 2000 Dec, 24(6), 869 - 82
AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer; Meyer S et al.; The cDNA corresponding to the open reading frame T17M13.3 from Arabidopsis chromosome II was isolated and the encoded protein was characterized as a member of a subgroup of higher plant sucrose transporters . The AtSUC3 (Arabidopsis thaliana sucrose transporter 3) open reading frame encodes a protein with 594 amino acid residues, being 81 and 82 residues longer than the previously described Arabidopsis sucrose carriers AtSUC1 and AtSUC2 . About 50 of these additional amino acids are part of an extended cytoplasmic loop separating the N-terminal from the C-terminal half of the protein . For functional characterization the AtSUC3 cDNA was expressed in baker's yeast . Substrate specificities, energy dependence and K(m) values of the recombinant protein were determined . Removal of the enlarged cytoplasmic loop and expression of the truncated cDNA caused no detectable change in the kinetic properties of the protein, suggesting a transport-independent function for this cytoplasmic domain . Immunolocalization with an AtSUC3-specific antiserum identified the protein in a cell layer separating the phloem from the mesophyll and in a single, subepidermal cell layer of the carpels that is important for pod dehiscence . These localizations suggest a possible role of AtSUC3 in the funnelling of sucrose from the mesophyll towards the phloem, and possibly in pod shatter.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 575 - 80
Commercial baker's yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices; Cerrutti P et al.; The effects of vacuum-drying and freeze-drying on the cell viability of a commercial baker's yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed . An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes . Higher viability values were observed in cells after vacuum-drying than after freeze-drying . Internal concentrations of trehalose in the range 10-20% protected cells in both dehydration processes . Endogenous trehalose concentrations did not affect the water sorption isotherm nor the Tg values . The effect of external matrices of trehalose and maltodextrin was also studied . The addition of external trehalose improved the survival of S . cerevisiae cells containing 5% internal trehalose during dehydration . Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 degrees C . The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step . The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins . The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 521 - 30
A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth; Van Dijck P et al.; The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance . This is disadvantageous for several biotechnological applications, e.g . the preparation of freeze doughs . We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance ('fil' mutants) using a heat shock selection protocol . We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase . It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity . The fil1 mutant displays a reduction in the glucose-induced cAMP increase, trehalase activation and loss of heat resistance . Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway . Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain . These results show that high stress resistance and high fermentation activity are compatible biological properties . Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.

Arch Histol Cytol, 2000 Oct, 63(4), 305 - 12
Lectin binding patterns in rat nasal-associated lymphoid tissue (NALT) and the influence of various types of lectin on particle uptake in NALT; Takata S et al.; We investigated the binding of four types of lectin to follicle-associated epithelium overlying the nasal-associated lymphoid tissue (NALT) of rats in order to identify M-cell specific surface markers and to determine the influence of lectin administration to NALT on the uptake of a particulate antigen . The NALT tissues were incubated with a panel of four types of lectin conjugated to horseradish peroxidase (HRP) . Ulex europaeus-1 (UEA-1) and Dolichos biflorus (DBA) lectin stained the surface of M-cells and goblet cells . Uniform staining by Triticum vulgaris (WGA) was detected in the M-cells, ciliated cells and goblet cells . In contrast, staining of Griffonia simplicifolia I isolectin-B4 (GSI-B4) was almost exclusively M-cell specific . The administration of M-cell specific lectin (GS I-B4) to NALT suppressed the uptake of baker's yeast particles administered later, whereas the non-specific one (UEA-1) had no influence on the uptake . These results indicate that GS I-B4 is a useful marker for the identification of rat NALT M-cells and that such a specific expression of surface glycoconjugates by M-cells may permit the targeting of vaccines and drugs to the antigen sampling sites of the nose . It also appears possible to block the uptake of pathogens by an administration of M-cell specific lectin to NALT.

Adv Biochem Eng Biotechnol, 2000, 69, 125 - 49
History of biotechnology in Austria; Roehr M; Austria has contributed significantly to the progress of the biotechnologies in the past and is actively engaged in doing so today . This review describes the early history of biotechnology in Austria, beginning with the Vienna process of baker's yeast manufacture in 1846, up to the achievements of the 20th century, e.g . the submerged vinegar process, penicillin V, immune biotechnology, biomass as a renewable source of fermentation products (power alcohol, biogas, organic acids etc.), biopulping, biopolymers, biocatalysis, mammalian cell technology, nanotechnology of bacterial surface layers, and environmental biotechnology.

Mol Cell Biol, 2000 Nov, 20(21), 7893 - 902
Saccharomyces cerevisiae expresses three functionally distinct homologues of the nramp family of metal transporters; Portnoy ME et al.; The baker's yeast Saccharomyces cerevisiae expresses three homologues of the Nramp family of metal transporters: Smf1p, Smf2p, and Smf3p, encoded by SMF1, SMF2, and SMF3, respectively . Here we report a comparative analysis of the yeast Smf proteins at the levels of localization, regulation, and function of the corresponding metal transporters . Smf1p and Smf2p function in cellular accumulation of manganese, and the two proteins are coregulated by manganese ions and the BSD2 gene product . Under manganese-replete conditions, Bsd2p facilitates trafficking of Smf1p and Smf2p to the vacuole, where these transport proteins are degraded . However, Smf1p and Smf2p localize to distinct cellular compartments under metal starvation: Smf1p accumulates at the cell surface, while Smf2p is restricted to intracellular vesicles . The third Nramp homologue, Smf3p, is quite distinctive . Smf3p is not regulated by Bsd2p or by manganese ions and is not degraded in the vacuole . Instead, Smf3p is down-regulated by iron through a mechanism that does not involve transcription or protein stability . Smf3p localizes to the vacuolar membrane independently of metal treatment, and yeast cells lacking Smf3p show symptoms of iron starvation . We propose that Smf3p helps to mobilize vacuolar stores of iron.

Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11319 - 24
Lineage-specific loss and divergence of functionally linked genes in eukaryotes; Aravind L et al.; By comparing 4,344 protein sequences from fission yeast Schizosaccharomyces pombe with all available eukaryotic sequences, we identified those genes that are conserved in S . pombe and nonfungal eukaryotes but are missing or highly diverged in the baker's yeast Saccharomyces cerevisiae . Since the radiation from the common ancestor with S . pombe, S . cerevisiae appears to have lost about 300 genes, and about 300 more genes have diverged by far beyond expectation . The most notable feature of the set of genes lost in S . cerevisiae is the coelimination of functionally connected groups of proteins, such as the signalosome and the spliceosome components . We predict similar coelimination of the components of the posttranscriptional gene-silencing system that includes the recently identified RNA-dependent RNA polymerase . Because one of the functions of posttranscriptional silencing appears to be "taming" of retrotransposons, the loss of this system in yeast could have triggered massive retrotransposition, resulting in elimination of introns and subsequent loss of spliceosome components that become dispensable . As the genome database grows, systematic analysis of coordinated gene loss may become a general approach for predicting new components of functional systems or even defining previously unknown functional complexes.

Yeast, 2000 Oct, 16(14), 1273 - 85
Purification, molecular and kinetic characterization of phosphofructokinase-1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition; Reuter R et al.; Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption of the cells with glass beads; (b) fractionated precipitation with polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q . The native enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equilibrium measurements . The apparent sedimentation coefficient was found to be s(20,c)=20.2+/-0.3 S . No significant dependence of the s-value on the protein concentration was observed in the range 0 . 07-0.7 mg/ml . Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100+/-5 kDa, forming an octameric structure . The N-terminus of the enzyme was found to be blocked . Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library . The Pfk-1 coding sequence of S . pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract . The kinetic analysis revealed less cooperativity to fructose 6-phosphate (n(H)=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast . Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate .

Mikrobiologiia, 2000 Jul-Aug, 69(4), 478 - 82
{Dependence of the effect of RNAase from Bacillus intermedius on the growth of baker's yeast on the concentration of the exogenous enzyme}; Kolpakov AI et al.; Bacillus intermedius RNase added at a low concentration (0.001 microgram/ml) stimulated yeast growth, while a high RNase concentration (1500 micrograms/ml) was inhibitory to yeast growth . The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells . The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.

FEBS Lett, 2000 Sep 22, 481(3), 293 - 8
Functional identification of the glycerol permease activity of Arabidopsis thaliana NLM1 and NLM2 proteins by heterologous expression in Saccharomyces cerevisiae; Weig AR et al.; NLM proteins (NOD26-like major intrinsic proteins) from plants contain amino acid sequence signatures which can be found in aquaporins including plant plasma membrane intrinsic proteins and tonoplast intrinsic proteins and glycerol permeases such as the Escherichia coli GlpF and the yeast FPS1 proteins . Heterologous expression of two members of the NLM subgroup from Arabidopsis thaliana (AtNLM1 and AtNLM2) in baker's yeast demonstrated the glycerol permease activity in addition to the previously described aquaporin activity of AtNLM1 . The transport was non-saturable up to 100 mM extracellular glycerol concentration . Longer-chain sugar alcohols did not compete with the transport of radiolabelled glycerol and hexoses were also not transported through the pore.

Prikl Biokhim Mikrobiol, 2000 Jul-Aug, 36(4), 479 - 83
{Effect of Bacillus intermedius ribonuclease on properties of the yeast Saccharomyces cerevisiae}; Kolpakov AI et al.; Effects of Bacillus intermedius ribonuclease on physiological, biochemical, and consumer properties of baker's yeast Saccharomyces cerevisiae were studied . This enzyme improved the yeast raising strength and increased the cell tolerance to various adverse factors . The antistress effect of RNase correlated with an earlier start of the stationary growth phase and increased trehalose pool.

Curr Opin Biotechnol, 2000 Aug, 11(4), 363 - 8
Organic transformations catalyzed by engineered yeast cells and related systems; Stewart JD; Asymmetric ketone reductions remain the most popular application of baker's yeast (Saccharomyces cerevisiae) in organic synthesis and data from the genome sequencing project is beginning to have an impact on improving the stereoselectivities of these reactions, augmenting traditional approaches based on selective inhibition . In addition, the catalytic repertoire of yeast has been expanded to include chiral ketone oxidations by overexpression of a bacterial Baeyer-Villiger monooxygenase.

