Yeast

Blood Purif, 1999, 17(2-3), 134 - 41
Uremic ultrafiltrate inhibits platelet-activating factor synthesis; Wratten ML et al.; Background: Several studies have suggested that uremic toxins may adversely affect phagocytic leukocytes of chronic renal failure patients . Platelet-activating factor (PAF) is produced by phagocytic leukocytes and is a potent mediator of inflammation which is produced by leukocytes upon appropriate stimulation . Methods: We added uremic or normal ultrafiltrate, ultrafiltrate fractionated by reverse phase HPLC or compounds eluting at the same retention time as the fractionated ultrafiltrate, to normal leukocytes . Complement-coated baker's yeast spores were added to stimulate phagocytosis . Total PAF was purified by thin layer chromatography and quantified by bioassay on rabbit platelets . The activities of two enzymes involved in the synthesis of PAF, phospholipase A2 (PLA2) and acetyltransferase, were measured in the presence of fractionated ultrafiltrate . Results: Ultrafiltrate from both healthy and uremic subjects inhibited PAF synthesis, but the inhibitory effect was more substantial for uremic subjects . Ultrafiltrate fractionated by HPLC showed high PAF inhibition for late eluting hydrophobic fractions . Addition of phenol or p-cresol, two uremic toxins with similar elution pattern as the late fractions, also inhibited PAF synthesis . The activity of PLA2 and acetyltransferase was decreased in the presence of uremic ultrafiltrate . Conclusions: We observed that uremic ultrafiltrate inhibits PAF synthesis upon stimulation with complement coated baker's yeast spores . The decrease in total PAF synthesis appears to be associated with an inhibition of phospholipase A2 and acetyltransferase activity, enzymes involved in the remodelling pathway for PAF synthesis.

Biochem J, 1999 Aug 1, 341 ( Pt 3), 669 - 78
The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice); Genschel U et al.; We have isolated a Lotus japonicus cDNA for pantothenate (vitamin B(5)) synthetase (PS) by functional complementation of an Escherichia coli panC mutant (AT1371) . A rice (Oryza sativum) expressed sequence tag, identified by sequence similarity to PS, was also able to complement the E . coli auxotroph, as was an open reading frame from Saccharomyces cerevisiae (baker's yeast) . The Lotus and rice cDNAs encode proteins of approx . 34 kDa, which are 65% similar at the amino acid level and do not appear to encode N-terminal extensions by comparison with PS sequences from other organisms . Furthermore, analysis of genomic sequence flanking the coding sequence for PS in Lotus suggests the original cDNA is full-length . The Lotus and rice PSs are therefore likely to be cytosolic . Southern analysis of Lotus genomic DNA indicates that there is a single gene for PS . Recombinant PS from Lotus, overexpressed in E . coli AT1371, is a dimer . The enzyme requires d-pantoate, beta-alanine and ATP for activity and has a higher affinity for pantoate (K(m) 45 microM) than for beta-alanine (K(m) 990 microM) . Uncompetitive substrate inhibition becomes significant at pantoate concentrations above 1 mM . The enzyme displays optimal activity at about 0.5 mM pantoate (k(cat) 0.63 s(-1)) and at pH 7.8 . Neither oxopantoate nor pantoyl-lactone can replace pantoate as substrate . Antibodies raised against recombinant PS detected a band of 34 kDa in Western blots of Lotus proteins from both roots and leaves . The implications of these findings for pantothenate biosynthesis in plants are discussed.

Medicina (B Aires), 1999, 59(2), 171 - 5
Cell differentiation increases astrocyte phagocytic activity . A quantitative analysis of both GFAP labeling and PAS-stained yeast cells; Iacono RF et al.; Since efficiency of phagocytic potential in activated astrocytes is still a subject of controversy, an attempt was made to quantify simultaneously phagocytic activity and astrocyte differentiation . Resorting to Junin virus, known to induce astrocyte activation, infected vs control samples of cultured rat astroglial cells were serially harvested up to day 12 post-inoculation (pi), and subjected to a triple staining procedure consisting in immunoperoxidase labeling of GFAP, periodic acid-Schiff (PAS) reaction in added baker's yeast cells and hematoxylin for nuclear staining of the whole cell monolayer . Adopting GFAP labeling as a specific marker of astrocyte differentiation, the immunoprecipitate development over time was measured . Direct calculation of the initial reaction rate was feasible given its linear behavior during the first 10 min, so that GFAP amount was regarded proportional to peroxidase activity . As determined by digital image analysis, mean optical density (MOD) values of GFAP in infected samples increased from 0.618 +/- 0.082 at day 1 pi to 0.825 +/- 0.125 at day 3, leveling off at 1.010 +/- 0.101 as from day 9, while control uninfected samples remained unchanged at roughly 0.6 during the entire observation period . In turn, phagocytosis was quantified by PAS staining densitometry, whose intensity varied according to wall degradation of yeast cells . MOD levels of PAS-stained phagocytized yeast cells were significantly lower (p < 0.05) in infected vs control cultures at 48 and 72 h following their addition to the astroglial monolayer . According to simultaneous quantification of two components of astrocyte response to viral infection, it is concluded that phagocytic activity increases with astrocyte differentiation.

Appl Environ Microbiol, 1999 Jul, 65(7), 3261 - 3
Development of bacterial contamination during production of yeast extracts; Barrette J et al.; Baker's yeast suspensions having bacterial populations of 10(6) and 10(8) CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE) . The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus) . Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan) . Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE . Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added . A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.

Appl Environ Microbiol, 1999 Jul, 65(7), 2841 - 6
Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial Baker's yeast; Shima J et al.; Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough . To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method . During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants . The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains . The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited . The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.

Mol Microbiol, 1999 Jun, 32(6), 1140 - 52
Genetic redundancy and gene fusion in the genome of the Baker's yeast Saccharomyces cerevisiae: functional characterization of a three-member gene family involved in the thiamine biosynthetic pathway; Llorente B et al.; Redundancy is a salient feature of all living organisms' genome . The yeast genome contains a large number of gene families of previously uncharacterized functions that can be used to explore the functional significance of structural redundancy in a systematic manner . In this work, we describe results on a three-member gene family with moderately divergent sequences (YOL055c, YPL258c and YPR121w ) . We demonstrate that two members are isofunctional and encode a hydroxymethylpyrimidine phosphate (HMP-P) kinase (EC 2.7.4.7), an activity required for the final steps of thiamine biosynthesis, whose genes were not previously known in yeast . In addition, we show that the three genes are each composed of two distinct domains, each corresponding to individual genes in prokaryotes, suggesting gene fusion during evolution . The function of the carboxy-terminal part of the proteins is not yet understood, but it is not required for HMP-P kinase activity . Expression of all three genes is regulated in the same way . Several other examples of gene fusions exist in the same biosynthetic pathway when eukaryotic genes are compared with prokaryotic ones.

Comp Biochem Physiol B Biochem Mol Biol, 1999 Mar, 122(3), 309 - 19
Identification, purification and properties of a beta-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions; Chen C et al.; A lectin specific for laminarin, a beta-1,3-glucan, agglutinating baker's yeast and enhancing prophenoloxidase activation by laminarin, has been purified from the cockroach, Blaberus discoidalis, serum . Purification involved gel filtration with Bio-gel P300 and affinity chromatography on blue Sepharose CL-6B and laminarin-Sepharose 4B . The purified lectin has a molecular mass estimate of 520 kDa determined by gel filtration, and approximately 80 and 82 kDa by SDS-PAGE, under non-reducing and reducing conditions, respectively . After isoelectric focusing the lectin focused as a single band at pH 4.9 . The purified lectin was stained by the periodic acid/Schiff's reagent showing that it is a glycoprotein, and was deglycosylated by endo-beta-N-acetylglu-cosaminidase F . Amino acid composition analysis showed the protein is similar to previously purified beta-1,3-glucan binding proteins from other invertebrates . In electron micrographs by negative staining, the protein formed large aggregates with 'Y'-shaped 'structural units' ca . 79 x 65 nm . Immunological tests confirmed that this lectin is not related to any other lectins previously purified from the same insect . This protein appears to be part of the hexamerin family of proteins . This is one of the first reports of a hexamerin-like molecule with lectin activity.

Curr Genet, 1999 Jun, 35(5), 491 - 8
Leu343Phe substitution in the Malx3 protein of Saccharomyces cerevisiae increases the constitutivity and glucose insensitivity of MAL gene expression; Higgins VJ et al.; To utilise maltose as a carbon source Saccharomyces cerevisiae needs one or more functional MAL loci that contain the MALx1 gene encoding maltose permease, MALx2 encoding maltase, and MALx3 encoding a transcriptional activator . Maltose causes a rapid MALx3-dependent induction of MAL gene transcription, and glucose represses this activation via Mig1p . A MALx3 gene conveying high MAL gene expression in the absence of maltose in a malx3 laboratory mutant strain has been isolated from baker's yeast . The construction of hybrid genes between the isolated gene and a highly regulated MALx3 gene showed that constitutivity was the result of multiple amino-acid alterations throughout the structural gene . The combined effect of these amino-acid alterations was shown to be stronger than the sum of their individual effects on constitutivity . Analysis in glucose-repressed conditions confirmed that increased MALx3 transcript levels increased the glucose insensitivity of MAL gene expression but did not affect constitutivity . Analysis of four mutations between aa 343 and 375, lying within a proposed negative regulatory domain, showed that the single mutation of Leu343Phe increased the glucose insensitivity of MAL gene expression by 30-fold . These results demonstrate that not only Mig1p modulation of MALx3 expression, but also the MALx3 protein structure, is involved in the glucose-insensitive expression of the MAL genes.

Biotechnol Prog, 1999 May, 15(3), 366 - 72
Immobilization of highly concentrated cell suspensions using the laminar jet breakup technique
Brandenberger HR, Widmer F.
The controlled breakup of a disturbed jet is a well-known technique for monodisperse particle production and has been well-investigated for Newtonian and viscoelastic fluids . For the immobilization of cells in beads of a hydrogel it has been observed that there is a maximum cell concentration beyond which a monodisperse bead distribution cannot be reached . Higher concentrations lead to irregular jet breakup and thus to droplet coalescence forming beads with double or triple volume . This maximum cell concentration has been investigated for different operating conditions using latex beads of different diameters as a model substance for cells and baker's yeast as a comparison . The maximum cell concentration where a monodisperse size distribution could still be achieved was increased using an electrostatic droplet dispersion system.

Biotechnol Prog, 1999 May-Jun, 15(3), 459 - 66
Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker's yeast strain; Nieto A et al.; The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain . To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus . High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette . We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium . This transformant also expressed and secreted high levels of alpha-amylase . Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain . The Rib-AMY transformant also was useful in retarding bread firming . This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

Trends Biotechnol, 1999 Jun, 17(6), 237 - 44
Engineering baker's yeast: room for improvement; Randez-Gil F et al.; Bread making is one of the oldest food-manufacturing processes . However, it is only in the past few years that recombinant-DNA technology has led to dramatic changes in formulation, ingredients or processing conditions . New strains of baker's yeast that produce CO2 more rapidly, are more resistant to stress or produce proteins or metabolites that can modify bread flavour, dough rheology or shelf-life are now emerging.

Acta Chem Scand, 1999 May, 53(5), 360 - 5
Synthesis of 1,3-dithianes and 1,3-dithiolanes . Baker's yeast reduction and lipase-catalyzed resolution for synthesis of enantiopure derivatives; Anthonsen T et al.; Three 1,3-dithiolanes and four 1,3-dithianes have been synthesised from 1-(1,3-dithiolan-2-yl)-2-propanone and 1-(1,3-dithian-2-yl)-2-propanone, respectively . Asymmetric reductions of these ketones using baker's yeast gave the corresponding enantiopure (S)-alcohols . Baker's yeast also reduced the double bond in 3-(1,3-dithian-2-yl)-3-buten-2-one enantioselectively to give (S)-3-(1,3-dithian-2-yl)-2-butanone . 3-(1,3-Dithian-2-yl)-3-buten-2-one was also reduced chemo-selectively and the resulting 3-(1,3-dithian-2-yl)-3-buten-2-ol was resolved by transesterification in organic solvent using lipase B from Candida antarctica to yield the (S)-alcohol and the (R)-acetate with very high enantiomeric ratio, E . Racemic 1-(1,3-dithiolan-2-yl)-2-propanol and 1-(1,3-dithian-2-yl)-2-propanol were also resolved under similar conditions to give the (S)-alcohols and the corresponding (R)-acetates.

Biotechniques, 1999 Apr, 26(4), 728 - 34
Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity; Karamohamed S et al.; A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed . The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system . In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast . In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively . The assay is sensitive and yields linear responses between 0.05-50 mU . The detection limit was found to be 0.05 mU of NDP kinase . The method was used to detect NDP kinase contamination in commercial enzyme preparations.

J Chromatogr A, 1999 Apr 30, 840(2), 195 - 204
Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker's yeast using an expanded bed adsorption system; Willoughby NA et al.; Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers' yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed . Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that charging with Zn2+ gave the highest recovery and the lowest EDTA concentration required for elution . These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA . The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively . Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers' yeast diluted to 10 mg/ml total protein content with a recovery of 80-100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.

Bioorg Khim, 1998 Feb, 24(2), 119 - 25
{Structural-functional characteristics of the Schizosaccharomyces pombe rpb8+ gene, coding the subunit of RNA polymerase I-III, specific only for eukaryotes}; Shpakovskii GV et al.; A full-length cDNA of the rpb8+ gene encoding a common subunit Rpb8 of nuclear RNA polymerases I-III only specific for Eucarya was isolated from an expression library of the fission yeast Schizosaccharomyces pombe . The primary structure of the corresponding fragment of the Sz . pombe genome was also established . The rpb8+ gene contains two short introns, 59 and 48 bp long . Only short segments of homology were found upon comparing the Rpb8 subunit homologs from various eukaryotic species, and substantial differences exist between the corresponding proteins of unicellular and multicellular organisms . Subunit Rpb8 of Sz . pombe proved to be the smallest one among the known related proteins: it lacks the 21-aa fragment corresponding to amino acids residues 68-88 of the central part of the homologous subunit ABC14.5 of Saccharomyces cerevisiae . Accordingly, subunit Rpb8 of the fission yeast was not capable of substituting in vivo subunit ABC14.5 in nuclear RNA polymerases of the baker's yeast.

J Inorg Biochem, 1999 Jan-Feb, 73(1-2), 109 - 15
Ternary complexes of palladium (II) with levamisole and L-amino acids; Nijasure AM et al.; The complexes of general formula {(LMS)2Pd(amino acid)}Cl with LMS = levamisole, and amino acid = L-alanine, L-phenylglycine, L-phenylalanine, L-valine, L-methionine, and L-proline, were synthesized by the interaction of {(LMS)2PdCl2} with the sodium salts of L-amino acids . The newly synthesized complexes are characterized by elemental analysis, conductivity, magnetic susceptibility, optical rotation measurements, and UV-Vis, IR and 13C NMR spectral data . Levamisole is coordinated to palladium via the N-7 nitrogen and the amino acids through the amino nitrogen and carboxylate oxygen, except for L-methionine which binds the metal via nitrogen and sulfur atoms . Optically active {(LMS)2Pd(amino acid)}Cl complexes are obtained when L-amino acids or D,L-amino acids are used for the synthesis of these complexes . L-Methionine and L-proline complexes induce new cell forms in Baker's yeast (Saccharomyces cerevisiae) cells.

Vopr Pitan, 1999, 68(1), 17 - 9
{Effect of biologically active food additives containing autolysate of baker's yeast and spirulina on intestinal permeability in an experiment}; Mazo VK et al.; Influence of bioactive food supplements (BFA) intake on intestinal barrier permeability to macromolecules of polyethylene glycol 4000 was studied in rats with intestinal anaphylaxis and after external gamma-irradiation . BFA studied included autolysed baker's yeast ("Vitasil") and edible algae Spirulina platensis . Intake of complex additive Vitasil + Spirulina resulted in significant diminution of permeability before irradiation and its partial normalization (24% decrease) after irradiation . Spirulina additive intake led to practically complete normalization of permeability (1.84 times decrease) in anaphylactic rats . It is concluded that Spirulina and Vitasil are promising BFA for organism general resistance elevation.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 133 - 8
A structured approach for selection among candidate metabolic network models and estimation of unknown stoichiometric coefficients; Vanrolleghem PA et al.; A metabolic network model is one of the cornerstones of the emerging Metabolic Engineering methodology . In this article, special attention is therefore, given to the phase of model building . A five-stage structured approach to metabolic network modeling is introduced . The basic steps are: (1) to collect a priori knowledge on the reaction network and to build candidate network models, (2) to perform an a priori check of the model, (3) to estimate the unknown parameters in the model, (4) to check the identified model for acceptability from a biological and thermodynamic point of view, and (5) to validate the model with new data . The approach is illustrated with a growth system involving baker's yeast growing on mixtures of substrates . Special attention is given to the central uncertainties in metabolic network modeling, i.e., estimation of energetic parameters in the network and the choice of the source of anabolic reducing equivalents NADPH .

Biotechnol Bioeng, 1998 Jun 20, 58(6), 587 - 94
A membrane bioreactor for biotransformations of hydrophobic molecules
Doig SD, Boam AT, Leak DI, Livingston AG, Stuckey DC.
The Membrane Bioreactor for Biotransformations (MBB) is based on the aqueous/organic two-phase system, and uses a tubular silicone rubber membrane to separate the two liquid phases . This avoids the key problem associated with direct contact two-phase processes, specifically, product emulsification . The baker's yeast mediated reduction of geraniol to citronellol was used as a model biotransformation to demonstrate MBB operation . Values for the overall mass transfer coefficient were determined for geraniol, (2.0 x 10(-5) ms-1), and for citronellol, (2.1 x 10(-5) ms-1) diffusion across the silicone rubber membrane . Using these values, and the specific activity of the biocatalyst (5 nmols-1g biomass-1), a suitable membrane surface area: biomass ratio was determined as 2.4 x 10(-3) m2g biomass-1 . The bioreactor was operated at this surface area: biomass ratio and achieved a product accumulation rate 90-95% that of a conventional direct contact two-phase system . The slight reduction in product accumulation rate was shown not to be due to mass transfer limitations with respect to reactant delivery or product extraction .

Eur J Biochem, 1999 Feb, 260(1), 284 - 90
Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p; Vollmer P et al.; Small GTPases of the Ypt/Rab family are regulators of vesicular protein trafficking in exo-and endocytosis . GTPase-activating proteins (GAP) play an important role as down regulators of GTPases . We here report the molecular cloning of a novel GAP-encoding gene (GYP7, for GAP for Ypt7) by high expression from a Saccharomyces cerevisiae genomic library . The GYP7 gene encodes a hydrophilic protein with a molecular mass of 87 kDa . Comparison of its primary sequence with that of the three other known GAPs for transport GTPases, the yeast Gyp6 and Gyp1 proteins and the Rab3A-GAP from rat brain, shows similarity between the yeast GAPs only . Like GYP6 and GYP1, GYP7 is not essential for yeast cell viability . Gyp7p was able to most effectively accelerate the intrinsic GTPase activity of Ypt7p . It was also active, but to a lesser extent, on Ypt31p, Ypt32p and Ypt1p . Ypt6p, Sec4p and the human H-Ras protein did not serve as substrates . We also report the identification and cloning of a gene from the dimorphic yeast Yarrowia lipolytica that encodes a protein whose primary structure and biochemical activity are significantly related to those of Gyp7p from baker's yeast.

Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1467 - 70
Crystallization and preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from Sacchromyces cerevisiae (baker's yeast); Gilboa R et al.; Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by which metabolic intermediates move between enzyme active sites during multi-step degradation or synthesis . An important enzyme for which a detailed three-dimensional structural analysis has been initiated is yeast glyceraldehyde 3-phosphate dehydrogenase (yGAP-DH) . This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinucleotide (NAD+) as cofactor . In this report, the crystallization and preliminary crystallographic characterization of several crystal forms of yGAP-DH are described . Of the five distinct crystal forms, the most suitable was found to contain the holo-enzyme, and the crystals were grown by the vapor-diffusion method using polyethylene glycol 6000 as precipitant, sodium acetate as buffer (pH 4.6), and NAD+ and dithiothreitol as additives . The crystals belong to the orthorhombic space group P21212, with cell dimensions of a = 87.33, b = 96.11 and c = 115.34 A . These crystals are mechanically strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 A resolution . A full 2.1 A resolution diffraction data set (98% completion) has been measured . The three-dimensional structures of related GAP-DH enzymes from several other sources have been determined and reported, and are available for a molecular replacement structure solution.

Biotechnol Appl Biochem, 1999 Apr, 29 ( Pt 2), 151 - 6
Rapid screening of textile dyes employed as affinity ligands to purify enzymes from yeast; Raya-Tonetti G et al.; A rapid method for screening potential dye ligands for use in affinity chromatography is described . Textile dyes were non-covalently coupled to a cross-linked polysaccharide Sepharose(R) matrix . Yeast alcohol dehydrogenase (ADH) was used as the model protein for evaluating the screening system . A homogenate from baker's yeast was used as the crude source of enzyme . Batchwise adsorption and elution were used to evaluate the individual dyes . The influence of pH and ionic strength in the binding and elution steps was evaluated . Batch isotherms were used to evaluate parameter characteristics . Experimental data obtained were fitted to Langmuir isotherms to determine the maximum binding capacity and the dissociation constant for each dye evaluated in this study . A dynamic binding capacity of 107.6 units of ADH/g of resin was determined for Procion Turquoise MXG dye by frontal analysis . Specific elution with NAD+ and non-specific elution with 50 mM Tris/HCl buffer, pH 8.5, were tested when Procion Turquoise MXG was used, giving purification factors of 53.5 and 4.4 respectively . This screening technique is inexpensive and can be performed in a few hours . It was possible to predict the performance of different reactive dyes in this way, and the influence of pH and salt on the binding behaviour was demonstrated.

Lett Appl Microbiol, 1999 Feb, 28(2), 148 - 52
Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding; Vincent SF et al.; Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity . To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology . Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture . Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

Biol Pharm Bull, 1999 Jan, 22(1), 21 - 5
S-(1,2-Dicarboxyethyl)glutathione in yeast: partial purification of its synthesizing enzyme; Tsuboi S et al.; S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) was found in Saccharomyces cerevisiae, but not in bacterial species nor in a unicellular alga (Acetabularia acetabulum) . The enzyme that catalyzes condensation of L-malate and glutathione (GSH) to form DCE-GS was partially purified from baker's yeast . It had a molecular mass of 49 kDa and was monomeric and the Km values were 2.2 and 1.4 mM for L-malate and GSH, respectively . The enzyme had a pH optimum of 7.5 . DCE-GS levels in yeast cells were significantly higher in aerobic cultures than in anaerobic ones . DCE-GS was synthesized in cells cultured between 20 and 35 degrees C.

J Immunol, 1999 Feb 1, 162(3), 1590 - 6
Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis; Wilson R et al.; Unlike vertebrates, insects do not have an Ab-based nonself recognition system, and must rely totally on innate immunity to defend themselves from microbial invaders . The most likely candidates for recognizing foreign material in insects are the lectins, which have already been shown to be important in mammalian innate immunity . The hemolymph of the cockroach, Blaberus discoidalis, contains multiple lectins, designated BDL1, BDL2, BDL3, and GSL (beta-1,3-glucan-specific lectin), two of which, namely BDL1 and GSL, have close similarities to acute phase reactants . These endogenous molecules, as well as Con A, wheat germ agglutinin, and Helix pomatia agglutinin, have been shown to induce an enhanced phagocytic response by B . discoidalis plasmatocytes . This effect is related to the carbohydrates presented on the surface of the microorganism and to the sugar specificities of the lectins . Thus, the mannose-specific lectins, BDL1 and Con A, both increase the phagocytosis of baker's yeast and Escherichia coli, whereas the N-acetyl-D-glucosamine/N-acetyl-D-galactosamine-specific lectins, BDL2, wheat germ agglutinin, and H . pomatia agglutinin, induce the phagocytosis of Bacillus cereus and E . coli . GSL, specific for beta-1,3-glucan, and the N-acetyl-D-galactosamine-specific BDL3, only enhance the phagocytosis of yeast and B . cereus, respectively . Phenylthiourea, an inhibitor of the prophenoloxidase system, caused either total, partial, or no inhibition of the lectin-induced increase in phagocytosis, indicating that this immune enhancement results, in some cases, from at least two closely linked mechanisms . These results show that the endogenous lectins in the cockroach hemolymph are capable of acting as nonself recognition molecules for a wide range of microorganisms, and thus obviate the necessity of Abs in these animals.