Gene, 2000 Aug 22, 254(1-2), 87 - 96
Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant; Lee J et al.; A polymerase chain reaction (PCR)-based strategy was used to isolate a mouse cDNA (mCtr1) encoding a Cu transport protein . The deduced mCtr1 protein sequence exhibits 92% identity to human Ctr1, and has structural features in common with known high affinity Cu transporters from yeast . The expression of mouse Ctr1 functionally complements baker's yeast cells defective in high affinity Cu transport . Characterization of the mCtr1 genomic clone showed that the mCtr1 coding sequence is encompassed within four exons and that the mCtr1 locus maps to chromosome band 4C1-2 . RNA blotting analysis demonstrated that mCtr1 is ubiquitously expressed, with high levels in liver and kidney, and early in embryonic development . Steady state mammalian Ctr1 mRNA levels were not changed in response to cellular Cu availability, which is distinct from the highly Cu-regulated transcription of genes encoding yeast high affinity Cu transporters . These studies provide fundamental information for further investigations on the function and regulation of Ctr1 in Cu acquisition in mammals.

Acta Crystallogr D Biol Crystallogr, 2000 Jul, 56 ( Pt 7), 915 - 7
Purification, co-crystallization and preliminary X--ray analysis of the natural aspartic proteinase inhibitor IA3 complexed with saccharopepsin from Saccharomyces cerevisiae; Badasso MO et al.; The vacuolar aspartic proteinase from baker's yeast, saccharopepsin, has been co-crystallized with its natural inhibitor I(A)3, found in the cytosol . The I(A)3-saccharopepsin complex crystals belong to the space group P6(2)22, with unit-cell parameters a = b = 192.1, c = 59 . 80 A and one molecule per asymmetric unit . The initial X-ray analysis of the complex indicates that the crystals diffract to 5.0 A, similar to native saccharopepsin crystals . This is probably a consequence in part of glycosylation of the native saccharopepsin . Full structural analysis of the complex crystal is in progress.

Eur J Biochem, 2000 Jul, 267(13), 3946 - 56
Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development; Owais M et al.; In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells . To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules . In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells . To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae) . The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used . The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment . This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes . Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses . It may be concluded that the liposomes made of lipids derived from S . cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.

Biotechnol Bioeng, 2000 Aug 20, 69(4), 370 - 6
Hydrolytic activity in baker's yeast limits the yield of asymmetric 3-oxo ester reduction; Chin-Joe I et al.; Microbial reductions of ketones hold great potential for the production of enantiopure alcohols, as long as highly selective redox enzymes are not interfered with by competing activities . During reduction of ethyl 3-oxobutanoate by baker's yeast (Saccharomyces cerevisiae) to ethyl (S)-3-hydroxybutanoate, a high enantiomeric excess (> 99%) can be obtained . However, reported yields do not exceed 50-70% . In this article, three main causes are shown to be responsible for these low to moderate yields . These are evaporation of the substrate and product esters, absorption or adsorption of the two esters by the yeast cells and hydrolysis of the two esters by yeast enzymes . The hydrolysis products are further metabolized by the yeast . By reducing the evaporation and absorption losses, the reduction yield can easily be improved to about 85% . Improvement of the efficiency of the reduction and hence the reduction/hydrolysis ratio should lead to a further increase in yield .

J Trace Elem Med Biol, 2000 Apr, 14(1), 43 - 7
Preparation of selenium yeasts I . Preparation of selenium-enriched Saccharomyces cerevisiae; Suhajda A et al.; Selenium (Se) is an essential micronutrient for human and animal organisms . Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable . Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds . It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast . Using a culture medium supplemented with 30 microg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200-1400 microg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method . In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast . As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content . The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast . A 0.40-0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality . Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5-6% at the expense, however, of approximately a 20% decrease in the final biomass.

Biotechnol Bioeng, 2000 Jun 5, 68(5), 517 - 23
Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast; van Hoek P et al.; High-cell-density fed-batch processes for bakers' yeast production will involve a low-average-specific growth rate due to the limited oxygen-transfer capacity of industrial bioreactors . The relationship between specific growth rate and fermentative capacity was investigated in aerobic, sucrose-limited fed-batch cultures of an industrial bakers' yeast strain . Using a defined mineral medium, biomass concentrations of 130 g dry weight/L were reproducibly attained . After an initial exponential-feed phase (mu = 0.18 h(-1)), oxygen-transfer limitation necessitated a gradual decrease of the specific growth rate to ca . 0.01 h(-1) . Throughout fed-batch cultivation, sugar metabolism was fully respiratory, with a biomass yield of 0.5 g biomass/g sucrose(-1) . Fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions with excess glucose) showed a strong positive correlation with specific growth rate . The fermentative capacity observed at the end of the process (mu = 0.01 h(-1)) was only half that observed during the exponential-feed phase (mu = 0.18 h(-1)) . During fed-batch cultivation, activities of glycolytic enzymes, pyruvate decarboxylase and alcohol dehydrogenase in cell extracts did not exhibit marked changes . This suggests that changes of fermentative capacity during fed-batch cultivation were not primarily caused by regulation of the synthesis of glycolytic enzymes .

Nahrung, 2000 Apr, 44(2), 85 - 8
Tarhana as a traditional Turkish fermented cereal food . Its recipe, production and composition; Daglioglu O; As a fermented product tarhana is the dry form of yogurt-cereal mixture and represents an important part of the diets of many people in Turkey . It is prepared by mixing wheat flour, yogurt, yeast and a variety of cooked vegetables (tomatoes, onions, green pepper etc.), salt, and spices (mint, paprika) followed by fermentation for one to seven days . Generally one part yogurt is mixed with two parts of wheat flour (w/w) . In commercial production there are two methods for tarhana making . First method is called straight method and ingredients in the recipe is mixed and kneaded, fermented, dried and finally sieved . Second method is called sour dough method that contains three steps, each one has a different recipe . Throughout fermentation lactic acid bacteria and yeast give the characteristic taste and flavour of tarhana by producing lactic acid, ethanol, carbondioxide and some other organic compounds . Organic acids composed in fermentation period lower the pH (3.4-4.2), and low moisture content (6-10%) is a poor medium for pathogens and spoilage organisms . The nutrient content of tarhana depends upon yogurt and flour ratios as well as some other ingredients, and it is also considered to be a useful high-protein dietary supplement with average 15% protein content . Addition of set yogurt due to high dry matter content and baker's yeast increase protein content and enhances it's amino acid composition.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 187 - 92
Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications; Van Dijck P et al.; In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture . A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells . A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period . We have isolated mutants that retain a better stress resistance during the initiation of fermentation . Some of these showed the same growth rate and fermentation capacity as the wild type cells . These mutants are called 'fil', for deficient infermentation induced loss of stress resistance . First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol . The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway . The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1 . GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway . In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol . Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale . The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage . It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.

Yeast, 2000 Apr, 16(6), 507 - 9
A comprehensive web resource on RNA helicases from the baker's yeast Saccharomyces cerevisiae; Linder P et al.; Members of the RNA helicase protein family are defined by several motifs that have been widely conserved during evolution . They are found in all organisms-from bacteria to humans-and many viruses . The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome . Recent progress in the functional analysis of various family members has confirmed the significance of RNA helicases for most cellular RNA metabolic processes . We have assembled a web resource that focuses on RNA helicases from the budding yeast Saccharomyces cerevisiae . It includes descriptions of RNA helicases and their functions, links to sequence- and yeast-specific databases, an extensive list of references, and links to non-yeast helicase web resources .

Appl Environ Microbiol, 2000 May, 66(5), 1974 - 9
Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus; Lamosa P et al.; Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L . O . Martins, R . Huber, H . Huber, K . O . Stetter, M . S . da Costa, and H . Santos, Appl . Environ . Microbiol . 63:896-902, 1997) . This solute was purified after extraction from the cell biomass . In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources . Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase . Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate . These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min . Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D . gigas and C . pasteurianum rubredoxins . In contrast, the stability of D . desulfuricans rubredoxin was not affected . These different behaviors are discussed in the light of the known structural features of rubredoxins . The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Appl Environ Microbiol, 2000 May, 66(5), 1970 - 3
Redirection of the respiro-fermentative flux distribution in Saccharomyces cerevisiae by overexpression of the transcription factor Hap4p; Blom J et al.; Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae . Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex . To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p . Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration . Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield . Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.

J Bacteriol, 2000 May, 182(10), 2865 - 8
Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature; Tokai M et al.; We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5 degrees C and return to their normal capacity at 25 to 40 degrees C . CSF1 was cloned by functional complementation of the Csf phenotype . CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids . The nucleotide sequence was identical to that of the YLR087C gene in the Saccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein . Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures . Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10 degrees C . The disruptant cells had the same glucose uptake rates as the parental cells at 30 degrees C, but three- to fivefold-lower rates than the parental cells at 10 degrees C . These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.

Biotechnol Prog, 2000 Mar-Apr, 16(2), 163 - 8
Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts; Schebor C et al.; Differential scanning calorimetry thermograms of various samples of commercial instant active dry yeasts revealed a clear glass transition typical of amorphous carbohydrates and sugars . The resulting glass transition temperatures were found to decrease with increasing moisture content . The observed glass curve was similar to that of pure trehalose, which is known to accumulate in large amounts in baker's yeast . The effect of heat treatment at various temperatures on the fermentative activity (as measured by the metabolic production of CO(2)) of dry yeast was studied . First-order plots were obtained representing the loss of fermentative activity as a function of heating time at the various temperatures assayed . Significant losses of fermentative activity were observed in vitrified yeast samples . The dependence of rate constants with temperature was found to follow Arrhenius behavior . The relationship between the loss of fermentative activity and glass transition was not verified, and the glass transition was not reflected on the temperature dependence of fermentative activity loss.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3520 - 5
The high copper tolerance of Candida albicans is mediated by a P-type ATPase; Weissman Z et al.; The pathogenic yeast Candida albicans has higher resistance than the baker's yeast Saccharomyces cerevisiae to elevated concentrations of copper . To understand the basis of this differential resistance, we performed a functional screen for C . albicans genes involved in copper detoxification . Here, we report the isolation of two such genes: a metallothionein, CaCUP1, and a copper-transporting P-type ATPase, CaCRP1 . Both genes are induced by extracellular copper . Gene disruptions indicated that the copper extrusion pump is responsible for the unusual resistance of C . albicans to copper, whereas the metallothionein is responsible for the residual copper resistance of the Cacrp1Delta mutant . We show further that under acidic and anaerobic conditions, such as prevail in the natural niche of C . albicans, the digestive tract of animals, CaCRP1 function becomes essential for survival in the presence of even very low copper concentrations . These observations suggest that copper in the gastrointestinal tract may present a toxic challenge to which enteric organisms had to adapt.