Biotechnol Prog, 1999 Jan, 15(1), 98 - 104
An unusual reversible sol-Gel transition phenomenon in organogels and its application for enzyme immobilization in gelatin membranes
Fadnavis NW, Koteshwar K.
An unusual phenomenon is observed for gelatin solutions (1.7-6.8%) in the microemulsion system of 0.3 M bis(2-ethylhexyl)sulfosuccinate sodium salt in isooctane and 14.5% distilled water . Highly viscous gels obtained at temperatures above 30 degreesC become free-flowing liquids at low temperatures (5-10 degreesC) . This reversible temperature-dependent sol-gel transition phenomenon is used to immobilize several enzymes, such as lipase from Candida rugosa, alcohol dehydrogenase from baker's yeast, mandelonitrile lyase from Sorghum bicolor, and horseradish peroxidase in the gelatin matrix by solubilizing the enzyme in a microemulsion-based gelatin solution at low temperature (<5 degreesC) and then cross-linking with glutaraldehyde . The enzymes retain 70-80% of their activity after immobilization and can be used in biotransformations in organic solvents without any changes in enantioselectivity . This work provides a unique low-temperature technique for enzyme immobilization in a biocompatible gelatin matrix with a great flexibility of size and shape.

Appl Environ Microbiol, 1999 Feb, 65(2), 680 - 5
Genetic evidence that high noninduced maltase and maltose permease activities, governed by MALx3-encoded transcriptional regulators, determine efficiency of gas production by baker's yeast in unsugared dough; Higgins VJ et al.; Strain selection and improvement in the baker's yeast industry have aimed to increase the speed of maltose fermentation in order to increase the leavening activity of industrial baking yeast . We identified two groups of baker's strains of Saccharomyces cerevisiae that can be distinguished by the mode of regulation of maltose utilization . One group (nonlagging strains), characterized by rapid maltose fermentation, had at least 12-fold more maltase and 130-fold-higher maltose permease activities than maltose-lagging strains in the absence of inducing sugar (maltose) and repressing sugar (glucose) . Increasing the noninduced maltase activity of a lagging strain 13-fold led to an increase in CO2 production in unsugared dough . This increase in CO2 production also was seen when the maltose permease activity was increased 55-fold . Only when maltase and maltose permease activities were increased in concert was CO2 production by a lagging strain similar to that of a nonlagging strain . The noninduced activities of maltase and maltose permease constitute the largest determinant of whether a strain displays a nonlagging or a lagging phenotype and are dependent upon the MALx3 allele . Previous strategies for strain improvement have targeted glucose derepression of maltase and maltose permease expression . Our results suggest that increasing noninduced maltase and maltose permease levels is an important target for improved maltose metabolism in unsugared dough.

Inflamm Res, 1998 Dec, 47(12), 476 - 81
Antiinflammatory potency of dehydrocurdione, a zedoary-derived sesquiterpene; Yoshioka T et al.; OBJECTIVE AND DESIGN: Dehydrocurdione, a sesquiterpene isolated from zedoary, was tested for in vivo and in vitro antiinflammatory actions . MATERIALS: Analgesic effect was tested in ICR mice by the acetic acid-induced writhing method . Antipyretic effect was studied in Sprague-Dawley rats treated with baker's yeast . Antiinflammatory activities were tested in Wistar rats with carrageenan-induced paw edema and adjuvant-induced chronic arthritis . In vitro analyses included the capabilities to inhibit cyclooxygenase activity, and to scavenge free radicals as determined by electron paramagnetic resonance (EPR) . RESULTS: Oral administration of dehydrocurdione (40 to 200 mg/kg) mitigated the writhing reflex . induced by acetic acid and the fever elicited by baker's yeast . A higher dose (200 mg/kg) of dehydrocurdione was required to inhibit the carrageenan-induced paw edema . Oral administration of dehydrocurdione at 120 mg/kg/day for 12 days significantly reduced chronic adjuvant arthritis . Unlike indomethacin (IC50: 0.1 microM), dehydrocurdione showed minimal cyclooxygenase inhibition . However, dehydrocurdione (100 microM to 5 mM) significantly reduced free radical formation from hydrogen peroxide and ferrous iron determined by EPR spectrometry using 5,5'-dimethyl-1-pyrroline-N-oxide as a spin trap agent . CONCLUSION: In addition to the well-known effect of zedoary as a stomachic, dehydrocurdione, the major component of Curcuma zedoaria Roscoe has antiinflammatory potency related to its antioxidant effect.

Skin Pharmacol Appl Skin Physiol, 1998, 11(4-5), 250 - 7
Dehydrogenation of 3-phenoxybenzyl alcohol in isolated perfused rabbit skin, skin homogenate and purified dehydrogenases; Bast GE et al.; The formation of 3-phenoxybenzoic acid from 3-phenoxybenzyl alcohol was determined in (a) rabbit ears, single-pass perfused with a protein-free buffer, pH 7.4; (b) the microsomal fraction and its supernatant from homogenized rabbit skin; and (c) purified alcohol dehydrogenase from horse liver and baker's yeast . The inhibition of product formation in (a) was about 60% by various 4-methylpyrazole concentrations, but metyrapone had no effect . Following ultracentrifugation, only the supernatant of homogenized skin showed product formation (apparent Vmay: 32 pmol/min per cm2 skin; apparent Km: 64 microM) . 3-Phenoxybenzyl alcohol and ethanol dehydrogenation was similar by alcohol dehydrogenase from horse liver (apparent Km: 0.7 vs . 0.4 mM; apparent Vmax: 0.3 vs . 0.2 U/ microg protein) . In baker's yeast, the apparent Km of 3-phenoxybenzoic acid formation was several times larger than that for ethanol dehydrogenation . The KI of 4-methylpyrazole for alcohol dehydrogenase from horse liver was 0.6 (3-phenoxybenzyl alcohol) vs . 0.04 microM (ethanol) . The KI for ethanol in baker's yeast was 470 microM . In conclusion dehydrogenation is an important metabolic pathway in the skin for xenobiotics with an aliphatic alcohol at a side chain.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 347 - 57
Yeast sphingolipids; Dickson RC et al.; Many advances in our understanding of fungal sphingolipids have been made in recent years . This review focuses on the types of sphingolipids that have been found in fungi and upon the genes in Saccharomyces cerevisiae, the common baker's yeast, that are necessary for sphingolipid metabolism . While only a small number of fungi have been examined, most contain sphingolipids composed of ceramide derivatized at carbon-1 with inositol phosphate . Further additions include mannose and then other carbohydrates . The second major class of fungal sphingolipids is the glycosylceramides, having either glucose or galactose attached to ceramide rather than inositol phosphate . The glycosylceramides sometimes contain additional carbohydrates . Knowledge of the genome sequence has expedited identification of S . cerevisiae genes necessary for sphingolipid metabolism . At least one gene is known for most steps in S . cerevisiae sphingolipid metabolism, but more are likely to be identified so that the 13 known genes are likely to grow in number . The AUR1 gene is necessary for addition of inositol phosphate to ceramide and has been identified as a target of several potent antifungal compounds . This essential step in yeast sphingolipid synthesis, which is not found in humans, appears to be an excellent target for the development of more effective antifungal compounds, both for human and for agricultural use.

Bioorg Med Chem Lett, 1998 Jun 2, 8(11), 1403 - 6
The first conversion of camptothecin to (S)-mappicine by an efficient chemoenzymatic method; Das B et al.; Camptothecin has been converted for the first time to (S)-mappicine via mappicine ketone, which is the sole product of the microwave irradiation of camptothecin . Baker's yeast reduction of mappicine ketone yielded (S)-mappicine in high optical purity.

Prikl Biokhim Mikrobiol, 1998 Sep-Oct, 34(5), 588 - 91
{The use of pancreatic RNAse in the production of baker's yeast}; Lutskaia AIu et al.; Microdoses of a preparation of pancreatic RNase were shown to stimulate the growth of Saccharomyces cerevisiae yeast . The effect was only retained at a certain inoculum/enzyme preparation ratio . Industrial tests of the preparation showed that the addition of RNase to the first reservoirs for culture accumulation (an inoculator and a seeding device) increased the yield of bakers' yeast and improved their quality.

Biotechnol Prog, 1998 Nov-Dec, 14(6), 931 - 42
Comparison of ultra- and microfiltration in the presence and absence of secondary flow with polysaccharides, proteins, and yeast suspensions; Gehlert G et al.; The purpose of this research was to show that controlled centrifugal instabilities-Dean vortices-produced by solutions and suspensions from typical biotechnology applications flowing through curved tubes can be used to reduce concentration polarization and/or fouling in pressure-driven ultrafiltration (UF) and microfiltration (MF) processes . Experiments were conducted to (i) evaluate the ultrafiltration performance of hollow fiber membranes in linear and helical configurations with dextran (low fouling) and bovine serum albumin (high fouling) solutions and (ii) compare the performance of linear and helical coiled UF hollow fiber modules with that of similar MF modules using baker's and beer yeast (Saccharomyces cerevisiae) suspensions as feed . Both constant transmembrane pressure (TMP) and constant permeation flux (J) experiments were utilized here . The membrane material was polyether sulfone . For the ultrafiltration experiments, the helical module performed consistently better than the linear module with dextran T500 and BSA solutions, resulting in performance improvements (helical versus linear) from 20 to 200% and up to 85%, respectively . For the comparative experiments between UF and MF, the helical module again performed better than the linear module for low concentration baker's yeast suspensions (0.5-1% dry wt) . At constant TMP, the flux improvements for UF were 30-120%, while at constant J, the capacity or loading was 4.5 times higher for the UF as compared to the MF membrane . At high beer yeast concentrations (5.1-6.8% dry wt), although flux improvements were not observed between the linear and helical modules for UF, the UF fluxes were 72% higher than that obtained with MF . Also, for MF, with the same high beer yeast concentrations, the helical module exhibited 30-90% higher fluxes than that obtained with the linear module . At constant flux (117-137 L m-2 h-1) and intermediate baker's yeast concentrations (0.65-2.7% dry wt), 10-20 times the capacity was obtained for the helical over the linear module . Yeast cells were the dominant foulant . For constant UF flux (70 L m-2 h-1) experiments at high beer yeast concentrations ((4.3-7.7) x 10(7) cells/mL or 5.1-6.8% dry wt), the capacity (loading) for the helical module was 10 times that of the linear module . Again, the yeast cells were the dominant foulant . A new mass-transfer correlation for ultrafiltration of dextran T500 solutions for laminar flow in a helical hollow fiber module was obtained, viz . Sh = 0.173Re0.55Sc0.33(a/Rc)0.07.

J Biotechnol, 1998 Oct 19, 65(1), 23 - 35
Principal component ANN for modelling and control of baker's yeast production; Kurtanjek Z; Modelling of baker's yeast production by the principal component based artificial neural networks (ANN) is presented . The models are derived for their application in adaptive control of fermentation by the internal model control (IMC) method . Modelling data are from industrial production in 40 m3 deep jet bioreactor and from computer simulations . The modelling effort is focused on selection of ANN structure and model verification . Principal component analysis of process variables results in projection of patterns to a space of low dimension, which enables determination of ANN structure, removes data colinearity and random components of measurement signals, and model degradation by over-training is eliminated . In view of IMC application, the models for prediction of the controlled variable (ethanol partial pressure) and the inverse model for manipulative variable (molasses feed rate) are determined . The models are tested for their predictability in the time horizon from 1 to 20 min . ANN models are derived with average relative errors for untrained patterns in the range from 1 to 10%.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1998 Oct, 120(3), 373 - 81
Effects of different protective agents on the phototoxicity of fluoranthene to Daphnia magna; Wernersson AS et al.; Some compounds, accumulated by organisms, are transformed into toxic forms when irradiated with UV light . The polycyclic aromatic hydrocarbon (PAH) fluoranthene is one such compound of environmental importance . In this study on Daphnia magna, fluoranthene toxicity increased significantly after a 2 h exposure to solar-simulating UV light, if organisms were allowed to accumulate the substance for 24 h prior to irradiation . Since no enhanced toxicity was observed if the solutions were irradiated before the daphnids were added and only a slight decrease in toxicity was observed if the daphnids were transferred to pure dilution water prior to exposure, it was concluded that the acute phototoxicity of fluoranthene was predominantly due to photoactivation of accumulated or adsorbed molecules . Thus, the enhanced toxicity of fluoranthene by UV light is thought to act through the production of either singlet oxygen or free radicals . Possible effects of different protective agents (antioxidants, free radical scavengers and UV-screening compounds) were examined in two cultured populations of Daphnia magna . One population received a synthetic diet and the other dried baker's yeast . The yeast-fed population became progressively more sensitive to the photoinduced toxicity of fluoranthene, and after 14 days it was significantly more sensitive than the population that received the synthetic feed . It was not obvious whether any of the additives influenced the UV-induced toxicity significantly, although, alpha-tocopherol, a known antioxidant, was the best candidate.

Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13597 - 602
Large-scale protein structure modeling of the Saccharomyces cerevisiae genome; Sanchez R et al.; The function of a protein generally is determined by its three-dimensional (3D) structure . Thus, it would be useful to know the 3D structure of the thousands of protein sequences that are emerging from the many genome projects . To this end, fold assignment, comparative protein structure modeling, and model evaluation were automated completely . As an illustration, the method was applied to the proteins in the Saccharomyces cerevisiae (baker's yeast) genome . It resulted in all-atom 3D models for substantial segments of 1,071 (17%) of the yeast proteins, only 40 of which have had their 3D structure determined experimentally . Of the 1,071 modeled yeast proteins, 236 were related clearly to a protein of known structure for the first time; 41 of these previously have not been characterized at all.

J Chromatogr A, 1998 Sep 25, 822(1), 59 - 66
Chromatographic determination of flavin derivatives in baker's yeast; Gliszczynska A et al.; The presence of flavin derivatives in baker's yeast was tested by high-performance liquid chromatography and thin-layer chromatography . In yeast samples, besides flavin adenine dinucleotide and flavin mononucleotide, small amounts of riboflavin and traces of 10-formylmethylflavin have been found . Total amount of flavins was calculated to be 17.9 +/- 2.9 micrograms/g of fresh yeast . The distribution of flavin adenine dinucleotide, flavin mononucleotide, riboflavin and 10-formylmethylflavin in total flavin content were estimated to be 71.5%, 25.8%, 1.7% and below 0.05%, respectively . In some samples we have additionally detected small amounts (0.8% of total flavins) of new flavin derivative which has been identified as 4',5'-riboflavin cyclic phosphate by means of its chromatographic and chemical behaviour . This compound seems to be a product of flavin adenine dinucleotide degradation and probably has been earlier mistaken for flavin mononucleotide . Its formation is dependent on pH conditions.

Hum Reprod, 1998 Oct, 13(1O), 2941 - 9
The phagocytic activity of human first trimester extravillous trophoblast; Choy MY et al.; It has been suggested previously that phagocytic activity in the human placenta is confined to cells of the macrophage lineage . However, earlier studies were hampered by the paucity and poor viability of cells inherent in primary trophoblast cell cultures, contamination by other cell types which themselves have phagocytic activity, lack of reliable markers of trophoblasts, and by limitations of methods available to demonstrate unequivocally the internalization of particulate material . We have overcome these limitations by using: (i) DNA transfection to provide unlimited supplies of pure trophoblast cell lines; (ii) human placental lactogen as a marker unique to trophoblast; and (iii) confocal microscopy to demonstrate unequivocally the intracellular locality of phagocytosed material . We found that both untransfected primary culture extravillous trophoblast cells, as well as the cell lines, had the capacity to phagocytose sheep red blood cells, Staphylococcus aureus and baker's yeast cells, and that this activity was inhibited by cytochalasin B and by culture at 4 degrees C . Phagocytic activity in trophoblast cells was less avid than that seen in a professional phagocyte . In physiological and pathological situations where tissue remodelling occurs, such as the rapid turnover in the periodontal ligament or during inflammation, epithelial cells and other cells that are not considered professional phagocytes actively phagocytose components of the extracellular matrix . We postulate that phagocytosis by human trophoblasts may play an important role in the extensive tissue remodelling that occurs during trophoblastic invasion of the decidua.

Appl Environ Microbiol, 1998 Nov, 64(11), 4226 - 33
Effect of specific growth rate on fermentative capacity of baker's yeast; Van Hoek P et al.; The specific growth rate is a key control parameter in the industrial production of baker's yeast . Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature . In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated . At specific growth rates (dilution rates, D) below 0.28 h-1, glucose metabolism was fully respiratory . Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol . g of biomass-1 . h-1 at D = 0.40 h-1 . A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h-1) . This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.025 h-1 to 20.5 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.28 h-1 . At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.40 h-1 . The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts . Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity . These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.

Mol Cell Biol, 1998 Nov, 18(11), 6340 - 52
Heat shock element architecture is an important determinant in the temperature and transactivation domain requirements for heat shock transcription factor; Santoro N et al.; The baker's yeast Saccharomyces cerevisiae possesses a single gene encoding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability . Yeast HSF (yHSF) contains two distinct transcriptional activation regions located at the amino and carboxyl termini . Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF . The results described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1 HSE (HSE1) functions to limit the magnitude of CUP1 transcriptional activation in response to heat and oxidative stress . The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA . Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the transcriptional activation of CUP1, via HSE1, in response to stress . In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1 . Limited proteolysis assays show that HSF adopts a distinct protease-sensitive conformation when bound to the CUP1 HSE1, providing evidence that the HSE influences DNA-bound HSF conformation . Together, these results suggest that CUP1 regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs . Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those of CUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.

Prog Nucleic Acid Res Mol Biol, 1998, 61, 133 - 79
Genetic regulation of phospholipid metabolism: yeast as a model eukaryote; Henry SA et al.; Baker's yeast, Saccharomyces cerevisiae, is an excellent and an increasingly important model for the study of fundamental questions in eukaryotic cell biology and genetic regulation . The fission yeast, Schizosaccharomyces pombe, although not as intensively studied as S . cerevisiae, also has many advantages as a model system . In this review, we discuss progress over the past several decades in biochemical and molecular genetic studies of the regulation of phospholipid metabolism in these two organisms and higher eukaryotes . In S . cerevisiae, following the recent completion of the yeast genome project, a very high percentage of the gene-enzyme relationships in phospholipid metabolism have been assigned and the remaining assignments are expected to be completed rapidly . Complex transcriptional regulation, sensitive to the availability of phospholipid precusors, as well as growth phase, coordinates the expression of the structural genes encoding these enzymes in S . cerevisiae . In this article, this regulation is described, the mechanism by which the cell senses the ongoing metabolic activity in the pathways for phospholipid biosynthesis is discussed, and a model is presented . Recent information relating to the role of phosphatidylcholine turnover in S . cerevisiae and its relationship to the secretory pathway, as well as to the regulation of phospholipid metabolism, is also presented . Similarities in the role of phospholipase D-mediated phosphatidylcholine turnover in the secretory process in yeast and mammals lend further credence to yeast as a model system.

Vopr Pitan, 1998, (3), 18 - 22
{Effect of a bioactive food supplement from selenium enriched baker's yeast autolysate on the status of the intestinal barrier in rats with anaphylaxis}; Golubkina NA et al.; Rat's intestinal barrier permeability disturbed in consequence of intestinal anaphylaxis reaction was almost completely normalized in animals fed with baker's yeast autolysate "Vitasil" enriched with selenium on a level of 3 mg Se/day during 29 days . These rats showed in comparison to Se-unsupplemented animals a significant elevation of Se level in red blood cells and plasma together with a decrease of intestinal mucosal TCA-soluble thiol compounds . Urinary Se excretion was significantly elevated in comparison to unsensitized rats both in Se-supplemented and unsupplemented animals with anaphylaxis . It's concluded that "Se-Vitasil" may be successfully used in antioxidative therapy of food allergy, malabsorption, inflammatory bowel diseases and intestinal infection.

Chromosoma, 1998 Sep, 107(4), 247 - 54
Meiotic pairing and segregation of translocation quadrivalents in yeast; Loidl J et al.; Meiotic pairing and segregation were studied in three different heterozygous reciprocal translocation strains of the baker's yeast, Saccharomyces cerevisiae . Pachytene translocation quadrivalents were identified by a combination of immunofluorescence and fluorescence in situ hybridization and the karyotypes of meiotic products were determined by pulsed-field gel electrophoresis . The translocations differed with respect to the relative sizes of the chromosomes involved and the positions of translocation breakpoints, and produced translocation quadrivalents of widely different shapes . This allowed us to study the influence of the morphology of quadrivalents on their segregation behaviour . In all cases alternate predominated over adjacent segregation . 3:1 disjunction of chromosomes was more frequent when translocation breakpoints were close to the centromeres . If a translocation breakpoint was distant from the centromere, the occurrence of an intervening chiasma influenced the pattern of segregation . In general, quadrivalent formation and segregation resembled the behaviour of translocation heterozygotes in most higher eukaryotes . We therefore conclude that, although chromosome condensation does not occur in yeast metaphase, centromere orientation and chromosome disjunction are governed in a way similar to that of higher eukaryotes.

Electrophoresis, 1998 Jul, 19(10), 1788 - 92
Detection of enzymes active on various beta-1,3-glucans after denaturing polyacrylamide gel electrophoresis; Trudel J et al.; Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing beta-1,3-glucans embedded as substrate . Lentinan, curdlan, paramylon, baker's yeast alkali-insoluble glucan, baker's yeast alkali-soluble glucan and carboxymethyl (CM)-pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying beta-1,3-glucanase activities . Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5-triphenyltetrazolium chloride (TTC) . For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures . No Lyticase enzyme was detected in either detection procedures for all tested substrates . For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM-pachyman as substrates . In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures . This was the same for the barley leaf extract (32, 36 and 39 kDa) . The Driselase extract showed one 42 kDa band . Many enzymes active on beta-1,3-glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS-PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers . However, some enzymes are only detected when proteins are denatured without reduction . Finally, the use of various polymeric beta-1,3-glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.

Mol Biol Evol, 1998 Aug, 15(8), 931 - 42
Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment; Brown CJ et al.; When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate . We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth . Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition . These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6 . Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high-affinity hexose transport loci, HXT6 and HXT7 . Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6 . We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.

Biotechnol Prog, 1998 Jul, 14(4), 588 - 93
Asymmetric reduction of acetophenone with calcium-alginate-entrapped Baker's yeast in organic solvents
Griffin DR, Yang F, Carta G, Gainer JL.
Baker's yeast cells entrapped in alginate beads are shown to catalyze reactions in organic solvents when a cofactor regeneration scheme is implemented . This study focused on the reduction of acetophenone to 1-phenylethanol, using baker's yeast as well as a cosubstrate to regenerate the cofactor . The product is a chiral alcohol, and it was desired to maintain a high enantiomeric excess . The effects of parameters such as the addition of a cosubstrate, water content, fermentation time, buffer pH, and bead diameter have been investigated . Such a general process may be quite useful when single enantiomers are needed, as well as for the production of other chemicals.

Appl Environ Microbiol, 1998 Aug, 64(8), 2952 - 7
Characteristics and efficiency of glutamine production by coupling of a bacterial glutamine synthetase reaction with the alcoholic fermentation system of baker's yeast; Wakisaka S et al.; Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker's yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation . Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors . By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration . The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization . Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield . However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures . A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol . In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.

J Inorg Biochem, 1998 Apr, 70(1), 63 - 9
Anti-inflammatory properties of diclofenac transition metalloelement complexes; Konstandinidou M et al.; As part of our research into understanding drug-metalloelement interactions, we have prepared complexes of Cu(II), Co(II), Ni(II), Mn(II), Fe(II), Fe(III), and Pd(II) with Diclofenac, in order to investigate their anti-inflammatory activity . Their inhibitory effects on rat or mouse paw edema induced by Carrageenan, Con-A, Nystatin, and Baker's yeast were compared with those of Diclofenac . Furthermore, the action of Diclofenac's metalloelement complexes on phagocytosis of yeast by rat peritoneal cells, as well as the capacity of some of the metalloelement complexes to inhibit lipid peroxidation of liver microsomal membranes was also investigated . These complexes exhibited a strong inhibitory effect on Carrageenan-, ConA-, and Nystatin-induced edemas (35-80% inhibition) comparable to the inhibition caused by Diclofenac (61-76% inhibition) . Furthermore, complexes with Co(II), Ni(II), Pd(II), and Mn(II) were found to have an anti-inflammatory profile (35-50% inhibition) superior to diclofenac (17% inhibition) when inhibiting inflammations due to Baker's yeast, the mechanism of which involves mainly the activation of lipoxygenase and/or complement system . Complexes of Ni(II) and Pd(II), which showed significant inhibition of induced-edemas in rats, were also tested in mice at lower and higher doses and showed a significant dose-dependent inhibition of edemas in mice . Some of these complexes also interfere with in vitro phagocytosis . The most active anti-inflammatory complexes Co(II), Pd(II), and Ni(II), also offered significant protection against lipid peroxidation in vitro, acting as antioxidant compounds, properties that are not demonstrated by Diclofenac . Finally, it is noted that almost all metalloelement complexes of Diclofenac showed high anti-inflammatory activity at molecular concentrations much lower than that of Diclofenac . From the present study it is suggested that the anti-inflammatory activity of Diclofenac is enhanced by the formation of coordination complexes with transition metalloelements.