Biol Reprod, 2000 Apr, 62(4), 1016 - 23
Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination; Yakirevich E et al.; The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, "tangled" sperm agglutination was used to isolate cDNAs encoding its cognate antigen . Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in lambdagt11 . Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3-kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen-36 (SA-36), composed of 558 deduced amino acids . SA-36 cDNA contained a 5' untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3' untranslated region of 1138 nt . SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA . This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from baker's yeast . Moreover, polyclonal, monospecific antibodies produced against beta-galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb . Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination.

J Agric Food Chem, 2000 Mar, 48(3), 958 - 61
Delignified cellulosic material supported biocatalyst as freeze-dried product in alcoholic fermentation; Iconomopoulou M et al.; Freeze-dried delignified cellulosic (DC) material supported biocatalyst is proposed as a suitable form of biocatalyst to be preserved . The alcoholic fermentation of glucose using freeze-dried immobilized cells is reported . Freeze-dried immobilized baker's yeast cells on DC material do not need any protective medium during freeze-drying . The effect of initial glucose concentration and temperature on the alcoholic fermentation kinetic parameters is reported in the present study . It was found that the freeze-dried immobilized cells ferment more quickly than free freeze-dried cells and have a lower fermentation rate as compared with wet immobilized cells . However, repeated batch fermentations showed freeze-dried immobilized cells to ferment at about the same fermentation rate as wet immobilized cells . The results indicate that the freeze-dried immobilized cells must be further studied to establish a process for the preservation of immobilized cells.

Yeast, 2000 Mar 30, 16(5), 463 - 74
Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis; Nissen TL et al.; Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively . One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD(+) . The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively . This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth . In anaerobic batch fermentations of strain TN5 (gpd2-Delta1), formation of glycerol was significantly impaired, which resulted in reduction of the maximum specific growth rate from 0.41/h in the wild-type to 0.08/h . Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products . The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth . Strain TN6 (gpd1-Delta1 gpd2-Delta1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD(+) by synthesis of glycerol . Also, strain TN23 (gpd1-Delta1 gpd2-Delta1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD(+) pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP(+) to NADPH and NAD(+) . Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-Delta1) and TN5 (gpd2-Delta1) without serious effects on the maximum specific growth rates or the biomass yields . Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-Delta1 gpd2-Delta1) resulted in a dramatic reduction in the maximum specific growth rate and in biomass formation . Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in micromax from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP(+) to NADH and NADP(+) . Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A . vinelandii in a S . cerevisiae strain with a double deletion in GPD1 and GPD2 .

Biotechnol Bioeng, 2000 Mar 20, 67(6), 671 - 90
Kinetics of product inhibition in alcohol fermentation . Reprinted from Biotechnology and Bioengineering, Vol . X, Issue 6, Pages 845-864 (1968)
Aiba S, Shoda M, Nagatani M.
The inhibitory effect of ethanol concentration p in a medium on the specific rates of growth mu and ethanol production nu of a specific strain of baker's yeast was studied in a chemostat, where except for ethanol as the product, only the concentration of glucose S was controlled to limit the metabolic activity of the yeast . This was designed to supplement the previous findings from the batch experiment, in which ethanol was added artificially and no substrate components were limiting the metabolism of the same yeast, that mu = mu(0)e(-k(1)p) and nu = nu(0)e(-k(1)p), where k(1) and k(2) are empirical constants and subscript the 0 denotes respective values at p = 0 . The effects of p on the values of mu and nu were confirmed by the Line-weaver-Burk plot to belong to noncompetitive inhibition . The formulas here for mu and nu as affected by p, if extrapolated to the case of no limiting substrates, were in good agreement in respective forms with those derived previously from the batch experiment, though the values of corresponding coefficients in these formulas were different . The differential equations for mu and nu as functions of both p and S and, in addition for the rate of glucose consumption as correlated by the yield factors either with the cell growth rate or the rate of ethanol production, were solved properly with a digital computer . A kinetic pattern calculated so far was discussed with reference to the data obtained in the batch experiment and those relevant to actual "sake" brewing.

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 265 - 270
Reductive biotransformation of diethyl beta-, gamma- and delta-oxoalkylphosphonates by cells of baker's yeast; Zyma&nacute;czyk-Duda E et al.; Enantiomerically pure hydroxyalkylphosphonates (over 95% of enantiomeric excess) were obtained by asymmetric reductive biotransformation of a variety of oxoalkylphosphonates catalyzed by baker's yeast . In the most cases the biotransformations were carried out in water under aerobic conditions using whole cell system . In the case of compounds unreactive under these conditions the anaerobic reduction was applied.