Carbohydr Res, 1998 Feb, 307(1-2), 83 - 95
Synthesis of alpha-glucosidase inhibitors: kojibiose-type pseudo-disaccharides and a related pseudotrisaccharide; Ogawa S et al.; Two kojibiose-type pseudo-disaccharides and a trisaccharide, containing a 5-amino-1,2,3,4-cyclopentanetetrol derivative or valienamine, linked by way of nitrogen bridges to the sugar residues, have been designed and synthesized as processing alpha-glucosidase I inhibitors . Synthesis of the pseudo-disaccharides was carried out starting from the coupling products of the sugar isothiocyanates and an aminocyclitol, respectively, by cyclization with mercury(II) oxide to the cyclic isoureas and subsequent deprotection . Pseudokojibiose was prepared in a poor yield by reaction of a protected valienamine and a sugar epoxide, followed by deprotection . Although the pseudooligosaccharides are all strong inhibitors of alpha-glucosidase (baker's yeast), they did not have any inhibitory potency against either sucrase isomaltase (rat intestine) or processing alpha-glucosidase (rat liver microsomes).

Am J Physiol, 1998 Jul, 275(1 Pt 1), G130 - 7
Kinetics of particle uptake in the domes of Peyer's patches; Beier R et al.; Uptake of particulate antigenic matter, including microorganisms and vaccine-bearing microspheres, by the intestinal mucosa takes place in the domes of the gut-associated lymphoid tissues and is achieved by membranous (M) cells, which continuously transport particles from the lumen to the underlying tissue where some particle components initiate immune reactions . Using yeast as tracer, we investigated the kinetics of particle uptake in the Peyer's patches of pigs . A suspension of baker's yeast (Saccharomyces cerevisiae) was injected into the gut lumen of anesthetized minipigs; the position of yeast cells in the tissue was determined after 1, 2.5, 4, and 24 h using fluorescence light- and thin-section electron microscopy . After 1 h, 18.5% of all M cells had taken up or were in close contact with yeast cells . The intercellular space of the epithelium contained a maximum of 60.8% of all yeast cells found in the tissue after 2.5 h, but only 1.3% had been phagocytosed by macrophages . After 4 h most yeast cells (77.8%) were found beneath the basal lamina, and most of these (89%) were found in macrophages . No yeast cells were detected in the Peyer's patch domes 24 h after application . The data show that transcytosis of yeast particles (3.4 +/- 0.8 micron in diameter) by M cells takes <1 h . Without significant phagocytosis by intraepithelial macrophages, the particles migrate down to and across the basal lamina within 2.5-4 h, where they quickly get phagocytosed and transported out of the Peyer's patch domes.

Proteins, 1998 Jun 1, 31(4), 445 - 52
Electrostatics, allostery, and activity of the yeast chorismate mutase; Lin SL et al.; The predicted active site of chorismate mutase of baker's yeast Saccharomyces cerevisiae has been studied by continuum electrostatics, molecular surface/volume calculations, and molecular modeling . Our study shows that despite being subject to an allosteric transition, the enzyme's active-site pocket neither decreased in volume nor deformed significantly in shape between the active R state and the inactive T state . We find that the polar atmosphere in the pocket is responsible for the enzyme's affinity . A single amino acid, Glu23, can adequately account for the atmospheric variation . This residue swings into the active-site pocket from the R state to the T state . In the R state, Glu23 on helix H2 doubly pairs with Arg204 and Lys208 of H11, which is packed against H2 . In the T state, a slide occurs between H11 and H2 such that Glu23 can no longer interact with Lys208 and competes with Asp24 for interacting with Arg204 . Consequently, Glu23 is found in the T state to couple with Arg157, an active-site residue critical to substrate binding . The tandem sliding of H11 in both monomers profoundly changes the interactions in the dimer interface . The loop between H11 and H12 demonstrates the largest conformational change . Hence, we establish a connection between the allosteric transition and the activity of the enzyme . The conformational change in the transition is suggested to propagate into the active-site pocket via a series of polar interactions that result in polarity reversal in the active-site pocket, which regulates the enzyme's activity.

Appl Microbiol Biotechnol, 1998 Apr, 49(4), 377 - 81
Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent; Kanda T et al.; To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization . In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells . It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection . Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation . Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 735 - 9
Trehalose 6-phosphate production with energy coupling fermentation by yeast cells; Doi J et al.; We tried a method for the production of trehalose 6-phosphate (T6P) with energy-coupling fermentation by baker's yeast . T6P was produced in a reaction mixture containing glucose, 5'-UMP, MgSO4, inorganic phosphate, and dried cells of baker's yeast as the enzyme preparation, T6P was isolated from the reaction mixture and identified by TLC, HPLC, GC-MS, and enzymatic methods . The reaction conditions suitable for T6P production were investigated . The formation of T6P and its precursors, glucose 6-phosphate and UDPglucose, at various pHs and concentrations of substrates was examined . Accumulation of T6P was maximum with a reaction mixture containing 1 M glucose, 20 mM 5'-UMP, 20 mM MgSO4, 400 mM sodium phosphate buffer (pH 6.2), and 100 mg/ml dried cells of baker's yeast shaken at 37 degrees C for 6 h . The yield of T6P as a percentage of glucose was 11% (mol/mol) under these reaction conditions.

Biochemistry (Mosc), 1998 Mar, 63(3), 303 - 11
Dissociative thermal inactivation, stability, and activity of oligomeric enzymes; Poltorak OM et al.; Results of kinetic studies on dissociative thermal inactivation of oligomeric enzymes are discussed . Dissociative thermal inactivation is the process in which the kinetically irreversible protein change is preceded by a reversible stage of oligomer dissociation . In experiments, this is demonstrated by the dependence of inactivation rate on total protein concentration . This paper gives the relations which allow the calculation from experimental data the following physicochemical constants which characterize the stability of oligomeric enzymes: the constant for the rate of irreversible change of monomeric protein, the equilibrium constant for dimer dissociation, and the rate constant for dimer dissociation . The problem of a "conformational lock", the contact between protein globules that admits a multistep destruction of active oligomer and explains the induction period occurring in kinetic thermal inactivation curves, is discussed . The X-ray structural analyses for several dimeric enzymes, i.e., alkaline phosphatase (EC 3.1.3.1) from E . coli, alcohol dehydrogenase (EC 1.1.1.1) from horse liver, and baker's yeast enolase (EC 4.2.1.11), explain why they lose catalytic activity during the dissociation of the protein into monomers and also provide a physically reasonable picture of the structure of their conformational lock . Also, these data support the kinetic scheme used to describe the dissociative inactivation of dimeric enzymes.

Biomed Sci Instrum, 1997, 34, 175 - 80
Modeling and optimising alcohol production by fermentation of dextrose-xylose mixed feed using a fluorosensor; Sundaram S et al.; Dextrose with differing amounts of xylose (mixed substrate medium) has been fermented at 28 degree Celsius with sacchromyces cerevisiae (Baker's Yeast) as seeding . The progress of the reaction was recorded by measuring the fluorescent signal due to intracellular reduced nicotinamide adenine di nucleotide (NADH) present in the cells with a Dr . Ingold (Switzerland) fluorosensor which has an excitation wavelength of 360 nm and measurement wavelength of 450 nm . The concentration of xylose in the xylose-dextrose feed was varied from 0.7% to 5.0% by weight . The optimum concentration of xylose at which the production of alcohol was a maximum was found to be 3.4 percent xylose . The fluorescent voltage data for different concentration of xylose fitted a first order model with an average absolute deviation of less than one percent . Development of this model is useful in design of model predictive controllers.

Biochim Biophys Acta, 1998 Apr 10, 1380(2), 163 - 76
A novel function of enolase from rabbit muscle; an immunoglobulin production stimulating factor; Sugahara T et al.; Rabbit muscle enolase stimulates immunoglobulin production by a human hybridoma line, HB4C5 cells under serum-free condition . IgM productivity of HB4C5 cells was enhanced more than 20-fold by this enzyme at 220 micro/ml . Human peripheral blood lymphocytes were also facilitated their IgM and IgG productivity in the serum-free medium . However, baker's yeast enolase was ineffective to accelerate immunoglobulin production by HB4C5 cells, in spite of the same specific enzymatic activity as rabbit muscle enolase . There were differences in sensitivities against heat treatment and trypsin digestion between IPSF and enzymatic activities of enolase . These results imply that the immunoglobulin production stimulating effect of rabbit muscle enolase is irrelevant to its enzymatic function and reaction products . This fact also means that this enzyme has another function other than enzymatic one in glycolysis . Rabbit muscle enolase enhanced IgM production of transcription-suppressed HB4C5 cells treated with actinomycin D . Cycloheximide treatment of HB4C5 cells was useless to inhibit the expression of immunoglobulin production stimulating activity . However, inhibition of post-transcriptional process by monensin invalidated the activity of enolase . These findings suggest that enolase from rabbit muscle accelerates the steps between translation and post-translational processes to enhance immunoglobulin productivity . In addition, laser confocal microscopic analysis revealed that enolase from rabbit muscle was subsequently incorporated by HB4C5 cells .

Electrophoresis, 1998 Apr, 19(4), 617 - 24
European Functional Analysis Network (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae genome; Dujon B; Less than two yeras after the sequence of its genome was completed, the baker's yeast, Saccharomyces cerevisiae, is a leading organism in the rapidly growing field of functional genomics . Two thousands novel protein coding genes, nearly all of them "orphans", have already been disrupted by the coordinated efforts of a large consortium of European laboratories, EUROFAN, and other initiatives . The mutants are submitted to many specialized functional assays, and studies are performed in parallel at the transcriptome and the proteome levels . With a central repository of mutant yeast strains, and a centralized database, EUROFAN lays the foundations for the future of genomics with yeast serving both as a model and a tool.

Crit Rev Biotechnol, 1998, 18(1), 25 - 83
The use of baker's yeast in the generation of asymmetric centers to produce chiral drugs and others compounds; Pereira Rde S; This review gives a general idea about the importance of chiral carbon in medicine and a way to obtain chiral building blocks with baker's yeast (Saccharomyces cerevisiae) or synthesis of medicaments and other organic compounds . Reactions with these microorganisms are cheaper and easier to be executed than with chemicals (for example, organometallics) . Examples of important and practical reactions catalyzed by enzymes inside Saccharomyces cerevisiae are given and probable mechanisms of action of these enzymes are shown . Although these microbes have advantages such as low cost and availability, there are some cares that are necessary to be taken, like NAD(P)H dosage to choose strains more adequate for reduction reactions.

Cytobios, 1997, 91(364), 7 - 13
The presence/absence of Bcl-2, Ca2+/calmodulin-dependent protein kinase IV, calretinin and p53 in baker's yeast and wheat germ; Kuo WN et al.; After removing the nonspecific immunoreactivities from crude extracts of Saccharomyces cerevisiae and wheat germ by immunoaffinity chromatography, the presence of Ca(2+)-related proteins was tested by Western blot analysis . Immunoreactivity for Bcl-2 was absent in the yeast, whereas the immunoreactivity was evident in wheat germ and remained unchanged after incubation for 4 h with or without actinomycin D . Such incubation caused the degradation of immunoreactive-peptides of Ca2+/calmodulin-dependent protein kinase IV (CaMPK IV) in the yeast and wheat germ . Calretinin and p53 were absent in the yeast and wheat germ . The level of cyclic AMP in the yeast increased 100% after incubation for 30 min with actinomycin D . These results suggest that actinomycin D may not affect intracellular levels of these calcium-related proteins in the yeast and wheat germ, and that Bcl-2 occurs in multicellular eukaryotes . Moreover, the cellular level of CaMPK IV may vary during the onset of cell division and differentiation.

Yeast, 1998 Mar 15, 14(4), 371 - 81
Glycosylation of human alpha 1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts; Kang HA et al.; Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues . To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris . The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S . cerevisiae showed that the recombinant alpha 1-AT secreted in S . cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues . Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT . The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S . cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S . cerevisiae . The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S . cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

Biochem Mol Biol Int, 1998 Mar, 44(3), 565 - 75
Investigation of the yeast mitochondrial unselective channel in intact and permeabilized spheroplasts; Manon S et al.; The existence of an activity corresponding to the nucleotide-induced Yeast Mitochondria Unselective Channel (YMUC2) of isolated mitochondria was investigated in permeabilized and intact spheroplasts of the baker's yeast Yeast Foam . In nystatin-permeabilized spheroplasts, ATP and GDP-beta-S induced a decavanadate-sensitive stimulation of the respiration only under conditions equivalent to those previously reported for isolated mitochondria (low phosphate concentration, presence of a salt) . On intact spheroplasts parallel measurements of respiration rate, {ATP}/{ADP} ratio and mitochondrial transmembrane potential allowed to show that the addition of the glucose analog 2-deoxyglucose decreased the permeability of the inner mitochondrial membrane owing to cellular ATP depletion . This strongly supports the hypothesis that Yeast Mitochondria Unspecific Channel is active in situ and inhibited by cellular {ATP} depletion.

Acta Chem Scand, 1998 Apr, 52(4), 461 - 8
A study of baker's yeast reduction of piperidone-carboxylates; Willert M et al.; The stereoselective baker's yeast reduction of various N-protected piperidone-carboxylic acids have been studied, and the enantioselectivity was found to be widely dependent on whether fermenting or non-fermenting conditions were employed . Thus reaction of N-tert-butoxycarbonyl-4-oxopiperidine-3-carboxylic acid ethyl ester (6) with fermenting baker's yeast gave almost racemic N-tert-butoxycarbonyl-4-hydroxypiperidine-3-carboxylic acid ethyl ester (7), however, with complete diastereoselectivity . Reduction of 6 with non-fermenting yeast gave 7 with a 24-41% enantiomeric excess . Similarly, reduction of N-tert-butoxycarbonyl-3-oxopiperidine-4-carboxylic acid ethyl ester (17) with fermenting baker's yeast gave racemic N-tert-butoxycarbonyl-3-hydroxypiperidine-4-carboxylic acid ethyl ester {(+/-)-18} diastereoselectively . A convenient method for determining the enantiomeric excess of the hydroxypiperidine carboxylic acids derivatives was found in the reaction with Sanger's reagent followed by HPLC on a chiral column.

Clin Exp Allergy, 1998 Jan, 28(1), 45 - 52
Expression of the house dust mite allergen Der p 2 in the baker's yeast Saccharomyces cerevisiae; Hakkaart GA et al.; BACKGROUND AND RESULTS: The major house dust mite allergen Der p 2 was expressed as a recombinant mature protein in the baker's yeast Saccharomyces cerevisiae . The yeast produces the protein fused to the invertase signal peptide, leading to the secretion of Der p 2 as a soluble protein into the culture medium . The signal peptide is hereby cleaved off, resulting in a mature allergen . In this system Der p 2 was produced in 7.6 (+/-2.9) mg/L growth culture . Purification of the recombinant allergen was achieved by a single gel filtration step, resulting in a purity > or = 95% . The yeast-derived Der p 2 was almost indistinguishable from natural Der p 2 with respect to IgE-reactivity and binding to the majority of Der p 2 specific MoAbs -- as was shown in RAST analysis (n = 168) and a sandwich ELISA and RIA analysis, respectively . Recombinant and natural Der p 2 also showed similar biological activity in histamine release assays (n = 4) . CONCLUSION: An expression system for Der p 2 was developed that enables the production of a soluble allergen in the culture supernatant with immunological characteristics similar to the natural allergen . In addition, yeast offers the advantage of the absence of endotoxin in comparison to E . coli . This might facilitate acceptance of recombinant allergens for in vivo applications as immunotherapy or skin-prick testing.

Allergy, 1998 Feb, 53(2), 165 - 72
Epitope mapping of the house-dust-mite allergen Der p 2 by means of site-directed mutagenesis; Hakkaart GA et al.; Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cerevisiae, was used as a tool to determine IgE- and monoclonal antibody (mAb)-binding sites on this allergen . For this purpose, mutant molecules were produced by application of site-directed mutagenesis . The amino-acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds . Charged residues in three predicted antigenic sites (residues 45-48, 67 + 69, and 88-90) were replaced by alanine residues, IgE- and mAb reactivity to these mutants was compared to that to "wild type" Der p 2 . Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb alpha DpX . Mutations in the areas adjacent to this loop (i.e., 67 + 69 and 88-90) had similar effects on this mAb (10- to 20-fold decreases in reactivity were observed), supporting the suggestion that these areas are involved in this antigenic structure . The area of residues 45-48 was shown to be involved in an epitope for mAb 2B12 . The reactivity of mAb 7A1 was influenced by substitutions of residues 45-48 as well as 88-90 . Deletion of the residues spanning cys21-cys27 resulted in decreased reactivity to three mAbs (10E11, alpha DpX, and 7A1) . From these observations, it may be concluded that binding of different mAbs is influenced by the same mutations and that the binding of single mAbs is influenced by two or more mutations scattered over the allergen molecule . These findings can point in two directions: minor amino-acid changes result in disruption of the overall conformation of the allergen, or distant sites are close together in the three-dimensional structure of the allergen . Decreased IgE reactivity was observed with all mutant molecules, varying between patients . The observed effects ranged from 5- to 1000-fold . Deletion of the amino-acid residues spanning cys21-cys27 and cys73-cys78 had the strongest effect on IgE reactivity, where decreases up to 1000-fold were observed . Such mutants might be useful tools to improve the safety of allergen-specific immunotherapy.

Arch Pharm (Weinheim), 1998 Feb, 331(2), 72 - 8
Anti-inflammatory properties of new adamantane derivatives . Design, synthesis, and biological evaluation; Antoniadou-Vyza E et al.; A series of adamantane-containing molecules consisting of two lipophilic centers which are linked by different bridges (oxime esters, oxime ethers, amides, and symmetric alcohols), were designed and synthesized as anti-inflammatory agents . Their anti-inflammatory activity was evaluated as their ability to inhibit phlogistic-induced mouse paw edema . Some of the tested compounds exhibited activity comparable to that of diclofenac, others had a weaker activity, while some oxime esters proved to enhance the inflammatory response . In all cases, activity was dose-dependent . The deacylated compound 10 was found to be the most active of the series, inhibiting inflammation due to Baker's yeast, the mechanism of which involves mainly the activation of lipoxygenase and/or complement systems, a property which is absent from most selective cyclooxygenase only inhibiting non-steroidal anti-inflammatory drugs (NSAIDs).

Izv Akad Nauk Ser Biol, 1997 Nov-Dec, (6), 728 - 34
{The destruction of microscopic organisms by their irradiation with a special form of UHF electromagnetic signals}; Antonov OE et al.; Electromagnetic signals of special form produced by an ultra-high frequency generator were used to destroy various microorganisms: baker's yeast; blue-green alga Nostoc muscorum; mold fungus; and two flagellates, plant flagellate Euglena gracilis and an animal flagellate parasitizing on humans . The control samples before irradiation and experimental samples damaged and destroyed by irradiation were examined on a microscope with a computer system of image analysis . The results are presented as computer graph images.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 181 - 4
Reduction of alkyl (2-oxocyclohexyl)acetates by baker's yeast; Ganaha M et al.; Baker's yeast reduction of methyl and ethyl (2-oxocyclohexyl) acetates proceeded with enantio- and diastereo-selectivity, affording the corresponding (2S)-trans-alcohols (major), (2S)-cis-alcohols (minor), and the unaltered (1S)-ketones with high optical purity.

Biochem J, 1998 Feb 1, 329 ( Pt 3), 433 - 48
Mitochondrial ribosomal proteins (MRPs) of yeast; Graack HR et al.; Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host . Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter . Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae) . To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher . Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences . Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described . The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research . Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans.

J Chromatogr A, 1997 Nov 28, 790(1-2), 83 - 91
Simple high-performance liquid chromatographic method for the determination of all seven vitamin B6-related compounds; Argoudelis CJ; A simple, sensitive, isocratic high-performance liquid chromatographic method has been developed for the separation of all seven vitamin B6-related compounds . The separation is accomplished using an ODS column and a mobile phase of 0.15 M sodium dihydrogenphosphate, adjusted to pH 2.5 with perchloric acid . The concentration of the compounds is determined with a fluorescence detector (excitation, 290 nm; emission, 389 nm) . Isopyridoxal is used as an internal standard . The fluorescence intensity of pyridoxal-5'-phosphate is enhanced by post-column derivatization with sodium bisulfite . All seven compounds are separated in less than 20 min at a flow-rate of 1 ml/min . Applications of this method to yeast cell-free culture media, baker's yeast extract, egg and milk are presented.

Nat Biotechnol, 1997 Dec, 15(13), 1351 - 7
Stress tolerance: the key to effective strains of industrial baker's yeast; Attfield PV; Application of yeasts in traditional biotechnologies such as baking, brewing, distiller's fermentations, and wine making, involves them in exposure to numerous environmental stresses . These can be encountered in concert and sequentially . Yeast exhibit a complex array of stress responses when under conditions that are less than physiologically ideal . These responses involve aspects of cell sensing, signal transduction, transcriptional and posttranslational control, protein-targeting to organelles, accumulation of protectants, and activity of repair functions . The efficiency of these processes in a given yeast strain determines its robustness, and to a large extent, whether it is able to perform to necessary commercial standards in industrial processes . This article reviews aspects of stress and stress response in the context of baker's yeast manufacturing and applications, and discusses the potential for improving the general robustness of industrial baker's yeast strains, in relation to physiological and genetic manipulations.

Appl Environ Microbiol, 1997 Dec, 63(12), 4800 - 6
Lysine-overproducing mutants of Saccharomyces cerevisiae baker's yeast isolated in continuous culture; Gasent-Ramirez JM et al.; Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected . The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC) . The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids . Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene . The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway . This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 245 - 56
1L-myo-inositol-1-phosphate synthase; Majumder AL et al.; 1L-myo-Inositol-1-phosphate synthase catalyzes the conversion of D-glucose 6-phosphate to 1L-myo-inositol-1-phosphate, the first committed step in the production of all inositol-containing compounds, including phospholipids, either directly or by salvage . The enzyme exists in a cytoplasmic form in a wide range of plants, animals, and fungi . It has also been detected in several bacteria and a chloroplast form is observed in alga and higher plants . The enzyme has been purified from a wide range of organisms and its active form is a multimer of identical subunits ranging in molecular weight from 58,000 to 67,000 . The activity of the synthase is stimulated by NH4Cl and inhibited by glucitol 6-phosphate and 2-deoxyglucose 6-phosphate . Structural genes (INO1) encoding the 1L-myo-inositol-1-phosphate synthase subunit have been isolated from several eukaryotic microorganisms and higher plants . In baker's yeast, Saccharomyces cerevisiae, the transcriptional regulation of the INO1 gene has been studied in detail and its expression is sensitive to the availability of phospholipid precursors as well as growth phase . The regulation of the structural gene encoding 1L-myo-inositol-1-phosphate synthase has also been analyzed at the transcriptional level in the aquatic angiosperm, Spirodela polyrrhiza and the halophyte, Mesembryanthemum crystallinum.

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 134 - 41
The phospholipid methyltransferases in yeast; Kanipes MI et al.; In fungal microorganisms including fission yeast, Schizosaccharomyces pombe and baker's yeast, Saccharomyces cerevisiae, two enzymes are required to catalyze the synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) . The genes encoding the class I and class II phospholipid N-methyltransferases (PLMTs) have been cloned from both yeasts . The class II PLMTs catalyze the first methylation step from PE to phosphatidyl-monomethylethanolamine (PMME) . Representatives of the class II type enzymes have been isolated only from yeast and the amino acid sequence of these enzymes contain regions of internal duplication . The class I PLMTs catalyze the last two methylation steps from PMME to PC . The class I PLMTs from both yeasts are homologous to the products of the phosphatidylethanolamine methyltransferase (PEMT) genes isolated from mouse and rat (described in the article by Vance et al . in this volume) . Like the mammalian PEMT gene products, the S . cerevisiae class I enzyme can catalyze all three methylation steps to PC biosynthesis . S . cerevisiae strains, in which either the class II or class I enzyme is deleted, grow slowly in the absence of choline and exhibit low levels of PC . However, in S . pombe, mutants lacking either one of the two PLMTs are choline auxotrophs . Thus, both enzymes are required in S . pombe for maximal growth in the absence of exogenous choline . The S . cerevisiae methyltransferase genes are regulated at the level of transcription in response to the soluble precursors, inositol and choline as well as to growth phase . The mechanism of regulation of the S . pombe methyltransferases is not yet understood but appears to occur post-transcriptionally in response to choline availability . In addition, the S . pombe PLMT genes are regulated transcriptionally in response to growth phase.

Biotechnol Appl Biochem, 1997 Oct, 26 ( Pt 2), 91 - 6
Application of a statistical technique to the production of Saccharomyces cerevisiae (baker's yeast); Alpbaz M et al.; The identification of a static model for Saccharomyces cerevisiae (baker's yeast) production under aerobic conditions has been realized . Two-level factorial experimental design was used to identify a statistical model . In order to find the optimal operating conditions by using the statistical model, Box-Wilson's steepest-ascent method was used {Box and Wilson (1951) J . R . Stat . Soc . Ser . B 13, 1} . Four independent variables which have an effect on aerobic yeast production were selected . These were temperature, pH, air flow rate and agitation rate . Yeast productivity was selected as the dependent variable . A first-order statistical model was considered to illustrate the dependence of the yeast productivity on the operating parameters . Optimum pH, temperature, air flow rate and agitation rate were determined as 5, 32 degrees C, 1 vol./vol . per min and 600 rev./min respectively.

EMBO J, 1997 Nov 3, 16(21), 6466 - 77
Conservation of a stress response: human heat shock transcription factors functionally substitute for yeast HSF; Liu XD et al.; Heat shock factors (HSF) are important eukaryotic stress responsive transcription factors which are highly structurally conserved from yeast to mammals . HSFs bind as homotrimers to conserved promoter DNA recognition sites called HSEs . The baker's yeast Saccharomyces cerevisiae possesses a single essential HSF gene, while distinct HSF isoforms have been identified in humans . To ascertain the degree of functional similarity between the yeast and human HSF proteins, human HSF1 and HSF2 were expressed in yeast cells lacking the endogenous HSF gene . We demonstrate that human HSF2, but not HSF1, homotrimerizes and functionally complements the viability defect associated with a deletion of the yeast HSF gene . However, derivatives of hHSF1 that give rise to a trimerized protein, through disruption of a carboxyl- or aminoterminal coiled-coil domain thought to engage in intramolecular interactions that maintain the protein in a monomeric state, functionally substitute for yeast HSF . Surprisingly, hHSF2 expressed in yeast activates target gene transcription in response to thermal stress . Moreover, hHSF1 and hHSF2 exhibit selectivity for transcriptional activation of two distinct yeast heat shock responsive genes, which correlate with previously established mammalian HSF DNA binding preferences in vitro . These results provide new insight into the function of human HSF isoforms, and demonstrate the remarkable functional conservation between yeast and human HSFs, critical transcription factors required for responses to physiological, pharmacological and environmental stresses.

Chin J Biotechnol, 1997, 13(2), 105 - 13
Fed-batch culture strategy for high yield of baker's yeast with high fermentative activity; Li Y et al.; Based on the determination of the operational conditions for batch culture and fed-batch culture of baker's yeast by process analysis and continuous culture, a correlation, which describes the relationship between fermentative activity (FA) and specific growth rate (mu), was obtained . Combining this correlation and the exponential fed-batch culture equation, a fed-batch culture strategy was developed to obtain high yield and high fermentative activity by controlling mu at different stages . The results showed that when the initial and residual sugar concentrations were controlled to be 15-30 g/L and 3-6 g/L, respectively, different stirring speeds were applied at different stages to provide optimal oxygen transfer conditions . When this proposed fed-batch culture strategy was used in a fed-batch culture, the yield of baker's yeast reached 0.432 g/g with high fermentative activity (1180 ml) . Thus, the combined bed-batch culture of baker's yeast with high yield and high fermentative activity was realized.

Arch Biochem Biophys, 1997 Oct 15, 346(2), 287 - 93
Redox-dependent conformational changes are common structural features of cytochrome c from various species; Calvert JF et al.; Discrepant results from X-ray crystallographic and physicochemical studies on the conformations of the two redox states of cytochrome c raise important questions about the nature of redox-dependent conformational changes and whether differences are common structural features of various cytochrome c species . Comparative studies of cytochrome c from 10 species (horse, cow, sheep, pig, dog, rabbit, chicken, pigeon, tuna, and baker's yeast) in aqueous solutions were carried out using Fourier transform infrared (FT-IR) spectroscopy . The second-derivative analysis revealed similar conformational changes in all 10 species upon reduction of the heme iron regardless of the differences in the amino acid sequences . The redox-dependent changes involve the amide I regions ascribed to extended beta-structure, beta-turn, and alpha-helix structures . Three species (cow, sheep, and pig) with identical amino acid sequences displayed nearly identical infrared spectra for the oxidized and reduced states, which rules out the possible contribution of experimental error . These results show unequivocally that redox-dependent conformational changes are common structural feature of various cytochrome c species and demonstrate the usefulness of FT-IR spectroscopy as a quick and inexpensive tool in comparative studies of functionally related conformational changes of proteins.

J Fam Pract, 1997 Oct, 45(4), 295 - 315; quiz 317-8
Hepatitis B virus infection, hepatitis B vaccine, and hepatitis B immune globulin; Zimmerman RK et al.; Hepatitis B virus (HBV) infection is a major health problem in the United States; in 1995, approximately 128,000 cases occurred . Transmission of HBV occurs primarily by blood exchange (eg, by shared needles during injection drug use) and by sexual contact . Persons infected early in life are much more likely to become chronically infected than those infected during adulthood: as many as 90% of infants infected perinatally develop chronic infection and up to 25% will die of HBV-related chronic liver disease as adults . Clinical signs of acute hepatitis occur in about 50% of infected adults but in only 5% of infected preschool-aged children . In the United States, hepatitis B vaccine is currently made by recombinant DNA technology using baker's yeast . Preexposure vaccination results in protective antibody levels in almost all infants and children (> 95%) and healthy adults younger than 40 years of age (> 90%) . The most common adverse event following administration of hepatitis B vaccine is pain at the injection site, which occurs in 13% to 29% of adult and 3% to 9% of children . A comprehensive hepatitis B vaccination policy is now recommended that includes (1) routine infant vaccination; (2) catch-up vaccination of 11- to 12-year-olds who were not previously vaccinated; (3) catch-up vaccination of young children at high risk for infection; (4) vaccination of adolescents and adults based on lifestyle or environmental, medical, and occupational situations that place them at risk; and (5) prevention of perinatal HBV infection.

J Mol Evol, 1997 Nov, 45(5), 485 - 98
Positional preferences of polypurine/polypyrimidine tracts in Saccharomyces cerevisiae genome: implications for cis regulation of gene expression; Raghavan S et al.; The complete genome of the baker's yeast S . cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly{pu/py}) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs) . The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions . Clear positional preferences are seen when these tracts occur in noncoding regions . These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs . Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames . The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags . A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Eur J Biochem, 1997 Aug 15, 248(1), 24 - 9
Carbohydrate protection of enzyme structure and function against guanidinium chloride treatment depends on the nature of carbohydrate and enzyme; Sola-Penna M et al.; Baker's yeast cells accumulate osmolytes as a response to several stress conditions such as high-temperature and low-temperature shifts, dehydration, or osmotic stress . One of the major osmolytes that accumulates is trehalose, which plays an essential role affecting the survival of yeast at the time of stress . In this report, we show that trehalose efficiently protects the function and the structure of two yeast cytosolic enzymes against chemical denaturation by guanidinium chloride . Other sugars tested also protected yeast pyrophosphatase and glucose-6-phosphate dehydrogenase structure against guanidinium chloride effects, but were not as efficient at protecting enzyme activity . The thermostable pyrophosphatase from Bacillus stearothermophilus was also protected by several sugars against the chaotropic properties of guanidinium chloride, but was only protected by trehalose against functional inactivation . The function of the membrane-embedded H+-ATPase from yeast could not be protected by any of the tested sugars, although all of the sugars protected its structure from guanidinium-chloride-induced unfolding . The results presented in this study suggest that several sugars are able to prevent protein unfolding induced by a chaotropic compound . However, prevention of functional inactivation depends on the nature of the sugar . Trehalose was the most efficient, being able to protect many cytosolic enzymes against guanidinium chloride . The efficiency of protection also depended on the nature of the protein tested . This might explain why trehalose is one of the osmolytes accumulated in yeast and also why it is not the only osmolyte to accumulate.

Microbiology, 1997 Sep, 143 ( Pt 9), 3033 - 44
A Candida albicans RAS-related gene (CaRSR1) is involved in budding, cell morphogenesis and hypha development; Yaar L et al.; Candida albicans, the most important human fungal pathogen, is a dimorphic fungus that can grow either as a yeast or as a hyphal form in response to medium conditions . A RAS-related C . albicans gene (CaRSR1) was isolated as a suppressor of a cdc24ts bud-emergence mutation of the baker's yeast, Saccharomyces cerevisiae . The deduced protein encoded by CaRSR1 is 248 amino acids long and 56% identical to that encoded by the S . cerevisiae RSR1 (BUD1) gene . Disruption of CaRSR1 in C . albicans indicated that CaRSR1 is involved in both yeast and hypha development . In the yeast phase, CaRSR1 is required for normal (polar) bud site selection and is involved in cell morphogenesis; in the yeast-mycelial transition it is involved in germ tube emergence; and in the development of the hyphae it is involved in cell elongation . The disruption of CaRSR1 leads to reduced virulence in both heterozygote and homozygote disruptants in a dose-dependent manner . The reduced virulence can be attributed to the reduced germination and shorter hyphae resulting from the disruption of CaRSR1.

FEMS Microbiol Rev, 1997 Aug, 21(1), 85 - 111
The molecular genetics of hexose transport in yeasts; Boles E et al.; Transport across the plasma membrane is the first, obligatory step of hexose utilization . In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins . In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified . Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known . The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources . In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters . Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis . However, the molecular genetics of hexose transport in many other yeasts remains to be established . The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.

Anal Chem, 1997 Aug 15, 69(16), 3267 - 71
Baker's yeast biomass (Saccharomyces cerevisae) for selective on-line trace enrichment and liquid chromatography of polar pesticides in water; Martin-Esteban A et al.; Baker's yeast cells (Saccharomices cerevisae) were successfully immobilized onto silica gel and used in the on-line isolation and trace enrichment of desisopropylatrazine, desethylatrazine, hydroxyatrazine, simazine, cyanazine, atrazine, carbaryl, propanil, linuron, and fenamiphos . Since humic and fulvic acids were not coextracted, no cleanup was necessary . The pesticides were spiked at 0.1-1 microgram L-1 in tap water, groundwater, and seawater and were preconcentrated using on-line solid-phase extraction into a yeast immobilized on silica gel precolumn followed by liquid chromatography with diode array detection . All the variables that affect the enrichment step, such as amount of yeast immobilized, dimensions of the precolumn, sample pH, and preconcentration flow rate, were optimized . The degree of selectivity was evaluated by comparing the chromatograms obtained after on-line sample preconcentration on the yeast precolumn with those obtained by on-line solid-phase extraction using a precolumn filled with C18 material . The relative standard deviation for the whole procedure in the determination of the selected pesticides at the 0.3 microgram L-1 concentration level ranged from 1 to 9%, depending on the pesticide and the type of water . Detection limits within the range 0.01-0.5 microgram L-1 were obtained by percolating only 25 mL of water sample without any additional cleanup step.

Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1133 - 7
Inactivation of food microorganisms by high-pressure carbon dioxide treatment with or without explosive decompression; Enomoto A et al.; In order to elucidate the sterilization mechanism underlying the explosive decompression system, baker's yeast was pressurized with CO2, N2O, N2, or Ar gas at 40 atm and 40 degrees C for 4h, and then explosively discharged . The survival ratio was markedly decreased only by the treatments with CO2 and N2O, which are relatively soluble gases in water, suggesting that the microorganisms' death may be highly correlated with gas absorption by the cells . Lower decompression rates to atmospheric pressure, however, led to neither any lower reduction of remaining cells nor any smaller release of total cellular proteins . Furthermore, operating with a longer treatment time and smaller number of repetitions was usually more lethal than with a shorter time and more frequent repetition . From these results, most of the yeast cells appear to have been sterilized during the pressurization process . The spore cells of B . megaterium are considered to have been killed in a somewhat different manner, because of their distinct sensitivity to the applied gases.

Appl Biochem Biotechnol, 1997 May, 66(2), 159 - 72
Enzymic activity of whole cells entrapped in reversed micelles . Studies on alpha-amylase and invertase in the entrapped yeast cells; Gajjar L et al.; Studies have been conducted on the enzymic activity of Baker's yeast and also of Brewer's yeast entrapped into the reversed micelles formed by cetyl pyridinium chloride (CPC1) in n-hexane . The activities of alpha-amylase and invertase enzymes in the entrapped cells have been estimated and compared with those in the control experiments where there was no entrapment . The following significant observations have been made: 1 . except for invertase, enzymes in Brewer's yeast, the entrapped yeast cells showed enhanced enzymic activities; 2 . when the yeast cells were entrapped inside the reversed micelles along with substrates of the two enzymes, alpha-amylase, and invertase, the activity of each of these enzymes showed a further enhancement in comparison to that showed in the experiments in which substrates of the individual enzymes alone were entrapped-the phenomenon of synergism; 3 . when the yeast cells and the respective substrates were entrapped inside separate reversed micelles and the solutions containing entrapped cells and entrapped substrates were mixed, the activities of the individual enzymes, alpha-amylase and invertase, showed further enhancement in comparison to the case in which the cells and the substrates were entrapped inside the same reversed micelle (in this case also the phenomenon of synergism was observed); and (4) In the case of experiments in which there was no entrapment, it was observed that the presence of substrates induced more release of enzymes from the yeast cells . These observations on yeast cells, which to the best of our knowledge have not been reported before, should be biotechnologically relevant.

Bioorg Med Chem, 1997 May, 5(5), 921 - 39
Synthesis and biological evaluation of potent glycosidase inhibitors: N-phenyl cyclic isourea derivatives of 5-amino- and 5-amino-C-(hydroxymethyl)-1,2,3,4-cyclopentanetetraols; Uchida C et al.; Twenty-four stereoisomers of 5-amino- and 5-amino-C-(hydroxymethyl)-1,2,3,4-cyclopentanetetraols and twenty-six of the corresponding N-phenyl cyclic isourea derivatives were assayed for inhibitory activity against six glycosidases . Among them, as has been expected for structure mimics of putative transition state glucopyranosyl cation for glycoside hydrolysis, 1L-(1,2,4,5/3)-5-amino-1-C-(hydroxymethyl)-1,2,3,4-cyclopentanetetrao l L-4 and its N-phenyl cyclic isourea derivative S-19 were shown to have strong inhibitory activity, IC50 4 x 10(-7) and 7.6 x 10(-9) M, respectively, against baker's yeast alpha-glucosidase . It has been analogously explained that compounds R,S-22 and R,S-26 possessed high inhibitory potency against Escherichia coli and bovine liver beta-galactosidases, respectively.

Biomed Chromatogr, 1997 May-Jun, 11(3), 180 - 4
Affinity chromatography of yeast alcohol dehydrogenase using immobilized monochlorotriazine colourless compounds; Li Y et al.; Four new symmetrical colourless biomimetics bearing two monochlorotriazine rings have been rationally designed and synthesized . These immobilized colourless ligands and one analogue of Cibacron Blue F3GA on Sepharose CL-4B were used to purify alcohol dehydrogenase from baker's yeast extract . Twenty-two-fold purification with 78% enzyme recovery was achieved in a single step with specific elution of NAD+ (5 mM) from the colourless absorbent comprised of two ortho-aminobenzene sulphonates as terminal rings, which is comparable to the result obtained from the analogue of Cibacron Blue F3GA . Differential spectra between ligand-enzyme complexes and free ligands were studied.

Gene, 1997 Apr 29, 190(1), 87 - 97
Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis -- a review; Gellissen G et al.; From the onset of gene technology yeasts have been among the most commonly used host cells for the production of heterologous proteins . At the beginning of this new development the attention in molecular biology and biotechnology focused on the use of the best characterized species, Saccharomyces cerevisiae, leading to an increasing number of production systems for recombinant compounds . In recent years alternative yeasts became accessible for the techniques of modern molecular genetics and, thereby, for potential applications in biotechnology . In this respect Kluyveromyces lactis, and the methylotrophs Hansenula polymorpha and Pichia pastoris have been proven to offer significant advantages over the traditional baker's yeast for the production of certain proteins . In the following article, the present status of the various yeast systems is discussed.

Biochemistry (Mosc), 1997 Apr, 62(4), 425 - 32
Determination of constants of substrate primary binding with baker's yeast transketolase by kinetic modelling; Selivanov VA et al.; A kinetic model of bisubstrate reaction catalyzed by baker's yeast transketolase is proposed . The model considers individual stages of substrates reversible primary binding . The model corresponds to the observed kinetics of product accumulation within a wide range of initial substrate concentrations . Kinetic parameters for the best simulation of the experimental data are defined . The equilibrium constants of the primary binding of both the initial and produced ketose and also the initial aldose were unequivocally determined by varying the initial substrate concentrations . The dissociation constants of the primary enzyme-substrate complex for the initial ketose (xylulose 5-phosphate) and the reaction product (sedoheptulose 7-phosphate) were found to differ by more than by two orders of magnitude . The result is discussed in the context of the hypothesis of flip-flop functioning of the transketolase active sites.

Biochemistry, 1997 Apr 1, 36(13), 4054 - 60
Cytochrome c/cytochrome c peroxidase complex: effect of binding-site mutations on the thermodynamics of complex formation; Erman JE et al.; The cytochrome c/cytochrome c peroxidase system has been extensively investigated as a model for long-range electron transfer in biology . Two models for the structure of the one-to-one cytochrome c/cytochrome c peroxidase complex in solution exist: one is based upon computer docking of the two proteins and the second is based upon the structure of the complex in the crystalline state . Titration calorimetry is used to investigate the interaction of horse ferricytochrome c with baker's yeast cytochrome c peroxidase and with six cytochrome c peroxidase mutants . Five of the six peroxidase mutants eliminate a negative charge in the cytochrome c binding site by replacing a side-chain carboxylate with an amide . The sixth mutation replaces a surface alanine residue with phenylalanine . The binding affinity between cytochrome c and the cytochrome c peroxidase mutants varies from no significant change in comparison to the wild-type enzyme to a 4-fold decrease in the equilibrium association constant . The pattern of decreasing cytochrome c binding affinity for the cytochrome c peroxidase mutants is consistent with the cytochrome c binding domain defined by X-ray crystallography {Pelletier, H., & Kraut, J . (1992) Science 258, 1748-1755} . For those mutants which have lower affinity for cytochrome c, the lower affinity is due to a decrease in the entropy change upon complex formation, consistent with the difference in hydration of carboxylate and amide groups.

Biochim Biophys Acta, 1997 Feb 21, 1324(1), 120 - 32
Conditions allowing different states of ATP- and GDP-induced permeability in mitochondria from different strains of Saccharomyces cerevisiae; Roucou X et al.; The effect of ATP and other nucleotides on the respiration of Saccharomyces cerevisiae mitochondria was investigated . It was observed that ATP induced a stimulation of the respiration rate only in the presence of a salt in mitochondria from the baker's yeast Yeast Foam, whereas an ATP-induced stimulation occurred even in the absence of salt in mitochondria from three different laboratory strains . In both cases, the stimulation was related to a collapse of the transmembrane potential, suggesting the opening of ion- and/or proton-conducting pathways . Not only ATP, but also GTP and CTP, induced these pathways . Moreover, a similar stimulation was obtained with GDP and its analog GDP-beta-S . The fact that, as opposed to NTPs, GDP did not induce any non-specific anion channel, allowed us to use it to demonstrate unambiguously that a proton-conducting pathway was opened through the inner mitochondrial membrane of laboratory strains but not of Yeast Foam . Three additional aspects of this nucleotide-induced permeability were investigated . (i) The proton-conducting pathway was insensitive to Mg2+, whereas the anion-conducting pathway was fully inhibited by 4 mM Mg2- . (ii) The proton-conducting pathway of mitochondria isolated from laboratory strains was opened by the action of nucleotides outside the mitochondrion, since it was fully insensitive to (carboxy)atractyloside, and fully active in mitochondria isolated from op1 and delta anc strains . On the other hand, the cation-conducting pathway of Yeast Foam mitochondria was partly sensitive to (carboxy)atractyloside and insensitive to bongkrekic acid, suggesting a role of the conformational state of ANC in this activity . (iii) Both the proton and cation-conducting pathways were inhibited by very low concentrations of vanadate, under conditions where this oxyanion was polymerized to decavanadate: a competitor to nucleotide-binding sites on some enzymes.

Neuropediatrics, 1997 Feb, 28(1), 18 - 20
Cross-species homology of the CLN3 gene; Taschner PE et al.; A murine cDNA clone was isolated by screening a mouse cDNA library with the human CLN3 cDNA . Sequence analysis indicates that the corresponding CLN3 proteins are highly homologous . We have compared these with recently identified CLN3 sequences from the dog, the nematode C . elegans, and baker's yeast S . cerevisiae . The CLN3 protein is remarkably conserved across eukaryotic species . Several protein modification sites which may be crucial for the function of the protein are conserved.

Pharmazie, 1997 Feb, 52(2), 87 - 91
Stereoselective syntheses and CNS activity of novel tricyclic amines; Wunsch B et al.; The enantiomerically pure amines (+)-5, (-)-9, (+)-11 and (+)-12 are stereoselectively prepared by reductive amination of the ketone (-)-4 {-->(+)-5}, LiAlH4 reduction of the oxime (-)-7 followed by reductive methylation {-->(-)-9}, SN2-substitution of the benzenesulfonate 10 {-->(+)-11} and reductive methylation of (+)-11 {-->(+)-12} . In the same way the racemic amines (+/-)-5, (+/-)-9, (+/-)-11 and (+/-)-12 are accessible starting from the racemic ketone (+/-)-4 . Kinetic resolution of the racemic ketone (+/-)-4 with baker's yeast leads to the dextrorotatory ketone (+)-4 (86% ec), which is transformed via the methanesulfonate 16 into the tricyclic amines (-)-11 and (+)-17 . Weak sedative effects are observed after application of the amines (+)-5, (+/-)-5, 0-9, (+/-)-9(+)-12, and (+/-)-12 to mice . Strong sedation is caused by (+/-)-11 with the dextrorotatory enantiomer (+/-)-11 being more effective than the levorotatory enantiomer (-)-11.

Kidney Int, 1997 Feb, 51(2), 507 - 13
Regulation of hypoxic gene expression in yeast; Zitomer RS et al.; Baker's yeast, Saccharomyces cerevisiae, can adapt to growth under severe oxygen limitation . Two regulatory systems are described here that control this adaptation . The first involves a heme-dependent repression mechanism . Cells sense hypoxia through the inability to maintain oxygen-dependent heme biosynthesis . Under aerobic conditions, heme accumulates and serves as an effector for the transcriptional activator Hap1 . The heme-Hap1 complex activates transcription of the ROX1 gene that encodes a repressor of one set of hypoxic genes . Under hypoxic conditions, heme levels fall, and a heme-deficient Hap1 complex represses ROX1 expression . As a consequence, the hypoxic genes are derepressed . The second regulatory system activates gene expression in response to a variety of stress conditions, including oxygen limitation . Oxygen sensing in this system is heme-independent . The same DNA sequence mediates transcriptional activation of each stress signal.

Chirality, 1997, 9(4), 321 - 4
Improvement of enantioselective syntheses and chiral high resolution gas chromatographic analyses of (+)-2-allyl-2-carboethoxy-cyclopentanol; Fraga CA et al.; The improvement of the biocatalytic reduction of 2-allyl-carboethoxy-cyclopentanone (2) to the corresponding cyclopentanol derivative (+)-(1R,2R)-(1) was accomplished employing baker's yeast in organic media . This chiral cyclopentanol derivative (1), analyzed by high resolution gas chromatography performed over beta-cyclodextrin stationary phase, was obtained in 38% yield (> 99% e.e.).

Biochem Mol Biol Int, 1997 Jan, 41(1), 209 - 15
Immunoreactivities of p11 and calcyclin in baker's yeast, wheat germ and lobster; Kuo WN et al.; After effectively eliminating the nonspecific cross-immunoreactivity with the affinity columns of anti-IgG agarose and IgG agarose, the potent immunoreactivities of p11 and calcyclin in wheat germ, lobster tail muscle, and three strains of baker's yeast were analyzed by Western blotting using mouse anti-p11 and rabbit anti-calcyclin . The occurrence of multiple bands may be due to either autolyses and/or the interactions between the p11 (or calcyclin) and other endogenous biological molecules . The results suggest not only a ubiquitous distribution and a universal Ca(2+)-mediating regulatory role of p11 and calcyclin in eukaryotes, but also an evolutionary conservation of these (S-100)-related proteins.