J Agric Food Chem, 2000 Jan, 48(1), 100 - 4
Inositol hexaphosphate hydrolysis by Baker's yeast . Capacity, kinetics, and degradation products; Turk M et al.; Phytases hydrolyze myo-inositol 1,2,3,4,5,6-hexaphosphate (IP(6)), yielding lower inositol phosphates and inorganic orthophosphate . Two commercial strains of baker's yeast (Saccharomyces cerevisiae), Y(1) and Y(2), were able to express phytase activity . This was determined by the capacity to grow in a synthetic medium with IP(6) as the sole phosphorus source . IP(6) hydrolysis was rapid for both strains, and after 24 h, all IP(6) was degraded . Control cultures contained inorganic orthophosphate (P(i)) and no IP(6) . Growth rate in IP(6) medium was for both strains essentially identical to growth in P(i) medium, indicating a well-adapted metabolism for utilization of phosphorus from IP(6) . There was some difference in growth yield (milligrams of biomass per milligram of glucose) between the two strains: 0.95 (Y(1)) and 1.35 (Y(2)) in IP(6) medium and 1.03 and 1 . 35, respectively, in P(i) medium . The phytases were of the 3-phytase type, forming mainly DL-Ins(1,2,4,5,6)P(5), DL-Ins(1,2,5,6)P(4), and DL-Ins(1,2,6)P(3).

Prikl Biokhim Mikrobiol, 1999 Nov-Dec, 35(6), 638 - 46
{Enzymatic synthesis of beta-NAD+ selectively marked with tritium at adenine and its use for determining the activity of poly(ADP-ribose) polymerase}; Shram SI et al.; A method of preparation of tritiated beta-HA {symbol: see text}+ from (adenine-2,8-(3)H)ATP and nicotinamide mononucleotide is suggested . This method is based on the baker's yeast (Saccharomyces cerevisiae) enzyme nicotinamide nucleotide adenylyltransferase . Experimental conditions for a nearly complete conversion of labeled ATP to NAD were determined . The tritiated NAD prepared by this method can be used in the quantitative assay of the chromatin enzyme poly(ADP-ribose) polymerase.

Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 291 - 5
Distantly related cousins of MAP kinase: biochemical properties and possible physiological functions; Miyata Y et al.; MAP kinases have been established to be key regulators of cellular signal transduction systems and are conserved from baker's yeast to human beings . Until now, three major types of mammalian MAP kinases (ERK, p38, and JNK/SAPK) have been reported and extensively studied . Advancement of genomic research as well as homology cloning techniques has revealed that there are several other protein kinase families that are structurally modestly related to those conventional MAP kinases . Indeed, most of them possess the TXY motif characteristic to MAP kinases in their activation loop, and can be regarded as members of the MAP kinase superfamily, yet some of them show closest overall similarity to Cdks . These kinases, all of mammalian origin, include MAK, MRK, MOK, p42KKIALRE, p56KKIAMRE, NLK, DYRK/Mnb, and Prp4 . Although most of their physiological roles remain unknown, recent progress starts shedding some light on their functions .

Syst Appl Microbiol, 1999 Sep, 22(3), 329 - 40
Genomic stability of Saccharomyces cerevisiae baker's yeasts; Gasent-Ramirez JM et al.; The objective of this study has been to gather data on genomic stability of baker's yeast strains during long-term mitotic growth under restrictive conditions so that comparisons could be made to other studies indicating genomic instability during meiosis . The work describes the analysis of mitotic stability of the nuclear and mitochondrial genomes in the baker's yeast strain V1 during incubation in continuous culture for 190 generations (300 days) . The cells were cultured in complete medium containing 2% glucose and 8 to 12% ethanol, as a mutagenic agent specific for mtDNA . The high concentration of ethanol severely limited the growth rate of the cells . DNA samples were monitored for chromosomal pattern, polymorphisms in selected nuclear genes (SUC2, MALIT, ADH1) and mobile genetic elements (Ty1 and Y'), and for RFLPs in mtDNA . The results show that both the nuclear and mitochondrial genomes of grande cells were very stable . However, the frequency of petite mutants in the population varied dramatically during the course of the experiment, reaching as high as 87% petite during the first 27 days of the experiment and declining to 5.8% petite at the end . This decline can be attributed to selection against petite mutants in media containing high concentrations of ethanol . Moreover, when samples and the parental strain were compared at the end of the experiment, no change could be observed in parameters such as their growth rate in different media, capacity to leave doughs, viability in ethanol or frequency of petite mutants . Results therefore indicated that the majority of the cells in the population were very similar to the parental throughout the experiments, with no apparent molecular or phenotypical changes.

J Mol Evol, 1999 Dec, 49(6), 789 - 97
Amino acid reiterations in yeast are overrepresented in particular classes of proteins and show evidence of a slippage-like mutational process; Mar Alba M et al.; Long amino acid repeats are often observed in eukaryotic proteins . In humans, several neurological disorders are caused by proteins containing abnormally long polyglutamines . However, no systematic analysis has attempted to investigate the relationship between reiterations of particular amino acids and protein function, the possible mechanisms involved in the generation of these regions, or the contribution of selection in restricting their genomic distribution, in a large collection of wild-type proteins . We have used baker's yeast open reading frames to study these questions . The most abundant amino acid repeats found in yeast proteins are repeats of glutamine, asparagine, aspartic acid, glutamic acid, and serine . Different amino acid repeats are concentrated in different classes of proteins . Acidic and polar amino acid repeats are significantly associated with transcription factors and protein kinases, while serine repeats are significantly associated with membrane transporter proteins . In most cases the codon structures encoding the repeats at the gene level show a significant bias toward long tracts of one of the possible codons, suggesting that trinucleotide slippage has played an important role in generating these reiterations . However, many, particularly those encoding serine repeats, do not show evidence of slippage . The distributions of codon repeats within proteins and between coding and noncoding regions of the genome, and of amino acids between proteins with different functions, suggest that repeats of these kinds are subject to strong selection.