FEMS Microbiol Lett, 1996 Dec 15, 145(3), 415 - 20
Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii; Watanabe Y et al.; An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii, which is resistant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 microns origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T . delbrueckii cells is active in both acidic and alkaline conditions . However, activity of phospholipase B gene (PLB1) in cells of disruption mutant (plb1:: URA3) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells . Over-expression of PLB1 with YEp plasmid vector in T . delbrueckii cells showed approximately 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells . Cells of plb1 delta mutant showed increased survival when cells of plb1 delta mutant and wild-type strain were incubated in water at 30 degrees C . Cells of PLB1-over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.

Bioorg Khim, 1996 Dec, 22(12), 938 - 40
{Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes}; Shpakovskii GV et al.; The rpb10+ cDNA from the fission yeast Schizosaccharomyces pombe was cloned using two independent approaches (PCR and genetic suppression) . The cloned cDNA encoded the Rpb10 subunit common for all three RNA polymerases . Comparison of the deduced amino acid sequence of the Sz . pombe Rbp10 subunit (71 amino acid residues) with those of the homologous subunits of RNA polymerases I, II, and III from Saccharomyces cerevisiae and Home sapiens revealed that heptapeptides RCFT/SCGK (residues 6-12), RYCCRRM (residues 43-49), and HVDLIEK (residues 53-59) were evolutionarily the most conserved structural motifs of these subunits . It is shown that the Rbp10 subunit from Sz . pombe can substitute its homolog (ABC10 beta) in the baker's yeast S . cerevisiae.

Curr Biol, 1996 Dec 1, 6(12), 1570 - 2
Cell biology: alternatives to baker's yeast; Glick BS; Saccharomyces cerevisiae is an excellent model organism for addressing questions in cell biology, but other yeast systems are also providing new insights into several fundamental cellular processes.

Appl Environ Microbiol, 1996 Dec, 62(12), 4401 - 4
Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs; Almeida MJ et al.; Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts . During storage of frozen doughs, strains of T . delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days . Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain . Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323 . These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions . The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S . cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T . delbrueckii (IGC 5321 and IGC 5323) . However, the strains of S . cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.

Immunopharmacol Immunotoxicol, 1996 Nov, 18(4), 597 - 608
Wortmannin inhibits the production of reactive oxygen and nitrogen intermediates and the killing of the Saccharomyces cerevisiae by isolated chicken macrophages; Yang M et al.; The direct effects of wortmannin (0 to 1280 nM) on several functions in cultured macrophages isolated from Sephadex-elicited Leghorn chicken peritonea were studied . Under concentrations not affecting cell viability, wortmannin, as low as 5 nM, inhibited lipopolysaccharide (LPS)-induced nitric oxide production (P < 0.01) . However, wortmannin (as high as 1280 nM) exposure 5 hours post LPS induction had no effect on nitric oxide production in macrophages, indicating a blockade of LPS-induction of a signaling pathway related to nitric oxide formation . Phorbol myristate acetate (PMA)-induced superoxide production was only inhibited (P < 0.001) by concurrent exposure to 1280 nM wortmannin . Prior exposure to 160 nM and higher of wortmannin for 24 hours reduced the average number of yeast cells ingested by or attached to a single macrophage (P < 0.001) and the ability of the macrophage to kill the baker's yeast (P < 0.05), while wortmannin itself did not affect the yeast . These data provide direct evidence for macrophages being the target cell of wortmannin and further support the notion that impaired macrophage functions are responsible for the immunosuppressive effect of wortmannin previously observed in birds.

Genetics, 1996 Nov, 144(3), 967 - 78
Saccharomyces cerevisiae S288C has a mutation in FLO8, a gene required for filamentous growth; Liu H et al.; Diploid strains of baker's yeast Saccharomyces cerevisiae can grow in a cellular yeast form or in filaments called pseudohyphae . This dimorphic transition from yeast to pseudohyphae is induced by starvation for nitrogen . Not all laboratory strains are capable of this dimorphic switch; many grow only in the yeast form and fail to form pseudohyphae when starved for nitrogen . Analysis of the standard laboratory strain S288C shows that this defect in dimorphism results from a nonsense mutation in the FLO8 gene . This defect in FLO8 blocks pseudohyphal growth in diploids, haploid invasive growth, and flocculation . Since feral strains of S . cerevisiae are dimorphic and have a functional FLO8 gene, we suggest that the flo8 mutation was selected during laboratory cultivation.

J Biotechnol, 1996 Oct 18, 51(1), 73 - 82
Model-based automation of baker's yeast production; Ringbom K et al.; An on-line model, estimating key state variables in bioprocesses, is utilized for control of fed-batch baker's yeast production . The state estimates are produced by balances and phenomenological expressions combined with on-line measurements . The goal of the control strategy is to maintain the highest possible glucose flux that can be entirely respiratively assimilated by the cells, resulting in the highest possible yeast growth without formation of metabolic products, such as acetic acid and ethanol . Stepwise improvement of the control algorithm is carried out in order to find a strategy to avoid undesired, irreversible metabolic pathways . In the final algorithm, such undesired changes in metabolism are predicted from an estimate of intracellular storage carbohydrates . A considerable decrease in the estimate indicates future metabolic changes at a time early enough to avoid them . At maximal yield, a growth rate near the highest possible is obtained in laboratory-scale Saccharomyces cerevisiae cultivations with the control strategy developed.

FEBS Lett, 1996 Sep 2, 392(3), 293 - 4
Localization of reactive tyrosine residues of baker's yeast transketolase; Kovina M et al.; Baker's yeast transketolase inactivated by tetranitromethane was digested with Staphylococcus aureus V8 protease . Four peptides absorbing at 360 nm were isolated by reverse-phase HPLC and sequenced . The modified tyrosines were identified as Tyr-184, Tyr-210 and Tyr-370.

Appl Biochem Biotechnol, 1996 Sep, 60(3), 229 - 44
Estimation of the optimal concentrations of residual sugar and cell growth rate for a fed-batch culture of Saccharomyces cerevisiae; He RQ et al.; Estimation of the optimal concentrations of residual sugar in medium for a fed-batch culture of Baker's yeast has been studied and practiced . The concentrations, however, depended on different species and targets of the biomass, which was expected to be made . Kinetic changes of the residual phosphate salt in the medium conformed to a logarithmic process until the fourth hour during an 11-h culture . The parabolic method (see ref . 9 later in article) might be qualified to maintain the concentrations of residual sugar around 0.15 g/L . It was demonstrated that cell growth followed a sigmoid process during a fed-batch culture, because the cells consumed the nutrient with two metabolic pathways, one was for cell conversion and another was for non-cell conversion . With the parabolic method, we can estimate kinetics of cell growth and cell growth rate during the culture.

Biotechnol Prog, 1996 Jul-Aug, 12(4), 510 - 8
Heat flux measurements for the fast monitoring of dynamic responses to glucose additions by yeasts that were subjected to different feeding regimes in continuous culture; van Kleeff BH et al.; Heat measurements have been successfully as an analytical tool for the study of the dynamics of energy metabolism of Saccharomyces cerevisiae and Candida utilis grown in continuous culture under fluctuating substrate supply . A low average dilution rate (D = 0.05 h-1) was maintained either by adding the medium as continuously (dropwise) as possible or (blockwise) by adding the medium at high speed during a short period (D = 0.5 h-1 for 40 s) and not at all during the following period (D = 0.00 h-1 for 360 s) . The resulting biological activity was monitored on-line with conventional (O2 and CO2) off-gas analyses, DOT measurements, and heat flux measurements . In C . utilis cultures, the biomass-specific maximum oxygen consumption rate (qO2,max), the biomass yield (Ys,x), and the dynamic responses to a glucose pulse and to a change in feeding regime were not significantly affected by different preceding feeding regimes . In contrast, S . cerevisiae grown in continuous culture with blockwise feed showed a 50% increase in qO2,max and a 25% drop in Ys,x compared to the culture grown with dropwise feed . The dynamic response to a glucose pulse (0.6 g L-1) was slower for the continuous (dropwise) than for the blockwise grown S . cerevisiae . With a second testing method for the dynamic response of the yeasts, the feeding regime was changed . The blockwise fed S . cerevisiae proved to be better "trained" to cope with sudden changes in glucose supply and, therefore, was more "shockproof" toward a change in feeding regime . This clearly points to major differences in the intracellular metabolic flux control between the yeasts . These findings are of relevance for industrial baker's yeast production, where reactor mixing times of one to several minutes are not uncommon . The observed, heat production, together with the dissolved oxygen concentration, appeared to give the fastest response to actual changes in the culture . It is suggested that heat measurements can be a very useful tool to monitor and control the growth of S . cerevisiae in laboratory and industrial fermenter operations.

J Chromatogr B Biomed Appl, 1996 May 17, 680(1-2), 65 - 70
Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling; Lu M et al.; Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride . A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed . The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature . This two-phase system has been used to purify 3-phosphoglycerate kinase from baker's yeast . The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature . The bottom-phase polymer (benzoyl dextran) could be recovered by addition of salt . Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system . After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase . The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na2SO4.

Appl Biochem Biotechnol, 1996 May, 59(2), 135 - 43
Observation of baker's yeast strains used in biotransformation by atomic force microscopy; Pereira Rde S et al.; Different strains of baker's yeast (Saccharomyces cerevisiae) were imaged with an atomic force microscope (AFM) . The images of uncoated and nonfixed samples were reproducible with high-constrast and nanometer-resolution . Molecules from the polysaccharide surface of the cell wall were pictured and the distance of atoms was measured . The preparation of samples was easy, suggesting that AFM is a useful tool in this type of analyses.

Plant Foods Hum Nutr, 1996 Apr, 49(3), 213 - 9
The suitability of African bush mango juice for wine production; Akubor PI; A good quality wine was produced from African bush mango (Irvingia var.gabonensis) . Analysis of the African bush mango juice showed that it contained 3.6% total sugar, 1.09% protein, 4.2 degrees Brix soluble solids (SS) 0.5% ash, 50.24% total solids (TS), 66.7 mg/100 ml ascorbic acid and pH 5.12 . The juice ameliorated to 23 degrees Brix was inoculated with 3% (w/v) Baker's yeast (Saccharomyces cerevisiae) and held at 30 +/- 2 degrees C for 28 days . SS and pH decreased while titratable acidity (TA) increased with increasing period of fermentation . Fermentation was 110% efficient . The wine produced had 8.12% (v/v) alcohol, 0.78% protein, 6.5% Brix SS, 0.64 g/100 ml TA, and a pH 3.10 . Sensory evaluation results showed that there was no significant difference (p = 0.05) in colour, mouthfeel, sweetness, flavour and general acceptability, between African Bush mango wine and a reference wine . The wine was generally accepted.

Genetics, 1996 Apr, 142(4), 1083 - 93
Rox3 and Rts1 function in the global stress response pathway in baker's yeast; Evangelista CC Jr et al.; Yeast respond to a variety of stresses through a global stress response that is mediated by a number of signal transduction pathways and the cis-acting STRE DNA sequence . The CYC7 gene, encoding iso-2-cytochrome c, has been demonstrated to respond to heat shock, glucose starvation, approach-to-stationary phase, and, as we demonstrate here, to osmotic stress . This response was delayed in a the hog1-delta 1 strain implicating the Hog1 mitogen-activated protein kinase cascade, a known component of the global stress response . Deletion analysis of the CYC7 regulatory region suggested that three STRE elements were each capable of inducing the stress response . Mutations in the ROX3 gene prevented CYC7 RNA accumulation during heat shock and osmotic stress . ROX3 RNA levels were shown to be induced by stress through a novel regulatory element . A selection for high-copy suppressors of a ROX3 temperature-sensitive allele resulted in the isolation of RTS1, encoding a protein with homology to the B' regulatory subunit of protein phosphatase 2A0 . Deletion of RTS1 caused temperature and osmotic sensitivity and increased accumulation of CYC7 RNA under all conditions . Over-expression of this gene caused increased CYC7 RNA accumulation in rox3 mutants but not in wild-type cells.

Biotechnol Appl Biochem, 1996 Apr, 23 ( Pt 2), 141 - 8
A non-isothermal bioreactor utilizing immobilized baker's-yeast cells: a study of the effect on invertase activity; Russo P et al.; The behaviour of the enzyme invertase, located on the cell wall of baker's-yeast cells and entrapped in a gelatin membrane, was studied under isothermal and non-isothermal conditions . The reaction rate linearly increased with the applied transmembrane temperature gradient, with reference either to the average temperature or to the temperature on the warm side of the catalytic membrane . These results were obtained both when the bioreactor was operated under conditions of closed volumes and when the substrate-containing solutions are recirculated . The mathematical relationships have been elaborated between the temperatures read in the working solutions and those on the two faces of the catalytic membrane . Since the temperature difference across the membrane is smaller than that indicated by the thermocouples, the observed effects are greater than expected . The potential advantages of the use of a non-isothermal bioreactor in processes of industrial interest are discussed.

Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 199 - 203
Transition rate kinetics from ethanol oxidation to glucose utilisation within a structured model of baker's yeast; Dantigny P et al.; The transition rate kinetics from ethanol oxidation to glucose utilisation, within a structured model of baker's yeast, described previously, were experimentally identified . The shift in metabolism has been assessed through glucose pulses during batch growth on ethanol . The influence of glucose concentration (between 0.25 g l-1 and 0.90 g l-1) and initial biomass concentration (between 0.61 g l-1 and 1.44 g l-1) on the transition rate was determined . The transition rate can not be described by a first-order saturation-type kinetics with respect to glucose only . A corrective term, which takes into account biomass concentration should be included.

Prikl Biokhim Mikrobiol, 1996 Mar-Apr, 32(2), 254 - 9
{Effect of Bacillus intermedius RNAase on growth of a Saccharomyces cerevisiae culture}; Kupriianova-Ashina FG et al.; The effects of RNase from Bacillus intermedius on proliferation of Saccharomyces cerevisiae were studied . The enzyme (0.01 microgram/ml) stimulated the yeast cell budding . This effect was dose-dependent and required an appropriate physiological stage of the growing culture cells . RNase produced maximal effects when added to exponentially growing cultures . Analysis of the age structure of the population showed that exogenous RNase stimulated the cell cycle at a stage preceding the initiation of DNA synthesis and budding of single yeast cells and cells occurring at the budding stage III . RNase did not decrease the buoyancy and osmotic sensitivity of baker's yeast.

Eur J Biochem, 1996 Feb 1, 235(3), 613 - 21
In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA; Das B et al.; Ribosomes from a number of prokaryotic and eukaryotic sources (e.g . Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HC1 prior to incubation with ribosomes . In this report, we present our observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E . coli, wheat germ and rat liver . The protein-folding activity of E . coli ribosomes was found to be present in 50S particles and in 23S rRNA . The 30S particle or 16S rRNA did not show any protein-folding activity . The protein-folding activity of 23S rRNA may depend on its tertiary conformation . Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA . This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.

Cytobios, 1996, 87(351), 251 - 63
Immunoreactivities of m-calpain, calpastatin, nitric oxide synthase, myelin basic protein and dynamin II in baker's yeast, wheat germ and lobster tail muscle; Kuo WN et al.; Vertebrate m-calpain, calpastatin, constitutive nitric oxide synthase, myelin basic protein, and dynamin I are substrates of protein kinase C (PKC) . The presence/absence of similar/related protein in nonvertebrate was investigated by immunological methods, including (1) affinity chromatography on agarose-secondary antibodies and agarose IgG for removal of nonspecific immunoreactivities from crude extracts; (2) omitting beta-mercaptoethanol treatment and boiling prior to SDS-PAGE to increase the immunoreactivity; (3) immunoreactivity comparisons of nonspecific IgG as controls with specific anti-(vertebrate PKC-substrates/related proteins) in Western blots . It was found that (a) m-calpain and dynamin I were absent in baker's yeast, wheat germ and lobster tail muscle, (b) m-calpain, nitric oxide synthase, myelin basic protein and dynamin II were present in all three samples, and (c) calpastatin was present in baker's yeast and lobster tail muscle . The presence and absence of these proteins suggest evolutionary conservation and divergence, respectively, of these PKC substrates.

Biosci Biotechnol Biochem, 1996 Jan, 60(1), 61 - 4
Impairment of the glycolytic system and actin in baker's yeast during frozen storage; Hatano S et al.; After frozen storage for 7 d, the viability and CO2 productivity of a conventional baker's yeast strain D greatly decreased . The viability of a freeze-tolerant strain, DFT, used for the frozen dough method slightly decreased after the same storage period, while the CO2 productivity greatly decreased . The CO2 productivity and DNase I inhibitory activity of actin of the cell-free extracts prepared immediately after thawing from 7-d frozen-stored cells markedly decreased in both strains . In DFT, however, the productivity and the inhibitory activity of the cell-free extract increased when the extract was prepared after incubation of the frozen-thawed cells at 30 degrees C . The increase in the inhibitory activity first occurred and then the increase in the CO2 productivity . Gel filtration patterns of actin and glycolytic enzymes were compared between cell-free extracts of both strains . Peaks of actin and activity peaks of hexokinase and pyruvate kinase decreased in the strain D after frozen storage, but only slightly in the strain DFT . After frozen storage, phosphofructokinase activity peak shifted to a lower molecular weight in strain D.

Chirality, 1996, 8(4), 305 - 10
Enantiofacial selective reduction of 2-allyl-2-carboethoxy-cyclopentanone mediated by baker's yeast; Fraga CA et al.; Enantioselective reduction of 2-allyl-2-carboethoxy-cyclopentanone (2) was accomplished in high enantiomeric excess (> 99%), using baker's yeast in the presence of CuO, to obtain the (+)-2-allyl-2-carboethoxy-cyclopentanol derivative (6) . This methodology also provides an entry to corresponding beta-keto ester (-)-(2), representing an important strategy to prepare chiral functionalized 2-oxabicyclic{3.3.0}octane and 2-oxabicyclic{4.4.0}nonane derivatives, useful synthons to access new bioactive compounds.

Biosystems, 1996, 39(1), 43 - 61
Oscillatory, stochastic and chaotic growth rate fluctuations in permittistatically controlled yeast cultures; Davey HM et al.; We describe a continuous culture system related to the turbidostat, but using a feedback system based on biomass estimation from the dielectric permittivity of the cell suspension rather than its optical density . It is shown that this system provides an excellent method of maintaining a constant biomass level within a fermentor . The computer-controlled system was able to effect the essentially continuous registration of growth rate by monitoring the rate of medium addition via the time-dependent activity of the pump . At some biomass setpoints for aerobically grown cultures of baker's yeast substantial time-dependent fluctuations in the growth rate of the culture were thereby observed . At some biomass setpoints, however, or under anaerobic conditions, or when using a non-Crabtree yeast, the growth rate was constant, indicating that the fluctuations were inherent to the biological system and not simply a property of the fermentor and control system . A variety of time series analyses (Fourier transformations, Hurst and Lyapunov exponents, the determination of embedding dimension, and non-linear time series predictions based on the methodology of Sugihara and May) were used to demonstrate, for the first time, that as well as stochastic and periodic components these fluctuations exhibited deterministic chaos . 'Trivial predictors' were unable to give accurate predictions of the growth rate in these cultures . The growth rate fluctuations were studied further by means of offline measurements of changes in percentage viability, bud count, and in the external ethanol and glucose concentrations; these data and other evidence suggested that the growth rate fluctuations were closely linked to the primary respiro-fermentative metabolism of this organism . The identification of chaotic growth rates in cell cultures suggests that there may be novel methods for controlling the growth of such cultures.

Microbios, 1996, 85(344), 139 - 44
Protein kinase C alpha, beta immunoreactivity in baker's yeast, lobster and wheat germ; Kuo WN et al.; The immunoreactivity of PKC alpha (protein kinase C alpha) and PKC beta in wheat germ, lobster tail muscle and three strains of yeast was analysed by Western blotting with mouse anti-PKC active fragments . The potency of the immunoreactivity of PKC alpha activity was much greater than that of PKC beta . The occurrence of multiple bands may be due to PKC self-interactions and/or the interactions between PKC and other molecules . The evolutionary conservation of PKC alpha and PKC beta implies that these PKC isoenzymes may play important roles in Ca2+/lipid-dependent signal transduction and cell growth in these eukaryotes.

Appl Biochem Biotechnol, 1996 Spring, 57-58, 593 - 8
Improvement of productivity of yeast cell with a novel airlift loop reactor; Liu D et al.; Two different strains of baker's yeast are cultivated using a fed-batch process with a novel airlift loop reactor . The reactor can be operated not only under steady-state conditions as the traditional airlift loop reactor, but also under forced periodically operational conditions in which the direction of liquid circulating flow is alternatively changed . Compared with the traditional steady-state operation, both the growth rate and yield of cells are much higher in the forced periodic operation.

J Biotechnol, 1995 Dec 15, 43(3), 213 - 20
Modeling of the aerobic growth of Saccharomyces cerevisiae on mixtures of glucose and ethanol in continuous culture; Dantigny P; A structured model dedicated to fed-batch growth of baker's yeast is detailed in steady-state conditions . The simulated results for aerobic growth on mixtures of glucose and ethanol are provided . The model differentiates and identifies glucose utilisation (either oxido-reductive or by oxidation), state 1 and ethanol oxidation, state 2 . Ethanol can be oxidised when glucose concentration is below a certain value, s(crit), only; ethanol is excreted when glucose concentration exceeds s(crit) . The amount of ethanol co-consumed with glucose is controlled by s(crit) through the transition rate from X1 to X2 . Two major novelties are introduced for modeling glucose metabolism . (1) The specific growth rate on glucose is constant, equal to D(crit), at low glucose concentrations, but follows Monod kinetics at high glucose concentrations . (2) Non-constant yields (i.e., Yx/s and Ye/s) are determined by means of dimensionless groups when the specific growth rate on glucose exceeds D(crit) . The model is developed on experimentally easily accessible parameters found in the literature for Saccharomyces cerevisiae H1022 . A remarkable prediction of the experimental data obtained by Rieger et al . (1983) (J . Gen . Microbiol . 129, 653-661) is highlighted . The simulations suggest that the specific growth rate on ethanol may be underestimated in limited respiratory capacity based models.

Int J Biol Macromol, 1995 Dec, 17(6), 323 - 6
Mitogenic activity of particulate yeast beta-(1-->3)-D-glucan and its water-soluble derivatives; Sandula J et al.; Particulate beta-D-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment . The residual water-insoluble glucan termed cerevan has a beta-(1-->3)-linked backbone with beta-(1-->6)-linked short side chains . In order to achieve water solubility of the glucan, various derivatives were prepared (carboxymethyl-,carboxyethyl-,hydroxyethyl-,sulfoethyl-), and the beta-glucan was oxidized to glucuronoglucan . Their solubility, degree of substitution (DS), and molecular weight distribution (Mw) were compared . The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes . Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan . Of the water-soluble derivatives, sulfoethylglucan was found to be the most active . Of the carboxymethyl derivatives of various DS, the preparation with DS = 0.75 exhibited the highest activity . Water-soluble carboxymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.

Biochemistry, 1995 Nov 28, 34(47), 15496 - 503
Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants; Alam SL et al.; The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ({N82A}CcPCN) has been studied by proton NMR spectroscopy . This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands . His52 is a residue critical to cytochrome c peroxidase's normal function . Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme . For {N82A}CcPCN, proton NMR spectra reveal two significant phenomena . First, similar to results published for the related mutant {N82D}CcPCN {Satterlee, J . D., et al . (1994) Eur . J . Biochem . 244, 81-87}, for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN . Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale . Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation . Varying temperature causes the two enzyme forms to interconvert . Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1 . The equilibrium constant at 20 degrees C is 1.5 . Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.

Biochem Mol Biol Int, 1995 Nov, 37(4), 805 - 11
Formation of fluorophore of yeast D-glyceraldehyde-3-phosphate dehydrogenase in the solution of potassium iodide; He RQ et al.; A new fluorescence property of yeast D-glyceraldehyde-3-phosphate dehydrogenase was detected after it had been incubated in KI solution . The fluorophore formed in Baker's yeast GAPDH fluoresced at 383 nm by excitation at 336 nm . The formation of the fluorophore depended on NAD+ . Modification of Cys-149 with iodoacetic acid influenced some properties of the fluorescence . It appears that the fluorophore may be situated at or near the active site.

Appl Biochem Biotechnol, 1995 Nov, 55(2), 123 - 32
Baker's yeast . Some biochemical aspects and their influence in biotransformations; Pereira Rde S; Baker's yeast is becoming an important reagent for organic synthesis . However, on many occasions, there are problems with the experimental reproducibility, which in general is the result of the different origins of baker's yeasts . In order to explain these differences, NAD (P)+ reduction inside intact living cells from different strains was measured . The method can select cells with better reduction power in a short period of time.