J Agric Food Chem, 1999 Feb, 47(2), 803 - 8
Expression of LIP1 and LIP2 genes from Geotrichum species in Baker's yeast strains and their application to the bread-making process; Monfort A et al.; Lipolytic baker's yeast strains able to produce extracellular active lipase have been constructed by transformation with plasmids containing the LIP1 and LIP2 genes from Geotrichum sp . under the control of the Saccharomyces cerevisiae actin promoter (pACT1) . Lipase productivity differed between both constructs, YEpACT-LIP1-t and YEpACT-LIP2-t, being higher for the strain bearing the LIP2 gene in all culture media tested . This result appeared not to be the consequence of a defect in the transcription of the LIP1 gene as revealed by Northern blot analysis . Replacing the signal sequence of LIP1 by that of LIP2 in the YEpACT-LIP1-t plasmid enhanced significantly the secretion of lipase 1, but the levels of lipase activity were still lower than those found for the YEpACT-LIP2-t transformant . Recombinant lipase 2 protein produced by baker's yeast exhibited biochemical properties similar to those of the natural enzyme . Fermented dough prepared with YEpACT-LIP2-t-carrying cells rendered a bread with a higher loaf volume and a more uniform crumb structure than that prepared with control yeast . These effects were stronger by the addition in the bread dough formulas of a preferment enriched in recombinant lipase 2.

J Agric Food Chem, 1999 Feb, 47(2), 550 - 3
Inhibitory effect of alpha-glucosidase inhibitors varies according to its origin; Oki T et al.; The inhibitory effect of alpha-glucosidase (AGH) inhibitors against its origins (baker's yeast and rat, rabbit, and pig small intestines) was investigated . All inhibitors used in this study showed quite different inhibitory activities according to AGH origins . Voglibose, acarbose and glucono-1,5-lactone strongly inhibited mammalian AGHs, whereas no or less inhibition was observed in yeast AGH . On the contrary, (+)-catechin, a good inhibitor against yeast AGH (IC(50) = 1.3 x 10(-)(1) mM) as well as voglibose (IC(50) = 2.6 x 10(-)(2) mM), did not retard the mammalian AGH activity . Subsequent inhibition study with various food components revealed that all of foods except for green (IC(50) = 0.735 mg/mL) and oolong teas (IC(50) = 1.34 mg/mL) showed no inhibitory activity against rat AGH, whereas they inhibited yeast AGH . Consequently, the magnitude of AGH inhibition was greatly affected by its origin, and more attention relating to AGH origin would be needed to evaluate in vitro AGH inhibitory effect.

Arch Biochem Biophys, 1999 Nov 15, 371(2), 277 - 83
Comparative study of the inhibition of alpha-glucosidase, alpha-amylase, and cyclomaltodextrin glucanosyltransferase by acarbose, isoacarbose, and acarviosine-glucose; Kim MJ et al.; Bacillus stearothermophilus maltogenic amylase hydrolyzes the first glycosidic linkage of acarbose to give acarviosine-glucose . In the presence of carbohydrate acceptors, acarviosine-glucose is primarily transferred to the C-6 position of the acceptor . When d-glucose is the acceptor, isoacarbose is formed . Acarbose, acarviosine-glucose, and isoacarbose were compared as inhibitors of alpha-glucosidase, alpha-amylase, and cyclomaltodextrin glucanosyltransferase . The three inhibitors were found to be competitive inhibitors for alpha-glucosidase and mixed noncompetitive inhibitors for alpha-amylase and cyclomaltodextrin glucanosyltransferase . The K(i) values were dependent on the type of enzyme and their source . Acarviosine-glucose was a potent inhibitor for baker's yeast alpha-glucosidase, inhibiting 430 times more than acarbose, and was an excellent inhibitor for cyclomaltodextrin glucanosyltransferase, inhibiting 6 times more than acarbose . Isoacarbose was the most effective inhibitor of alpha-amylase and cyclomaltodextrin glucanosyltransferase, inhibiting 15.2 and 2.0 times more than acarbose, respectively .

Yeast, 1999 Sep 30, 15(13), 1299 - 305
Construction of a lactose-assimilating strain of baker's yeast; Adam AC et al.; A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently . This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making . The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively . To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K . lactis genes . Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power . The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey .

Int Rev Cytol, 2000, 194, 197 - 238
The petite mutation in yeasts: 50 years on; Chen XJ et al.; Fifty years ago it was reported that baker's yeast, Saccharomyces cerevisiae, can form "petite colonie" mutants when treated with the DNA-targeting drug acriflavin . To mark the jubilee of studies on cytoplasmic inheritance, a review of the early work will be presented together with some observations on current developments . The primary emphasis is to address the questions of how loss of mtDNA leads to lethality (rho 0-lethality) in petite-negative yeasts and how S . cerevisiae tolerates elimination of mtDNA . Recent investigation have revealed that rho 0-lethality can be suppressed by specific mutations in the alpha, beta, and gamma subunits of the mitochondrial F1-ATPase of the petite-negative yeast Kluyveromyces lactis and by the nuclear ptp alleles in Schizosaccharomyces pombe . In contrast, inactivation of genes coding for F1-ATPase alpha and beta subunits and disruption of AAC2, PGS1/PEL1, and YME1 genes in S . cerevisiae convert this petite-positive yeast into a petite-negative form . Studies on nuclear genes affecting dependence on mtDNA have provided important insight into the functions provided by the mitochondrial genome and the maintenance of structural and functional integrity of the mitochondrial inner membrane.