J Biotechnol, 1995 Oct 16, 42(3), 255 - 69
Population balance models of autonomous microbial oscillations; Hjortso MA et al.; Autonomous oscillations in continuous microbial cultures is well documented for the case of baker's yeast, Saccharomyces cerevisiae, for which it has been observed under a range of operating conditions . We have found that autonomous microbial oscillations can be modeled by unstructured population balance models in which a key cell cycle parameter is a function of the environmental conditions, e.g., the concentration of a substrate or product . Although these models are remarkably simple, they can display a wide range of dynamic behaviors . These behaviors include, for binary fission organisms, solutions containing a single synchronous population and, for budding yeasts, two synchronized subpopulations with a period of oscillation similar to that of the cell cycle length, a pattern that has been observed experimentally in S . cerevisiae . Numerical simulations of the model equations also show that complex periodic solutions with periods very different from the cell cycle length are possible . The ability of the population balance approach to accurately describe the available data of yeast culture dynamics will be discussed.

Biochem Mol Biol Int, 1995 Oct, 37(3), 423 - 30
Immunoreactivities of PKC delta, eta, zeta in baker's yeast, lobster and wheat germ; Kuo WN et al.; Varied immunoreactive bands of protein kinase C delta (PKC delta), PKC eta, and PKC zeta were detected in crude extracts of wheat germ, lobster tail meat, and three strains of baker's yeast by analysis of Western blots . Protease-deficient and Fleischmann's Active Dry yeasts exhibited immunoreactivity of PKC delta, whereas wheat germ and Fleischmann's RapidRise yeast displayed immunoreactivities of both PKC delta and PKC zeta . Lobster tail meat showed immunoreactivities of PKC eta and PKC zeta . These positive and negative immunoreactivities reflected evolutionary conservation and divergence, respectively, of these PKC isozymes in eukaryotes.

Curr Genet, 1995 Oct, 28(5), 499 - 501
New alleles of mgm1: a gene encoding a protein with a GTP-binding domain related to dynamin; Backer JS; Three previously described genes that affect baker's yeast (Saccharomyces cerevisiae) mitochondrial DNA (mtDNA) or mitochondrial RNA, tpm2-1, mna1-1, and mgm-1-1, are shown to be alleles of the same gene . This report demonstrates that tpm2-1 does not affect recombination of mtDNA . Therefore, there is no evidence that this dynamin-like protein is involved in movement of mtDNA within a cell.

Appl Environ Microbiol, 1995 Oct, 61(10), 3604 - 8
The relationship between viability and intracellular pH in the yeast Saccharomyces cerevisiae; Imai T et al.; The relationship between viability (cell proliferation activity) and intracellular pH in the yeast Saccharomyces cerevisiae was investigated by using cells that had been deactivated by low-temperature storage, ethanol treatment, or heat treatment . The intracellular pH was measured with a microscopic image processor or a spectrofluorophotometer . At first, the intracellular pH measurements of individual cells were compared with slide culture results by microscopic image processing . A clear correlation existed between the proliferation activity and intracellular pH . Moreover, by spectrofluorophotometry analysis, it was found that there was a relationship between the viability and intracellular pH of brewing yeast under conditions of low external pH (n = 15, r = 0.960, P = 0.001) . This relationship was also observed in baker's yeast (n = 13, r = 0.950, P = 0.001) . On the other hand, when the fluorescein staining method was used in these experiments, the relationship between viability and staining percentage was not observed . From these results, intracellular pH was found to be a sensitive factor for estimating yeast physiology . The possible role of cell deterioration is also discussed.

Arch Biochem Biophys, 1995 Sep 10, 322(1), 39 - 42
Kinetics of the reaction of baker's yeast glucose-6-phosphate dehydrogenase with 5,5'-dithiobis(2-nitrobenzoic acid); Adediran SA et al.; The kinetics of the reaction of baker's yeast glucose-6-phosphate dehydrogenase with excess 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were studied at pH 8.5 and 30 degrees C and at constant ionic strength of 0.01 and in the absence and in the presence of NADP+ or glucose 6-phosphate . The reaction follows pseudo-first-order kinetics irrespective of whether these substrates are absent or present . The observed pseudo-first-order rate constant is reduced in the presence of NADP+ or glucose 6-phosphate but on a molar basis, glucose 6-phosphate is more effective than NADP+ in protecting the sulfhydryl groups of the enzyme against the reaction with DTNB . In the presence of NADP+, the observed pseudo-first-order rate constant decreases to a constant, but finite, value at a saturating coenzyme concentration . The effect of NADP+ on the rate constant is consistent with the presence of noninteracting coenzyme binding sites in baker's yeast glucose-6-phosphate dehydrogenase.

Obstet Gynecol, 1995 Sep, 86(3), 326 - 9
Saccharomyces cerevisiae vaginitis: transmission from yeast used in baking; Nyirjesy P et al.; OBJECTIVE: To determine whether vaginitis due to Saccharomyces cerevisiae can be caused by exposure to exogenous sources of baker's yeast . METHODS: Eight women with S cerevisiae vaginitis were identified from a cohort of women referred for the evaluation of chronic vaginal symptoms . In those with high-level exposure to exogenous sources of S cerevisiae, isolates from the vagina and those sources were sent in a blinded fashion for contour-clamped homogeneous electric-field electrophoresis . RESULTS: Four women from a cohort of approximately 750 referred patients had high-level exposures to S cerevisiae . In one of these patients, electrophoresis analysis revealed similarities between the strains isolated from her vagina, her husband's fingers, and the yeast he used in his pizza shop . CONCLUSION: Saccharomyces cerevisiae vaginitis can be the result of the inoculation of this yeast from exogenous sources.

Experientia, 1995 Aug 16, 51(8), 834 - 7
Baker's yeast, an attractant for baiting traps for Chagas' disease vectors; Guerenstein PG et al.; We tested the attraction of volatile compounds, produced by the aerobic growth of Saccharomyces cerevisiae on saccharose for Triatoma infestans . For these tests, we exploited the behavioural characteristic of these haematophagous insects of dropping when searching for food . In olfactometer assays, yeast cultures activated and attracted bugs as effectively as a mouse . The attraction of the cultures was significantly reduced when the carbon dioxide released was partially eliminated using potassium hydroxide . Yeast cultures were also tested as lures in a novel trap device . A baited device for trapping Chagas' disease vectors using the behavioural peculiarities of T . infestans and this simple attractant is described.

Biochem Mol Biol Int, 1995 Aug, 36(5), 957 - 63
Immunoreactivity of PKC gammalambda and RACK1 in baker's yeast, lobster and wheat germ; Kuo WN et al.; Varied patterns of immunoreactive bands of protein kinase C gamma (PKC gamma) and receptor for activated C-kinase-1 (RACK1) were detected by analysis of Western blots in crude extracts of wheat germ, lobster tail meat, and three strains of baker's yeast . Anti-PKC lambda also reacted with wheat germ and yeast extracts, but failed to react with the lobster extract . The findings may implicate a regulatory role and an evolutionary conservation of these PKC isoenzymes and their receptor proteins in eukaryotes.

Biokhimiia, 1995 Jul, 60(7), 1089 - 94
{Some properties of multiple forms of transketolase from baker's yeast}; Filippov MIu et al.; It has been shown that transketolase A does not differ from the enzyme earlier described in the literature by a number of properties (lack of intersubunit disulfide bonds, identical number of sulfhydryl groups, two values of Km for thiamine pyrophosphate and identity of their absolute values) . Transketolase C subunits are linked together by disulfide bonds; their total number in the enzyme molecule is five (transketolase C-1) or six (transketolase C-2) . The Km values of transketolase C-1 for thiamine pyrophosphate are commensurate with those of transketolase A . Transketolase C-2 has only one Km value for thiamine pyrophosphate which is close to one of the two Km values for transketolase A . The maximal rate values for transketolases C-1 and C-2 with dihydroxyacetone as substrate differ by more than one order of magnitude.

Biochem Mol Biol Int, 1995 Jul, 36(3), 669 - 77
Covalent immobilization of invertase and horseradish peroxidase on concanavalin A-Seralose via carbohydrate moieties; Husain S et al.; Invertase from Baker's yeast (Saccharomyces cerevisiae) and Horseradish peroxidase (HRP) were covalently immobilized on Concanavalin A precoupled to Seralose via carbohydrate moieties . Covalent coupling of glycoenzymes was achieved by periodate induced aldehydic groups of glycosyls with amino groups of Concanavalin A, at different pH values . A bifunctional reagent such as glutaraldehyde crosslinks the glycoenzymes with lectin both intra and intermolecularly . Therefore, attempts were made to introduce covalent linkages between glycoenzymes and Concanavalin A-Seralose without intramolecular crosslinking in either . The immobilized preparations of glycoenzymes exhibited high yield of immobilization and eta value . About 90 and 85% covalent coupling could be observed in invertase and HRP at pH 7.0 respectively, as determined by treatment with 0.5 M methyl alpha-D-mannopyranoside . All immobilized glycoenzyme preparations exhibited marked stabilization towards thermal inactivation.

Prikl Biokhim Mikrobiol, 1995 Jul-Aug, 31(4), 458 - 62
{The effect of autolysis on characteristics of amino acid mixtures, obtained using ethanol-assimilating yeasts}; Belousova NI et al.; The influence of the enzyme preparation - macerate, concentration of the yeast biomass in the reaction medium, and chloroform on the autolysis of the ethanol assimilating yeast Saccharomyces cerevisiae BKM-Y-2656, BKM-Y-2465, and the baker's yeast r . L-2 was studied . Amino acid mixtures were isolated from the yeast autolysates . The use of macerate and a decrease in the biomass concentration were shown to increase the yield of the amino acid mixtures . The use of chloroform positively influenced the baker's yeast autolysis . Conditions of the joint action of these factors on the intensification of the process of the amino acid mixtures preparation based on the autolysis were determined.

Appl Environ Microbiol, 1995 Jun, 61(6), 2113 - 21
Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose; Gasent-Ramirez JM et al.; Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized . The strains, which originally lacked alpha-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV1-SMR1 allele of the ILV2 gene and a MEL1 gene . The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media . The alpha-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel . The yield, YX/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel . When molasses was used, YX/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls . Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci . Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n . Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Mycol, 1995 Jun, 19(2), 137 - 52
Endoplasmic reticulum subcompartments in a plant parasitic fungus and in baker's yeast: differential distribution of lumenal proteins; Bachem U et al.; His-Asp-Glu-Leu (HDEL)-bearing proteins were quantified in different endoplasmic reticulum (ER) subcompartments of Saccharomyces cerevisiae and the plant parasite Uromyces viciae-fabae by immuno-electron microscopy (immuno-EM) . In both fungi, the immunogold labeling of these proteins within the ER was three times greater than within the nuclear envelope . In U . viciae-fabae, the ER in germinating uredospores differed from the ER in fungal structures produced within the plant, e.g., haustoria . In haustoria, the cisternal ER differentiated large tubular-vesicular complexes (TVC) . TVC contained higher levels of HDEL-bearing proteins than ordinary ER cisternae . ELISA readings also indicated an increased concentration of these proteins in isolated haustoria compared to germinating uredospores . In S . cerevisiae, the ER was differentiated into cortical and internal regions . Immuno-EM revealed that labeling of the binding protein (BiP) was lower in the ER of the cell cortex . Heat shock increased BiP signals, but the relative distribution within the ER did not change . Our results suggest that ER subcompartments can be differentiated by immunogold labeling of proteins with a retention signal . In special cases, such as in the parasitic phase of rust fungi, these proteins accumulate to higher levels in ER subcompartments, probably as a response to plant-induced stress.

Anal Biochem, 1995 May 20, 227(2), 351 - 62
Effects of solutes on optical properties of biological materials: models, cells, and tissues; Chance B et al.; Perturbations of scattering background for absorbance measurements by photon diffusion techniques complicate algorithms used for determining the concentration of blood and the saturation of hemoglobin in tissues . In order to better define these perturbations, we have undertaken a study of the effect of solutes, some of physiological importance, upon the scattering of three types of model systems: a lipid vessel suspension (Intralipid), a cell suspension (Baker's yeast), and a tissue (perfused liver) . A simple formula relates absorbancy change to proportional changes of the input/output separation rho and the square root of mu a and mu's in relation to a relevant model system . Thus, absorbance changes at 850 nm slopes and intercepts are measured as a function of rho; the absorption and reduced scattering coefficient, mu a and mu's, are calculated . We studied each of these cases as a function of the perturbation induced by low-molecular-weight polyhydroxy solutes, generally sugars (mannitol, fructose, sucrose, and glucose), alcohols (propanediol and methanol), and electrolytes (sodium and potassium chloride) . Dilatometric studies indicate volume changes of the solvent system and afford correction factors . The slopes are approximately +/- 0.5 x 10(-4) OD/mM solute per centimeter separation of input/output per percent scatter (yeast or Intralipid) and indicate possible physiological detection of solutes in tissues in the millimolar range . The large optical effects of temperature upon the solute effect on model systems and of osmotic and perfusion pressures on the perfused liver further complicate the possibility of quantitative in vivo studies of these solutes.

Biochem Mol Biol Int, 1995 May, 36(1), 1 - 12
Changes in RNA of Saccharomyces cerevisiae during thermal cellular dehydration; He RQ et al.; Changes in RNA of Baker's yeast have been studied during a thermal dehydration, through which the cellular moisture decreased from 70% to 9%, followed by a mortality of cells being less than 8%, and by consumption of the cellular glycogen . Simultaneously, the RNA of 4.2s and 5.8s increase, however, the RNA of 12.0s and 18.0s decrease during the thermal cellular dehydration . The contents of the low molecular mass RNA may not be affected by incubation of chloromycetin and penicillin in media during the fed-batch culture . It suggests that RNA play a part during the cellular thermal dehydration.

Int J Immunopharmacol, 1995 May, 17(5), 385 - 92
The effects of colony stimulating factors on human monocyte cell function; Bober LA et al.; We used a panel of functional assays to compare directly the pattern and potency of GM-CSF and M-CSF on monocyte activity associated with cell-mediated immune defense . GM-CSF and M-CSF were found to be equivalent both in their capacity to stimulate human monocyte functions in vitro and in their pattern of monocyte activation . The two CSFs were effective in inducing monocyte chemotaxis towards either fMLP or LTB4 at equivalent concentrations across a panel of donors . GM-CSF and M-CSF demonstrated equipotency in the induction of monocyte phagocytosis of heat-killed baker's yeast and in the regulation of the hexose-monophosphate shunt (NBT reduction) . Both were also found to be equivalent in preventing steroid (dexamethasone)-induced suppression of monocyte anti-bacterial (Candida albicans) and anti-fungal (Staphylococcus aureus) phagocytic capacities . GM-CSF was somewhat more effective than M-CSF in stimulating monocyte C . albicans killing at a lower E:T ratio.

Biosci Biotechnol Biochem, 1995 Apr, 59(4), 602 - 9
Inhibitory activity of 8-azadecalin derivatives towards 2,3-oxidosqualene:lanosterol cyclases from baker's yeast and pig's liver; Hoshino T et al.; The inhibitors of 2,3-oxidosqualene:lanosterol cyclase were investigated by comparative studies between pig's liver and Baker's yeast . The fundamental skeleton of the inhibitors was 8-azadecalin . To the nitrogen atom, an isoprenoid-like chain {nerylacetone (Z-form), geranylacetone (E-form) or its hydrogenated form} was attached by the reaction of reductive amination with NaCNBH3 . Among the three forms, the Z-isomer was the most potent inhibitors toward both the pig's liver and yeast cyclases . To examine the effect of carbon chain length (lipophilicity), various fatty acids (C6-C18) were appended to the 8-azadecalin derivatives . Strong inhibitory activity was observed for those compounds having carbon chains around C12 . Interestingly, the amide compounds (not the carbocationic intermediate) exhibited remarkably strong inhibition toward the liver cyclase, whereas they had an insignificant effect on the yeast cyclase (about 10(2)-fold less active) . The yeast cyclase needed the amine functionality (carbocationic intermediate), which was prepared by using LiAlH4 from the corresponding amides, to exhibit potent inhibition . We found that N-dodecyl-8-aza-4,4,10 beta-trimethyl-trans-decal-3 beta-ol (7i) was the most potent inhibitor (IC50 = 1 microM) toward the yeast cyclase amongst any known material . Kinetic studies showed that the inhibition pattern was dependent only on whether the side chains on the 8-azadecalin were linear or branched; the compounds having isoprenoid-like chains were non-competitive inhibitors, while those having linear hydrocarbon chains (amides or amines) were competitive inhibitors.

Res Commun Mol Pathol Pharmacol, 1995 Apr, 88(1), 123 - 6
Metabolism of 3-acetylcoumarin in rat 9000xg supernatant fraction (S-9)and baker's yeast; Takeshita M et al.; In the incubation of 3-acetylcoumarin in rat liver supernatant fraction (S-9), four metabolites were isolated, and two of which were inseparable diastereomeric mixture as a major product . However, when 3-acetylcoumarin was fermented with baker's yeast (Saccharomyces cerevisiae), only two compounds were obtained as the same as the above mixture . The structures of those metabolites were confirmed by NMR and Mass spectra . However, the above metabolites in rat 9000xg supernatant fraction were not induced by phenobarbital, nor inhibited with SKF-525A at all.

Anal Chem, 1995 Feb 15, 67(4), 750 - 4
Speciation of methylmercury and Hg(II) using baker's yeast biomass (Saccharomyces cerevisiae) . Determination by continuous flow mercury cold vapor generation atomic absorption spectrometry; Madrid Y et al.; Baker's yeast cells (Saccharomyces cerevisiae) were successfully used to selectively separate methylmercury and Hg(II) . Several parameters affecting the degree of biosorption and the binding kinetics of methylmercury and Hg(II) were evaluated: solution pH, temperature, incubation time, amount of biomass and analyte, and presence of foreign ions . Methylmercury is immediately bound to the yeast cells over a wide pH and temperature range . The fraction of methylmercury bound was in all cases 100% and was unaffected by the parameters mentioned above . Hg(II) has less affinity for yeast cells and remains in solution, although the percentage of Hg(II) bound to the cell does increase at high incubation time (3 h) and biomass . Of the foreign ions tested, chloride at high concentrations strongly increases the Hg(II) binding efficiency . Methylmercury and Hg(II) are quantitatively separated under optimum conditions, i.e., 30 min incubation time at pH 7.0 and 37 degrees C . The results were compared with those obtained using a purified S . cerevisiae isolate, and no significant differences were observed . Our work suggests that the cell rapidly reduces CH3Hg+ to more volatile species, such as Hg(I) or Hg0, whereas Hg2+ is slowly bound and reduced, perhaps because of the different toxicities of the two species . The method was applied to the selective determination of CH3Hg+ and Hg(II) in spiked water samples . In all cases good recoveries were obtained.

Liver, 1995 Feb, 15(1), 39 - 44
Mannan-binding protein and complement dependent opsonization in alcoholic cirrhosis; Homann C et al.; Mannan-binding protein is synthesized by the liver and functions in first-line host defence by opsonizing mannose-rich microorganisms due to activation of the classical complement pathway independent of Clq, and by an intrinsic ability to opsonize and mediate phagocytosis . We have investigated whether the increased susceptibility to bacterial infections in patients with cirrhosis could be explained by low plasma concentrations of mannan-binding protein and impaired complement-dependent opsonization . We examined 51 patients with compensated alcoholic cirrhosis, 34 who were decompensated and 16 healthy controls . Irrespective of group, we found a significant correlation (p < 0.05) between plasma mannan-binding protein concentration and deposition of the complement opsonin C4 on mannan from baker's yeast . In contrast to what was expected, this kind of opsonization and plasma levels of mannan-binding protein were significantly increased in the patients with decompensated cirrhosis (p = 0.01 and p = 0.007, respectively) . A significant correlation (0 < 0.05) was found between mannan-binding protein and erythrocyte sedimentation rate, fibrinogen and haptoglobin in these patients . Though the correlations were weak (rho = 0.49, rho = 0.48 and rho = 0.40, respectively), the elevated levels of mannan-binding protein in the patients with decompensated cirrhosis may reflect an acute phase reaction . It is concluded that plasma levels of mannan-binding protein are increased in patients with decompensated cirrhosis and that complement-dependent opsonization of mannan does not seem to be compromized in patients with alcoholic cirrhosis.

J Dairy Sci, 1995 Feb, 78(2), 397 - 403
Baker's yeast effluent as a liquid feed for dairy cows and heifers; Blauwiekel R et al.; Liquid effluent from baker's yeast production was fed to dairy cows to determine whether effluent is an acceptable feed and whether it affects milk yield or composition . Effluent averaged 5.4% DM and 6.1% N (DM basis) . In experiment 1, 20 Holstein cows were offered effluent free choice or no effluent for 8 wk . Milk yield, composition, and group feed intake were measured . In Experiment 2, 20 cows were fed effluent blended into the TMR (11.3 L/d per cow) or no effluent for 4 wk . In Experiment 3, six groups of 6 heifers were offered free choice or no effluent . Free choice intake of effluent peaked at 15 L/d per cow at wk 3 but declined to 5.3 L/d per cow by wk 6 . Milk and 3.5% FCM yields were not affected by effluent regardless of feeding system . Milk protein and fat percentages were higher for cows offered effluent free choice . Milk protein percentage and yield were lower with effluent in the TMR . Intake of effluent by heifers was 1.05 L/d, and effluent did not affect DMI or weight gain . Acceptability of yeast effluent fed free choice is poor, but when yeast is blended into the TMR, cows consume effluent without adverse effects on milk yield or DMI.

Appl Environ Microbiol, 1995 Feb, 61(2), 639 - 42
Isolation of a cold-sensitive fermentation mutant of a baker's yeast strain and its use in a refrigerated dough process; Kyogoku Y et al.; Conventional baker's yeast converts sugars in dough into CO2 and ethanol to a significant extent when the dough is stored for days at 5 degrees C . We have isolated Csf (cold-sensitive fermentation) mutants of a commercial baker's yeast by a selection method including as the critical step a nystatin treatment to mutagenized cells at 10 degrees C in the presence of antimycin A . The fermentative activity of mutant strain CSF3 was substantially zero at 5 degrees C and one-fifth that of the parent at 10 degrees C but was restored to the same level as the parental activity at 25 to 40 degrees C . In contrast with the parent, the mutant strain normally produced white bread dough and butter roll dough even after the dough was stored for a week at 5 degrees C.

Appl Environ Microbiol, 1995 Feb, 61(2), 630 - 8
Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts; Codon AC et al.; To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken . When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6% . Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase . Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C . Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected . Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts . When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters . This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Annu Rev Genet, 1995, 29, 651 - 74
Yeast transcriptional regulatory mechanisms; Struhl K; Transcriptional regulation directly influences many biological phenomena such as cell growth, response to environmental change, development of multicellular organisms, and disease . Transcriptional regulatory mechanisms are fundamentally similar in eukaryotic organisms (93) . Components of the basic RNA polymerase II (Pol II) machinery are highly conserved and, in some cases, functionally interchangeable . Transcription factors with similar structures and DNA-binding specificities are found throughout the eukaryotic kingdom, and acidic activation domains stimulate transcription across a wide range of species . Complex promoters with multiple protein binding sites are typical in all eukaryotic organisms, and efficient transcription generally requires the combinatorial and synergistic action of activator proteins that function at long and variable distances from the mRNA initiation site . Molecular mechanisms of eukaryotic transcriptional regulation have been elucidated from the studies that involve a wide variety of genes, promoters, proteins, organisms, and experimental approaches . This review focuses on transcriptional regulatory mechanisms in the baker's yeast Saccharomyces cerevisiae . Studies in yeast have emphasized powerful genetic approaches that are not available in other eukaryotic organisms . As a consequence, yeast is particularly amenable for analyzing transcriptional regulatory mechanisms in vivo under true physiological conditions . Furthermore, classical and molecular yeast genetics has permitted the discovery and functional characterization of transcriptional regulatory proteins that were not identified in biochemical studies . Thus, genetic analysis in yeast has often generated information complementary to that obtained from biochemical studies of transcription in vitro, and it has provided unique insights into mechanisms of eukaryotic transcriptional regulation.

Appl Environ Microbiol, 1995 Jan, 61(1), 109 - 15
Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells; Van Dijck P et al.; The trehalose content in laboratory and industrial baker's yeast is widely believed to be a major determinant of stress resistance . Fresh and dried baker's yeast is cultured to obtain a trehalose content of more than 10% of the dry weight . Initiation of fermentation, e.g., during dough preparation, is associated with a rapid loss of stress resistance and a rapid mobilization of trehalose . Using specific Saccharomyces cerevisiae mutants affected in trehalose metabolism, we confirm the correlation between trehalose content and stress resistance but only in the absence of fermentation . We demonstrate that both phenomena can be dissociated clearly once the cells initiate fermentation . This was accomplished both for cells with moderate trehalose levels grown under laboratory conditions and for cells with trehalose contents higher than 10% obtained under pilot-scale conditions . Retention of a high trehalose level during fermentation also does not prevent the loss of fermentation capacity during preparation of frozen doughs . Although higher trehalose levels are always correlated with higher stress resistance before the addition of fermentable sugar, our results show that the initiation of fermentation causes the disappearance of any other factor(s) required for the maintenance of stress resistance, even in the presence of a high trehalose content.

Appl Microbiol Biotechnol, 1995 Jan, 42(5), 797 - 806
Biosorption of heavy metals by Saccharomyces cerevisiae; Volesky B et al.; Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions . Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4-5 . Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu approximately (Cd) > U . Non-living brewer's yeast biomass accumulated 0.58 mmol U/g . The best biosorbent of zinc was non-living baker's yeast (approximately 0.56 mmol Zn/g) . Dead cells of S . cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures . Biosorption of uranium by S . cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact . Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine needle-like crystals both on the inside and outside of the S . cerevisiae cells.

C R Acad Sci III, 1995 Jan, 318(1), 43 - 50
{G1 cyclin degradation and cell differentiation in Saccharomyces cerevisiae}; Barral Y et al.; The baker's yeast Saccharomyces cerevisiae can undergo pseudohyphal differentiation upon limited starvation for nitrogen . This differentiation is characterized by a hyperpolarized cell growth that gives rise to elongated cells . These elongated cells can form chains that penetrate an agar surface . The study of the grr1 mutant, affected in the degradation of the G1 cyclins, showed that the stabilization of Cln1 and Cln2 leads to a similar hyperpolarized cell growth . We suggest that G1 cyclin stability is a key element controlling cellular morphogenesis . Examination of G1 cyclin turnover during pseudohyphal growth strongly supports this hypothesis . Saccharomyces cerevisiae is thus an interesting model for studying the interconnections between cell cycle control and cellular differentiation.

Cytobios, 1995, 81(325), 103 - 8
Immunoreactivity of S-100 protein in baker's yeast, lobster, and wheat germ; Kuo WN et al.; The immunoreactivity of S-100 proteins in three strains of yeast and wheat germ was observed by analysis of Western blotting with rabbit anti-bovine S-100 . A high level of activity was exhibited in wheat germ, whereas a very low level was found in lobster tail meat . The occurrence of multiple bands may be due to the interactions between S-100A and S-100B and/or other molecules . The highly evolutionally conserved S-100 may play an important role in cellular signal transduction and cell growth in yeast and wheat germ.

Cytobios, 1995, 81(326), 175 - 80
Calpain immunoreactivity in baker's yeast, lobster and wheat germ; Kuo WN et al.; The immunoreactivities of mu-calpain and m-calpain in wheat germ, lobster tail meat, and three strains of yeast were analysed by Western blotting using mouse anti-mu-calpain and rabbit anti-m-calpain . The occurrence of multiple bands may be due to either autolyses or the interactions between the calpains and other molecules . The results suggest not only a ubiquitous distribution and a universal regulatory role of calpain in eukaryotes, but also an evolutional conservation of calpain.

J Chromatogr B Biomed Appl, 1994 Nov 18, 661(2), 193 - 204
Separation and characterization of the main methylated nucleobases from nuclear, cytoplasmic and poly (A)+ RNA by high-performance liquid chromatography and mass spectrometry; Cong HN et al.; We were able to detect nine methylated nucleobases (3-methyluracil, 1-, 2-, 3- and 7-methylguanine, 1-, 2-, 3- and 6-methyladenine) in RNA from rat and calf liver, baker's yeast, Torula and Euglena cells by using reversed-phase high-performance liquid chromatography and thermospray mass spectrometry . Total cellular, nuclear, cytoplasmic and poly (A)+ RNA from rat liver showed marked methylation, mainly of 1- and 3- methylguanine, and 3- and 2-methyladenine . These bases were especially abundant in nuclear RNA and, to a lesser extent, in poly (A)+ RNA . In contrast, 7-methylguanine and 6-methyladenine were poorly represented in poly (A)+ RNA.

FEMS Microbiol Lett, 1994 Nov 15, 124(1), 29 - 34
Cloning and sequencing of phospholipase B gene from the yeast Torulaspora delbrueckii; Watanabe Y et al.; The extracellular phospholipase B gene from baker's yeast Torulaspora delbrueckii was cloned and sequenced . Analysis of DNA sequence data revealed an open reading frame (ORF) encoding a 649-amino acid protein, that included amino acid sequences obtained from the purified enzyme . Comparison of these sequence data with the N-terminal amino acid sequence of the enzyme indicated that this secreted protein is synthesized as a large precursor with a 21-amino acid N-terminal extension to the mature enzyme of 628 amino acids . A homology search was carried out between phospholipase B from T . delbrueckii and Penicillium notatum . The deduced amino acid sequence of the cloned phospholipase B was homologous (about 50% identity) to phospholipase B from P . notatum, and contained six conserved regions . The transcriptional level of mRNA of the phospholipase B gene was higher in the cells from early exponential and stationary phases.

Plant J, 1994 Nov, 6(5), 697 - 706
A phloem-specific sucrose-H+ symporter from Plantago major L . supports the model of apoplastic phloem loading; Gahrtz M et al.; In this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H+ symporter from Plantago major is described . This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non-vascular tissue . A cDNA library was constructed from poly(A)+ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs . The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues . The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters . Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae . The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells . Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200-fold . This transport was sensitive to uncouplers or SH-group inhibitors . Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-c-oxidase . In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H+ symporter . The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels . The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H+ symporter.

Prep Biochem, 1994 Nov, 24(3-4), 289 - 96
Rapid purification of yeast cytoplasmic fumarate reductase by affinity chromatography on blue sepharose CL-6B; Muratsubaki H et al.; The rapid and effective purification of soluble fumarate reductase from baker's yeast achieved by Blue Sepharose CL-6B chromatography . Cibacron Blue F3GA, the chromophore of Blue Sepharose, inhibited the activity of fumarate reductase . The enzyme bound to the column was selectively eluted by flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin . The purified enzyme was essentially homogeneous as indicated by polyacrylamide gel electrophoresis under nondenaturing conditions and under denaturing conditions in sodium dodecylsulfate . By this procedure, the enzyme could be rapidly purified with high yield from yeast cells.

Biochem Mol Biol Int, 1994 Nov, 34(5), 1049 - 54
Intersubunit disulfide bonds in the molecule of baker's yeast transketolase; Solovjeva ON et al.; It has previously been shown that baker's yeast trans ketolase has no intersubunit disulfide bonds and is able to dissociate reversibly into subunits at a low apoprotein concentration in solution . By contrast, in the present work it was found that in the molecule of transketolase C (a newly discovered form of the enzyme) subunits are bound to each other by disulfide bonds.

J Chromatogr A, 1994 Oct 28, 684(1), 45 - 54
Interaction of Cibacron blue with polymers: implications for polymer-shielded dye-affinity chromatography of phosphofructokinase from baker's yeast; Galaev YuI et al.; Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena . Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the dye . The difference spectra for the PVP-dye complex was typical of "electrostatic interaction spectra" at low ionic strength and typical of "hydrophobic interaction spectra" in the presence of 1.5 M KCl . The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites . One dye molecule was bound by a PVP segment with a molecular mass of 1000-1300 . Regardless of the size of the polymer molecules, the binding constants were in the micromolar range . Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP . One dye molecule was bound by a polymer segment with a molecular mass of about 10,000 . The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded dye-affinity chromatography . This concept was successfully applied to the chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast . Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.

Appl Environ Microbiol, 1994 Oct, 60(10), 3499 - 502
Construction from a single parent of baker's yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs; Nakagawa S et al.; From a freeze-tolerant baker's yeast (Saccharomyces cerevisiae), 2,333 spore clones were obtained . To improve the leavening ability in lean dough of the parent strain, we selected 555 of the high-maltose-fermentative spore clones by using a method in which a soft agar solution containing maltose and bromocresol purple was overlaid on yeast colonies . By measuring the gassing power in the dough, we selected 66 spore clones with a good leavening ability in lean dough and a total of 694 hybrids were constructed by crossing them . Among these hybrids, we obtained 50 novel freeze-tolerant strains with good leavening ability in all lean, regular, and sweet doughs comparable to that of commercial baker's yeast . Hybrids with improved leavening ability or freeze tolerance compared with the parent yeast and commercial baker's yeasts were also obtained . These results suggest that hybridization between spore clones derived from a single parent strain is effective for improving the properties of baker's yeasts.

Biofizika, 1994 Sep-Oct, 39(5), 919 - 22
{Detection of nitric oxide formed from L-arginine in the murine stomach in vivo by EPR}; Mikoian VD et al.; Suspension of baker's yeast loaded with a specific trap of nitric oxide (NO), a complex of Fe2+ with diethyldithiocarbamate (DETC), was used for the detection of NO formed in mouse stomach at its adaptive relaxation or under action of ethanol in vivo . NO formation was determined by the increase of intensity of the EPR signal due to mononitrosyl iron complex (MNIC) with DETC which appeared in yeast cells infused into the stomach . An increase of signal intensity was observed in stomach preparations isolated from mice when the doses of yeast suspensions injected p/o into mouse stomach for 40 min increased up to 1.5 ml or when 25% ethanol solutions were added p/o to the stomach . These effects were attenuated when NO-synthase inhibitor, NG-nitro-L-arginine was i/p injected into mice.

Clin Exp Allergy, 1994 Sep, 24(9), 836 - 42
Immediate hypersensitivity to bakery, brewery and wine products in yeast-sensitive atopic dermatitis patients; Kortekangas-Savolainen O et al.; Ultrafiltered (> 1000 Da) samples of beer, aged red wine, young white wine, sparkling wine and extracts of fresh wheat bread and dried rye bread were analysed by skin-prick test (SPT), radioallergosorbent test (RAST) inhibition, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to find out if they contain Saccharomyces cerevisiae (S . cerevisiae, baker's yeast) allergens . Serum pool consisting of S . cerevisiae positive sera was used in the assays . The results were compared with freeze-dried reference S . cerevisiae and cereal antigens . The beer, bread, red wine and sparkling wine extracts elicited immediate reactions . However, no evident correlation with suspected symptoms was observed . White wine extract caused reactions in four out of six atopic dermatitis (AD) patients with symptoms, and in five out of seven symptom-free AD patients and in two of the 24 controls . In SDS-PAGE, protein bands were found in wheat and rye bread extracts and beer . In IgE immunoblotting, however, no staining was seen with the S . cerevisiae positive sera suggesting that they were of cereal origin . In white wine and champagne extracts a non-specific staining was seen in the region 20 kDa representing, e.g . lectin-like activity . No baker's yeast antigen could be detected in brewery and bakery products with IgE-immunoblotting even in the excessively concentrated extracts . The IgE mediated allergy to baker's yeast alone should thus not lead to denial of bakery, brewery and wine products.

Eur J Biochem, 1994 Aug 15, 224(1), 81 - 7
The effect of the Asn82-->Asp mutation in yeast cytochrome c peroxidase studied by proton NMR spectroscopy; Satterlee JD et al.; Proton NMR studies of the mutant of baker's yeast cytochrome c peroxidase-cyanide with the Asn 82-->Asp mutation ({N82D}cytochrome c peroxidase-CN) are presented and compared to the wild-type enzyme . This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands . His52 is an important participant in the initial hydrogen peroxide decomposition step of cytochrome c peroxidase . In wild-type cytochrome c peroxidase-CN, His52 hydrogen bonds to the neighboring Asn82 peptide carbonyl group and to heme-coordinated cyanide . His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety . The principal result from this study is the observation that three hyperfine-shifted resonances disappear from the spectrum of {N82D} cytochrome c peroxidase-CN compared to the wild-type enzyme . All three absent resonances in {N82D}cytochrome c peroxidase-CN belong to His52 and this leads to the conclusion that the result of the mutation has been elimination of the His52-Asn82 and His52-heme-coordinated cyanide hydrogen bonds.

Biochem J, 1994 Aug 15, 302 ( Pt 1), 95 - 101
Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c; Schejter A et al.; Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast . This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule . The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8 . The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude . In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type . The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2 . These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine {Luntz, Schejter, Garber and Margoliash (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 3524-3528}, because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues . The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

Plant J, 1994 Aug, 6(2), 225 - 33
Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae; Stolz J et al.; Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae . Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells . The C-terminal modification caused no loss of transport activity compared with the wild-type protein . Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana . Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase . Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with {3H}tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of D-glucose into the energized vesicles . STP1-His6 protein is functionally active after solubilization with octyl-beta-D-glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity . Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni(2+)-NTA columns . The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.

Anal Chem, 1994 Jul 15, 66(14), 2420 - 3
Enzymatic profiling of immobilized cells using CZE; Miller KJ et al.; Baker's yeast cells (Saccharomyces cerevisiae) were immobilized on a CZE capillary using a midcapillary frit of novel design . The frit was constructed using a single 50-microns particle with "through pores" . Cells trapped on the capillary were profiled using different amino acid beta-naphthylamide-aminopeptidase substrates . These substrates were introduced sequentially and metabolized for 1 min . After incubation the hydrolysis products were withdrawn by electrophoresis and detected by laser-induced fluorescence . An aminopeptidase profile was produced using just 500 cells . The new CZE-based method was found to have several significant advantages over the older cuvette/fluorometer method.

Plant J, 1994 Jul, 6(1), 67 - 77
SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein; Sauer N et al.; An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H+ symporter . This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2 . Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner . The KM-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors . The VMAX but not the KM-values of sucrose transport depend on the energy status of transgenic yeast cells . The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2 . The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices . A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S . cerevisiae . The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS-polyacrylamide gels.

Acta Chem Scand, 1994 Jun, 48(6), 506 - 10
Stereoselectivity of Baker's yeast reduction of 2-propanones: influence of substituents; Waagen V et al.; The stereoselectivity of Baker's yeast reduction of prochiral alpha-oxygenated 2-propanones has been studied by varying the substrate structure . The 1-hydroxy-3-methoxy-3-propanone 1a was reduced to the corresponding alcohol (R)-2a with 88% enantiomeric excess . Replacing the hydroxy group in 1a with phenoxy or benzyloxy (1b and 1c) gave the alcohols (S)-2b and (S)-2c with 53 and 32% ee, respectively . Reduction of the methyl ketone 1d gave the alcohol (S)-2d with 91% ee . Attempts to improve the enantioselectivity of the reduction of 1c by lowering the substrate concentration or addition of selective reductase inhibitors had only small effect on the enantioselectivity.

Bioorg Med Chem, 1994 Jun, 2(6), 433 - 7
Effect of cyclodextrin on improvement of enantioselectivity in the reduction of ketopantolactone with baker's yeast; Nakamura K et al.; Addition of beta-cyclodextrin improves enantioselectivity dramatically in the reduction of ketopantolactone mediated by baker's yeast . It has been found that the selectivity increases with the decrease in concentration of ketopantolactone in bulk solvent, and beta-cyclodextrin controls its effective concentration . The role of beta-cyclodextrin is discussed.

Bioorg Med Chem, 1994 Jun, 2(6), 395 - 401
Reduction of bicyclo{3.3.1}nonane-2,8-diones with baker's yeast; Mori K et al.; Reduction of bicyclo{3.3.1}nonane-2,8-dione (1) and its homologues 2 and 3 with baker's yeast affords (1R,5S,8S)-8-hydroxybicyclo{3.3.1}nonan-2-one (4) and its higher homologues 5 and 6 with ca 97% e.e . in 72-86% yields . The absolute configuration of 5 was confirmed by the X-ray crystallographic analysis of its camphanic ester 22.

Ann N Y Acad Sci, 1994 May 2, 721, 365 - 73
Two-step cell disruption for the extraction of membrane-associated recombinant protein from Saccharomyces cerevisiae; Chi WK et al.; The use of rDNA technology to express heterologous proteins has been very successful during the last several years . Choice of an expression host is very important in order to retain the biological activity of recombinant proteins . Baker's yeast, Saccharomyces cerevisiae, is a eucaryotic GRAS organism suitable for the expression of biologically active proteins . Specifically, hepatitis B surface antigen (HBsAg) is expressed in baker's yeast . Because the yeast cells need to be disrupted for the recovery of bioactive intracellular proteins and because the protein HBsAg is hydrophobic and has a tendency to become associated with cell membranes, the use of detergent increases the recovery yield . In order to remove most of the contaminants from yeast, a two-step disruption/extraction scheme has been developed that facilitates downstream processing . Furthermore, it also has the advantage of minimizing proteolytic actions on the recombinant protein by removing most of the contaminants and proteases into the supernatant during the first disruption step, while keeping the desired protein in the pellet fraction . Final recovery is then achieved by the extraction process . Parameters affecting the disruption/extraction processes have been discussed.

Biochem J, 1994 Apr 15, 299 ( Pt 2), 347 - 50
The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants; Koshy TI et al.; The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied . Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1 . Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol . These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.

Biochem Mol Biol Int, 1994 Apr, 32(6), 1157 - 60
EPR evidence for nitric oxide formation via L-arginine-dependent way in stomach of mice in vivo; Mikoyan VD et al.; Suspension of baker's yeast loaded with a specific trap of nitric oxide (NO), a complex of Fe2+ with exogenous diethyldithiocarbamate (DETC), was used for the detection of NO formed in mouse stomach at its adaptive relaxation in vivo . NO formation was determined by the increase of intensity of the ERP signal due to trapping of NO in mononitrosyl iron complex with DETC (MNIC-DETC) which appeared in yeast cells infused into the stomach . An increase in signal intensity was observed in stomach preparations isolated from mice when the doses of yeast suspensions injected p/o into mouse stomach for 40 min were increased . The intensity of this signal which was proportional to the concentration of MNIC-DETC in yeast cells was diminished when the NO-synthase inhibitor, NG-nitro-L-arginine was injected i/p into mice.

Chem Pharm Bull (Tokyo), 1994 Apr, 42(4), 802 - 5
Synthesis of optically active alpha-phenylpyridylmethanols with baker's yeast; Takemoto M et al.; We have synthesized optically active alpha-phenylpyridylmethanols by using free baker's yeast (FBY) in water, immobilized baker's yeast (IMBY) in water, and IMBY in hexane.

J Inorg Biochem, 1994 Mar, 53(4), 273 - 80
Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a spectroscopic and thermodynamic study; Ascenzi P et al.; Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated quantitatively by EPR and absorbance spectroscopy . The X-band EPR spectra and the absorption spectra in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c display the same basic characteristics reported for the beef heart cytochrome a3 in cytochrome c oxidase, and horseradish and baker's yeast cytochrome c peroxidase, as well as the high affinity form of oxygen carrying proteins . Values of the dissociation equilibrium constant for nitrosylation of ferrous native and carboxymethylated cytochrome c are 8.2 x 10(-6) M and < or = 5 x 10(-8) M, respectively, at pH 7.0 and 10 degrees C . The results here reported represent clearcut evidence for the nitric oxide-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and allow estimation of the free energy associated to the heme-iron sixth coordination bond (> 10 kJ mol-1, at 10 degrees C).

Mycopathologia, 1994 Mar, 125(3), 129 - 41
Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans; Alloush HM et al.; This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeast Candida albicans . UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration . Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions . SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa . The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg++ concentration was 2 mM . The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate . Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction . 'Ping pong' mechanism has been previously reported for other phosphoribosyltransferases . The enzyme has a low affinity for its substrates (Km = 70.5 and 186 microM for uracil and PRPP, respectively) as compared with those of E . coli and baker's yeast . Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.

Clin Exp Allergy, 1994 Mar, 24(3), 257 - 62
Thermal and storage stability of Saccharomyces cerevisiae (baker's yeast) allergens; Kortekangas-Savolainen O et al.; The effect of storage and high temperatures on the stability of Saccharomyces cerevisiae allergens was studied by immunoblotting . Saccharomyces cerevisiae allergic serum pool and 125I- and galactosidase-labelled anti-IgE were used in the assays . Freeze-dried extracts were reconstituted with saline and with 50% glycerol and then stored at room (+20 degrees C) and refrigerator temperature (+6 degrees C) for different time periods . The stability was better in 50% glycerol at +6 degrees C than at room temperature without glycerol . However, after 1 month, two of the most important allergens of S . cerevisiae, the 48 and 32 kDa protein allergens, lost their IgE-binding capacity even in the extracts stored with 50% glycerol at +6 degrees C . The 45 kDa allergen was, on the other hand, quite stable after storage for 9 months at +6 degrees C . Although the beneficial effect of 50% glycerol was clear, storage at +6 degrees C, even with 50% glycerol should not exceed 1 month for S . cerevisiae extracts . Two commercially available S . cerevisiae extracts in solution with valid expiry dates were also analysed . They had only little allergenic potency, while a freeze-dried extract stored for 8 years showed good allergenic potency . Heating S . cerevisiae extracts resulted in precipitation, the precipitated fraction contained almost all the specific proteins as judged by electrophoresis and IgE detection . The supernatant fraction contained only a few allergens.

Biochem J, 1994 Feb 1, 297 ( Pt 3), 603 - 8
The amino acid sequence of the small monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe; Nairn J et al.; The amino acid sequence of the monomeric 2,3-bisphosphoglycerate (BPG)-dependent phosphoglycerate mutase (PGAM) from the fission yeast Schizosaccharomyces pombe has been determined . Amino acid sequencing of proteolytic fragments of the enzyme showed the S . pombe mutase to be similar in sequence to the tetrameric enzyme of baker's yeast (Saccharomyces cerevisiae) . An S . pombe cDNA library was screened using a PCR fragment generated from two oligonucleotides complementary to sequences encoding the regions at the two active-site histidine residues . The 0.63 kb cDNA encoded an open reading frame of 210 amino acids . This sequence agreed completely with sequences of peptides derived from the purified protein . The amino acid sequence of S . pombe PGAM is 43% identical with that of S . cerevisiae PGAM and shows an equally high degree of identity with BPG-dependent PGAMs from other sources . However, the sequence of the S . pombe enzyme differs from other BPG-dependent enzymes in three important ways: (i) it does not contain the alanine- and lysine-rich sequence of amino acids at the C-terminus which have been proposed to constitute a flexible tail involved in catalysis; (ii) the sequence spanning residues 122-146 (S . cerevisiae PGAM numbering) is not present in the S . pombe PGAM sequence; in the S . cerevisiae PGAM crystal structure this stretch of sequence has been shown to occur as an extended loop, part of which is involved in inter-subunit interactions; (iii) the amino acid sequence in the region of a second S . cerevisiae inter-subunit contact (residues 74-78) shows radical mutations in the S . pombe enzyme.

Eur J Biochem, 1994 Jan 15, 219(1-2), 187 - 93
The role of trehalose synthesis for the acquisition of thermotolerance in yeast . II . Physiological concentrations of trehalose increase the thermal stability of proteins in vitro; Hottiger T et al.; In baker's yeast (Saccharomyces cerevisiae), accumulation of the non-reducing disaccharide, trehalose, is triggered by stimuli that activate the heat-shock response . Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance . Genetic evidence in support of this view is presented in an accompanying paper {De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T . & Wiemken, A . (1993) Eur . J . Biochem . 219, 179-186} . In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance . Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose-6P-dehydrogenase, phosphoglucose-isomerase) as well as enzymes of non-yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro . Trehalose also reduced the heat-induced formation of protein aggregates . The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes . Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose-6P was devoid of any protective effect . Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate . The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.

J Biotechnol, 1994 Jan 15, 32(1), 45 - 57
Transient behaviour of baker's yeast during enforced periodical variation of dissolved oxygen concentration; Abel C et al.; The aim of the investigation was to find out the influence of the variation of the dissolved oxygen concentration in the microenvironment of yeast cells on their physiological behaviour in small laboratory reactors and estimate their behaviour in large industrial reactors . Since the morphology of the laboratory and industrial yeasts differed considerably, their transient behaviour was investigated and compared . For this purpose, the strain Saccharomyces cerevisiae H620 and an industrial strain were cultivated on synthetic as well as on complex medium in batch operation during periodical variation of the dissolved oxygen concentration, monitoring the most important key parameters . Also the yeast was cultivated in batch as well as in continuous operation, the cell-containing culture medium was recirculated through a nonaerated loop at different recirculation rates (residence times of the cells in the loop), and the key operation variables were monitored . It was found that the transient behaviour of laboratory and industrial yeasts differed slightly . Since cells growing in batch culture are more sensitive to dissolved oxygen concentration variation than cells growing in continuous culture, the transient behavior of cells cultivated in batch operation varied from those in continuous operation . If the anaerobic phase was longer than 1 min, ethanol was produced . However, it was consumed during the aerobiosis again, provided that phase was considerably longer than the anaerobic phase . This means that the yeast cultivation was not influenced by the periodic operation of the dissolved oxygen . Judging from the measurements in large stirred tank and airlift tower loop reactors, in general, the cells would spend more time in the aerobic than in the anaerobic flow region, and they would spend less than 1 min in the anaerobic flow region . Therefore, no considerable effect of the periodically varied dissolved oxygen concentration on the cell cultivation can be expected in large-scale reactors . In the stirred tank-loop-system at high pumping rates/high frequencies of periodically varied dissolved oxygen concentration, unexpectedly, the formation of ethanol was observed, which might be caused by stress imposed on the cells.