Biochem J, 1999 Oct 1, 343 Pt 1, 159 - 68
Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression; Kraakman LS et al.; In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression . Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression . Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels . However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast . In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling . We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering . Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained . However, this was not sufficient to cause cAMP activation nor catabolite repression . Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression . Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity . We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme . In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function . The implications of this model are discussed.

Microbiol Mol Biol Rev, 1999 Sep, 63(3), 554 - 69
Function and regulation of yeast hexose transporters; Ozcan S et al.; Glucose, the most abundant monosaccharide in nature, is the principal carbon and energy source for nearly all cells . The first, and rate-limiting, step of glucose metabolism is its transport across the plasma membrane . In cells of many organisms glucose ensures its own efficient metabolism by serving as an environmental stimulus that regulates the quantity, types, and activity of glucose transporters, both at the transcriptional and posttranslational levels . This is most apparent in the baker's yeast Saccharomyces cerevisiae, which has 20 genes encoding known or likely glucose transporters, each of which is known or likely to have a different affinity for glucose . The expression and function of most of these HXT genes is regulated by different levels of glucose . This review focuses on the mechanisms S . cerevisiae and a few other fungal species utilize for sensing the level of glucose and transmitting this information to the nucleus to alter HXT gene expression . One mechanism represses transcription of some HXT genes when glucose levels are high and works through the Mig1 transcriptional repressor, whose function is regulated by the Snf1-Snf4 protein kinase and Reg1-Glc7 protein phosphatase . Another pathway induces HXT expression in response to glucose and employs the Rgt1 transcriptional repressor, a ubiquitin ligase protein complex (SCF(Grr1)) that regulates Rgt1 function, and two glucose sensors in the membrane (Snf3 and Rgt2) that bind glucose and generate the intracellular signal to which Rgt1 responds . These two regulatory pathways collaborate with other, less well-understood, pathways to ensure that yeast cells express the glucose transporters best suited for the amount of glucose available.

J Mol Evol, 1999 Sep, 49(3), 376 - 84
Intron-genome size relationship on a large evolutionary scale; Vinogradov AE; The intron-genome size relationship was studied across a wide evolutionary range (from slime mold and yeast to human and maize), as well as the relationship between genome size and the ratio of intervening/coding sequence size . The average intron size is scaled to genome size with a slope of about one-fourth for the log-transformed values; i.e., on the global scale its increase in evolution is lower than the increase in genome size by four orders of magnitude . There are exceptions to the general trend . In baker's yeast introns are extraordinarily long for its genome size . Tetrapods also have longer introns than expected for their genome sizes . In teleost fish the mean intron size does not differ significantly, notwithstanding the differences in genome size . In contrast to previous reports, avian introns were not found to be significantly shorter than introns of mammals, although avian genomes are smaller than genomes of mammals on average by about a factor of 2.5 . The extra-/intragenic ratio of noncoding DNA can be higher in fungi than in animals, notwithstanding the smaller fungal genomes . In vertebrates and invertebrates taken separately, this ratio is increasing as the increase in genome size . Two hypotheses are proposed to explain the variation in the extra-/intragenic ratio of noncoding DNA in organisms with similar numbers of genes: transition (dynamic) and equilibrium (static) . According to the transition model, this variation arises with the rapid shift of genome size because the bulk of extragenic DNA can be changed more rapidly than the finely interspersed intron sequences . The equilibrium model assumes that this variation is a result of selective adjustment of genome size with constraints imposed on the intron size due to its putative link to chromatin structure (and constraints of the splicing machinery).

J Ind Microbiol Biotechnol, 1999 Jun, 22(6), 627 - 632
Enhancement of maltose utilisation by Saccharomyces cerevisiae in medium containing fermentable hexoses; Hazell B et al.; Some industrial strains of Saccharomyces cerevisiae are unable to maintain high rates of fermentation during transition from catabolism of hexoses to maltose . This phenomenon, termed 'maltose lag', presents problems for the baking, brewing and distilling industries, which rely on yeast catabolism of mixtures of hexoses and maltose . Maltose utilisation requires the presence of maltose permease and alpha-glucosidase (maltase), encoded by MAL genes . Synthesis of these is induced by maltose and repressed by glucose . One strain of baker's yeast used in this work exhibited a marked maltose lag, whereas a second strain exhibited a shorter lag during conversion from hexose to maltose metabolism . The extent of the lag was linked to the levels of maltose permease and maltase in cells at the time of inoculation into mixed sugar medium . This view is supported by results showing that pulsing yeast with maltose to induce expression of MAL genes prior to inoculation into mixed sugar medium, enhanced sugar fermentation . Maltose pulsing of yeasts could therefore be useful for enhancing some fermentations relevant to baking and other yeast industries.

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