Carbohydr Res, 1994 Jan 3, 251, 89 - 98
A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization, and mode of action; Maruyama Y et al.; A 1,2-alpha-D-mannosidase was purified to homogeneity from the culture supernatant of Bacillus sp . M-90, which was isolated from soil by enrichment culture on baker's yeast mannan . The purified enzyme had M(r) 380,000 Da, and was comprised of two apparently identical 190,000 Da subunits . It had a neutral optimum pH (7.0) and an isoelectric point of 3.6 . The enzyme was highly specific for alpha 1,2-linked D-mannose oligosaccharides . An N-linked high-mannose type oligosaccharide, Man9GlcNAc2, was a good substrate, yielding Man5GlcNAc2, and the alpha 1,2-linked side chains of Saccharomyces cerevisiae mannan were also specifically hydrolyzed by the enzyme . p-Nitrophenyl alpha-D-mannopyranoside and 1,2-alpha-D-mannobiitol were not hydrolyzed at all . Calcium ion, 1-deoxyman-nojirimycin, and swainsonine had no effect on the enzyme, but the activity was completely inhibited by EDTA . The mode of action on alpha 1,2-linked mannotetraose indicated that the enzyme is an exo-1,2-alpha-D-mannanase.

J Clin Microbiol, 1994 Jan, 32(1), 17 - 23
Diagnostic value of anti-Candida enolase antibodies; van Deventer AJ et al.; An immunodominant antigen with enolase enzyme activity was purified and used for the development of an assay to detect antibodies directed against this antigen in sera from patients with either invasive candidiasis or Candida colonization . The Au enzyme-linked immunosorbent assay established with the Candida enolase antigen was able to discriminate significantly between invasive candidiasis and colonization in both immunocompetent and immunodeficient groups of patients . The test had a sensitivity of 50% and a specificity of 86% in the immunocompetent patient group . In the immunodeficient patient group, a sensitivity of 53% and a specificity of 78% were established . Antibody levels determined by a counterimmunoelectrophoresis assay with the same set of sera resulted in a better sensitivity for sera from the immunocompetent patient group but a lower specificity, i.e., 80 and 29%, respectively . The counterimmunoelectrophoresis assay of sera from the immunodeficient patient group was not able to discriminate significantly between invasive candidiasis and colonization . With the use of more serum from each patient, the sensitivity of the antibody detection assays increased, while the specificity was maintained . The increase, however, was not statistically significant . Combining the results of the antibody assays with antigen titers obtained by the Cand-Tec assay did not improve the predictive value with respect to invasive candidiasis, as determined by multivariance regression analysis . Furthermore, it was demonstrated by performance of Western blots (immunoblots) that sera from patients as well as a rabbit antiserum cross-reacted with the Candida enolase and baker's yeast enolase enzyme . However, by tandem crossed immunoelectrophoresis it was demonstrated that the antibodies were directed toward different epitopes of the antigen.

Digestion, 1994, 55(1), 40 - 3
Lymphocyte proliferation response to baker's yeast in Crohn's disease; Young CA et al.; The etiology of Crohn's disease is still unknown . The present study served to test the hypothesis that baker's yeast (Saccharomyces cerevisiae) plays a role in Crohn's disease . Blood samples were obtained from 12 patients and 15 healthy controls . Peripheral blood leukocytes were isolated and incubated alone or with different concentrations of baker's yeast . After 3 days, the cultures were pulsed with tritiated thymidine . None of the lymphocyte cultures from healthy controls, including 3 bakers, proliferated in response to yeast . In striking contrast, all 9 patients with Crohn's disease in remission, on no medication, showed a threefold increase in their lymphocyte proliferation rate . Lymphocytes from 3 patients on 1.5 g of olsalazine maintenance therapy failed to respond . These results are consistent with previous findings that showed increased titers of IgG and IgA antibodies to baker's yeast in patients with Crohn's disease as compared to healthy controls . They confirm the suspicion that baker's yeast itself or a related antigen play a role in Crohn's disease and suggest that anti-inflammatory agents may act, in part, by inhibiting lymphocyte proliferation.

J Okla Dent Assoc, 1994 Spring, 84(4), 24 - 8
The effectiveness of a u-v toothbrush sanitizing device in reducing the number of bacteria, yeasts and viruses on toothbrushes; Glass RT et al.; Sixty-six sterile toothbrushes were exposed to one of the following microorganisms: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillis subtilis, Serratia marcescens and Baker's yeast . The Pollenex DS60 Daily Dental Sanitizer was found to be effective in substantially reducing the number of retained bacteria and yeasts as compared to contaminated toothbrushes not treated with such a device . Different toothbrush types had different response rates . Seventy-two sterile toothbrushes were exposed to Herpes Simplex Virus, Type I and seventy-two sterile toothbrushes were exposed to Parainfluenza Virus, Type III . The Pollenex DS60 Daily Dental Sanitizer consistently killed both viruses on all of the toothbrushes treated . Both viruses were consistently retained on non-treated toothbrushes for at least 24 hours.

Chin J Biotechnol, 1994, 10(3), 211 - 7
Model-based quality control of the baker's yeast Saccharomyces cerevisiae; Yuan J et al.; This paper deals with the optimal control of storage stability for compressed baker's yeast by minimizing the fraction of budding cells (FBC) based on a metabolic and cell cyclic model system for Saccharomyces cerevisiae . Three experiments of quality control were successfully carried out . The experimental data revealed that under optimal operation conditions, the final FBC-values approached the theoretical minimum and that storage stability was enhanced.

Biochim Biophys Acta, 1993 Dec 12, 1153(2), 283 - 8
ATP activation of plasma membrane yeast H(+)-ATPase shows complex kinetics independently of the degree of purification; Berberian G et al.; ATP stimulation of plasma membrane H(+)-ATPase activity from a wild baker's yeast (Saccharomyces cerevisiae) was followed under conditions of progressive degrees of purification . A particular emphasis was put to cover a wide range of concentrations which went from 2 microM up to 3000 microM ATP . The preparations used were (i) crude membrane fraction, (ii) untreated plasma membrane fraction obtained by differential centrifugation, (iii) residual plasma membrane treated with Triton X-100, (iv) enzyme solubilized with either Zwittergent 3-14 alone or after Triton X-100 treatment . Under all conditions the fitting of the dose-response curves required an equation composed by the sum of two Michaelian terms . Depending on the treatment, the Km values and Vmax values varied . The fitted curves displayed a high affinity-low Vmax (Km values of 7-60 microM and Vmax values of 0.03-0.50 mumol P(i)/mg per min) and a low affinity-high Vmax component (Km values of 408-1960 microM and Vmax values of 0.26-5.82 mumol P(i)/mg per min) . The complex ATP activation curve of the yeast plasma membrane H(+)-ATPase is in line with similar behavior found for the H(+)-ATPase of higher plants and all known animal cation transport ATPases.

Biotechnol Appl Biochem, 1993 Dec, 18 ( Pt 3), 401 - 8
Immobilization and stabilization of invertase using specific polyclonal antibodies; Jafri F et al.; Antisera raised in rabbits to baker's-yeast invertase significantly activated the enzyme in vitro . The antisera contained precipitating antibodies, a significant fraction of which appeared to be directed against the glycosyl residues of the enzyme . Invertase could be immobilized as insoluble enzyme antibody adducts or by binding to a Sepharose matrix precoupled with the gamma-globulin fraction derived from the antisera . The immobilized invertase preparations exhibited high enzyme activity and had markedly enhanced thermal stability, which could be further improved by cross-linking with glutaraldehyde.

Biokhimiia, 1993 Nov, 58(11), 1820 - 9
{Crystallization of three multiple forms of transketolase from baker's yeast}; Kuimov AN et al.; Three forms of baker's yeast transketolase have been revealed . These forms differed in thermal stability and elution profiles during chromatography on a phosphocellulose column and migrated with identical rates during electrophoresis in the presence of sodium dodecyl sulfate . The same forms in yeast, pig and rat liver and in different organs and tissues of the rabbit were found to be similar in their thermal stability and chromatographic properties . The relative amounts of the forms appeared to depend on the physiological state of the organism . Crystals of the three pure forms were grown using ammonium sulfate as the precipitating agent . These crystals differed morphologically and by stability upon storage . The possibility of interconversion of the transketolase forms is discussed.

Eur J Biochem, 1993 Oct 15, 217(2), 527 - 33
Limited proteolysis of yeast phosphofructokinase . Sequence locations of cleavage sites created by the actions of different proteinases; Kopperschlager G et al.; Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteolysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions . In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme . The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis . Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual alpha and beta polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate . The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components . The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the alpha chain and Leu192 in the beta chain . The truncated phosphofructokinase retains its octameric structure . The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the alpha chain involving the peptide bond preceding Val914 . This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers . The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase . It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphofructokinase are not directly involved in catalysis or its allosteric regulation . However, the last 80 residues of the alpha polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.

Gastroenterology, 1993 Oct, 105(4), 1061 - 8
Evaluation of liquid yeast-derived sucrase enzyme replacement in patients with sucrase-isomaltase deficiency; Treem WR et al.; BACKGROUND: No enzyme replacement therapy exists for patients with congenital sucrase-isomaltase deficiency (CSID) . A by-product of the manufacture of baker's yeast is a liquid preparation containing high sucrase activity . The aim of the present study was to investigate the activity and stability of this preparation and its effect on breath hydrogen excretion and gastrointestinal symptoms after sucrose ingestion in 14 patients with CSID . METHODS: The homogeneity of yeast sucrase was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its activity was measured . Stability at various temperatures and pH ranges and in the presence of gastric aspirate, pepsin, and bovine serum albumin was assessed . Fourteen patients with CSID underwent double-blind placebo-controlled breath tests with yeast sucrase . They then completed an 8-week dose response study that used different enzyme concentrations while consuming a sucrose-containing diet . RESULTS: Liquid yeast sucrase is highly glycosylated, contains no lactase activity, and is stable at 4 degrees C and over a wide range of pH . Pepsin digestion of the enzyme in vitro can be blunted by bovine serum albumin and by increasing the pH . Yeast sucrase reduces breath hydrogen excretion in patients with CSID who are given a sucrose load (P < 0.001) and allows most patients to consume a sucrose-containing diet . CONCLUSIONS: Liquid yeast sucrase offers effective enzyme replacement therapy for patients with CSID.

Plant J, 1993 Oct, 4(4), 601 - 10
A sink-specific H+/monosaccharide co-transporter from Nicotiana tabacum: cloning and heterologous expression in baker's yeast; Sauer N et al.; A cDNA clone for a monosaccharide transporter (MST1) was isolated from tobacco, which is most strongly expressed in the various sink tissues of mature tobacco plants: roots, flowers, and young leaves . An open reading frame of 1569 bp codes for a protein with 523 amino acids and a calculated molecular weight of 57,717 Da . The protein is homologous to a group of other plant monosaccharide transport proteins from Arabidopsis thaliana and Chlorella kessleri, to human glucose transporters and to Saccharomyces cerevisiae and several bacterial sugar transport proteins . As with these other transporters, the MST1 protein is extremely lipophilic and has 12-putative membrane-spanning domains . Heterologous expression of the MST1 cDNA clone in Saccharomyces cerevisiae allowed its characterization as a putative H+/monosaccharide co-transporter, catalyzing the uptake of hexoses (e.g . D-glucose and D-galactose) or pentoses (e.g . D-xylose) and the energy dependent and uncoupler sensitive accumulation of non-metabolizable substrates (e.g . D-xylose or 3-O-methyl-glucose) . Polyclonal antibodies were raised against a fusion protein of beta-galactosidase and the last 27 amino acids of the C-terminus of the MST1 protein . In SDS extracts of transformed yeast cells these antibodies recognize a polypeptide with an apparent molecular weight of 42 kDa, which is absent in extracts from untransformed control cells.

J Pharmacol Exp Ther, 1993 Oct, 267(1), 51 - 7
BAY X1005, a new inhibitor of leukotriene synthesis: in vivo inflammation pharmacology and pharmacokinetics; Muller-Peddinghaus R et al.; (R)-2-{4-(quinolin-2-yl-methoxy)phenyl}-2-cyclopentyl acetic acid) (BAY X1005) is an orally active inhibitor of the synthesis of the leukotrienes B4 and C4 in selected animal models that effectively reduces the vascular phenomena of inflammation, i.e., edema formation and leukocyte immigration . The arachidonic acid-induced mouse ear inflammation test allowed the evaluation of the antiedematous effects of BAY X1005 after topical (ED50, 18 micrograms/ear) and oral (ED50, 48.7 mg/kg) administration . Profound inhibition of myeloperoxidase activity as a marker for phagocyte infiltration was seen (ED50, 3 micrograms/ear topically and 7.9 mg/kg p.o.) even 5 hr after application . The platelet-activating factor-induced death of mice was statistical significantly and dose-dependently reduced (100 mg/kg p.o.; mean, 51%) . BAY X1005 had no analgesic properties in the phenyl-benzoquinone writhing test in mice and only limited efficacy in the baker's yeast-induced hyperalgesia test in the rat (ED50, 90 mg/kg p.o.), although cyclooxygenase inhibitors (indomethacin ED50, 1.7 mg/kg p.o.) are very potent . In another cyclooxygenase-sensitive test, the carrageenan-induced edema and the baker's yeast-induced fever in the rat, BAY X1005 was virtually devoid of any activity . The rat whole blood ex vivo leukotriene B4 inhibition assay demonstrated that BAY X1005 was potent (ED50, 11.8 and 6.7 mg/kg p.o . at 1 and 5 hr, respectively) and had a long duration of action (16-hr ED40, 70 mg/kg p.o.) . Similarly, inhibition of the zymosan-induced exudate leukotrienes B4 and C4 inhibition confirmed these data (ED50, 8.3 and 10.5 mg/kg p.o., respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1993 Oct, 306(1), 16 - 21
Antioxidant defenses of baker's yeast against free radicals and lipid peroxides in rat brain; Santiago LA et al.; We examined through the electron spin resonance spectrometry/spin trapping technique the antioxidant activity of baker's yeast Saccharomyces cerevisiae which is known to have both enzymatic and nonenzymatic antioxidants . The components in baker's yeast were separated by differential filtration/centrifugation using centrifuge-type filter tubes, yielding four nonenzymatic thermostable fractions of varied molecular weights which scavenged 85-95% of 1,1-diphenyl-2-picrylhydrazyl in organic solution, of hydroxyl and superoxide radicals in aqueous solution, and of lipid carbon-centered radicals induced by equimolar mixture of ascorbic acid and Fe(II) salt in rat brain homogenate . Baker's yeast also inhibited the thiobarbituric acid-reactive substance formation in the cortex, midbrain, pons-medulla oblongata, cerebellum, hippocampus, and striatum . Partial characterization of the antioxidative defenses in baker's yeast against free radicals and lipid peroxides in the brain confirmed the presence of superoxide dismutase, catalase, peroxidase, and glutathione.

Eur J Biochem, 1993 Sep 15, 216(3), 841 - 8
Purification of trehalose synthase from baker's yeast . Its temperature-dependent activation by fructose 6-phosphate and inhibition by phosphate; Londesborough J et al.; A trehalose synthase purified from baker's yeast contained 56-kDa, 102-kDa and 123-kDa polypeptides as its main components . The 102-kDa polypeptide was isolated and shown to be a specific trehalose-6-phosphatase . The trehalose-6-phosphate synthase (Tre6P synthase) activator described by Londesborough and Vuorio {(1991) J . Gen . Microbiol . 137, 323-330} was shown to be phosphoglucoisomerase and to function entirely by generating fructose 6-phosphate . Below 35 degrees C, fructose 6-phosphate is a powerful activator of the Tre6P synthase activity of intact trehalose synthase, especially at physiological phosphate concentration, but does not affect its trehalose-6-phosphatase activity nor the Tre6P synthase activity of truncated trehalose synthase containing truncated versions of the 123-kDa polypeptide . At 50 degrees C, activation by fructose 6-phosphate and inhibition by phosphate are greatly decreased, resulting in an unusually high temperature-dependence for the Tre6P synthase activity at a physiological phosphate concentration (2 mM).

Arch Environ Contam Toxicol, 1993 Aug, 25(2), 250 - 4
A comparison of microbial bioassays for the detection of metal toxicity; Codina JC et al.; Heavy metal toxicity was studied by assaying six microbiological toxicity tests, both in solution and wastewater . Pseudomonas fluorescens and baker's yeast (Saccharomyces cerevisiae) were used; growth and respirometric determinations were performed . In addition, the Microtox test was employed as a reference method . The Microtox test is the most sensitive assay for detecting toxicity of zinc, copper, and mercury but not for cadmium, chromium, and nickel . Wastewater increases the sensitivity threshold (EC20) and EC50 values of the metals in most of the assays, which is correlated to the presence of organic and inorganic compounds that can reduce the bioavailability of the metals, leading to a general loss of sensitivity . All the above-mentioned assays are potentially useful in the detection of chemical toxicity of metals . However, each test shows different sensitivities to each metal, which is related to different sensitivities of the organisms used in the assays, as well as to other factors . Therefore, it would be advisable to use a battery of tests for biological evaluation of metal toxicity.

J Mol Biol, 1993 Jul 20, 232(2), 701 - 3
Crystallization and preliminary crystallographic characterization of aspartic proteinase-A from baker's yeast and its complexes with inhibitors; Badasso M et al.; The aspartic proteinase from yeast vacuoles, proteinase-A, has been crystallized with and without non-hydrolysable transition-state analogue inhibitors . The native enzyme crystals belong to the space group I2(1)2(1)2(1), with two molecules per asymmetric unit . The inhibitor complex crystals are trigonal with space group P3(2)21 and with one molecule in the asymmetric unit . Preliminary X-ray analysis of both native enzyme and its complexes indicate that the complexes diffract to higher resolution than the native crystals . This is probably due to reduced flexibility in the enzyme-inhibitor complex.

Biochem Mol Biol Int, 1993 Jul, 30(3), 499 - 504
Expression of oryzacystatin cDNA using yeast artificial chromosome under ADH promoter in baker's yeast; Emori Y et al.; We constructed an expression vector into a yeast artificial chromosome (YAC) harboring the cDNA for oryzacystatin, a cysteine proteinase inhibitor from rice, under the control of the yeast ADH promoter . When the expression vector was introduced into Saccharomyces cerevisiae in the form of either an artificial chromosome or a circular plasmid, transformants carrying the DNA grew well in a selective medium . However, the content of the introduced DNA decreased significantly during passages in non-selective YPD medium . The stability of the introduced DNA was enhanced in selected clones obtained as colonies viable in selective medium after many passages in YPD medium . The stable transformants thus obtained expressed the mRNA for oryzacystatin at levels as high as those of intrinsic yeast ADH.

Appl Environ Microbiol, 1993 Jul, 59(7), 2244 - 50
Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens; Huang Z et al.; Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively . The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography . The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry . The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies . Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere.

Appl Environ Microbiol, 1993 Jul, 59(7), 2022 - 8
Combined effects of sulfites, temperature, and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae; Gardner N et al.; Analysis of variance was used to evaluate the simultaneous effects of strain, incubation temperature (15 to 25 degrees C), agitation time (0 to 24 h), and initial sulfite concentration (100 to 300 ppm) on glycerol production in grape juice by Saccharomyces cerevisiae . Fourteen strains were studied to determine their growth patterns in the presence of sulfites and ethanol . Baker's yeast strains were more sensitive to sulfite than wine strains, and little growth occurred at initial sulfite levels greater than 150 ppm . Sensitivity to sulfite increased with increasing levels of ethanol . Three strains exhibiting the best growth in the presence of sulfites and ethanol were selected for interaction studies . Fermentations were carried out until the solids content had decreased to less than 6 degrees Brix, which was the point that glycerol content became stable . For the three strains used, the greatest level of glycerol production was observed in the presence of 300 ppm of sulfite for most incubation temperatures and agitation times . There was significant interaction between the strain, incubation temperature, and agitation time parameters for glycerol synthesis, and a response surface method was used to predict the optimal conditions for glycerol production . Under static conditions, the highest level of glycerol production was observed at 20 degrees C, while incubation at 25 degrees C gave the best results when the cultures were agitated for 24 h . Response surface equations were used to predict that the optimum conditions for glycerol production by S . cerevisiae Y11 were a temperature of 22 degrees C, an initial sulfite concentration of 300 ppm, and no agitation, which yielded 0.68 g of glycerol per 100 ml.

Clin Exp Allergy, 1993 Jul, 23(7), 587 - 90
Gastrointestinal stability of baker's yeast allergens: an in vitro study; Kortekangas-Savolainen O et al.; An in vitro model was established to study the stability of baker's yeast (Saccharomyces cerevisiae) allergens in conditions simulating the gastrointestinal tract . The protocol consisted of 2 hr incubation under gastric conditions (pH 1.2, +37 degrees C and gastric enzymes) and 2 hr incubation under duodenal conditions (pH 6.8, +37 degrees C and duodenal enzymes) . These were studied together and separately, as well as under pure acidic conditions without gastric enzymes . The yeast extracts contained equal amounts of allergen and were analyzed by IgE-immunoblotting . The acidic conditions had partly an enhancing and slightly degrading effect on the yeast allergens, whereas the gastric enzymes destroyed several allergens, including the important intermediate allergens of 31 and 45 kD . After treatment under both gastric and duodenal conditions most of the yeast allergens were destroyed, except mannan and a 10 kD protein component . The findings suggest that the allergen exposure caused by baker's yeast takes place mainly on the mucosal surfaces orally and oesophageally and through viable baker's yeast organisms that manage to pass the stomach and duodenum and possibly lead to intestinal growth of the organism . Patients with IgE production against the 10 kD allergen and mannan are, however, moderately exposed to allergens consisting of soluble antigenic material only.

Biochemistry, 1993 Jun 8, 32(22), 5792 - 9
Catalytic mechanism of yeast adenosine 5'-monophosphate deaminase . Zinc content, substrate specificity, pH studies, and solvent isotope effects; Merkler DJ et al.; Adenosine 5'-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide {Merkler, D . J., & Schramm, V . L . (1990) J . Biol Chem . 265, 4420-4426} . The enzyme contains 0.98 +/- 0.17 zinc atom per subunit . The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor {Wilson, D . K., Rudolph, F . B., & Quiocho, F . A . (1991) Science 252, 1278-1284} . Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue . On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn(2+)-activated water molecule to attack C6 of AMP with the displacement of NH3 . The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4 . This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns {Merkler, D . J., Wali, A . S., Taylor, J., Schramm, V . L . (1989) J . Biol . Chem . 264, 21422-21430} . The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 +/- 0.2 and 7.7 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1993 Jun, 303(2), 443 - 50
Factors affecting the species-homologous and species-heterologous binding of mitochondrial ATPase inhibitor, IF1, to the mitochondrial ATPase of slow and fast heart-rate hearts; Rouslin W et al.; We examined the effects of a variety of conditions upon the IF1-mediated inhibition of the ATPase in both intact and sonicated mitochondria and in IF1-depleted submitochondrial particles (SMP) in species-homologous and species-heterologous combinations of IF1 and ATPase . IF1-mediated ATPase inhibition occurred in intact rabbit heart mitochondria at low matrix pH and low membrane potential, but not in intact pigeon and rat heart mitochondria under the same conditions . IF1-mediated ATPase inhibition was, however, demonstrable in both the rabbit and pigeon heart systems in sonicated mitochondria incubated at low ionic strength . The rat heart system failed to exhibit significant IF1-mediated ATPase inhibition in either intact or sonicated mitochondria due to the low amount of IF1 present . When rabbit heart IF1-containing extracts were incubated with IF1-depleted rabbit heart SMP over a range of KCl concentrations, increasing the {KCl} to 100 mM had little effect on IF1-mediated ATPase inhibition . When pigeon heart IF1-containing extracts were incubated with IF1-depleted pigeon heart SMP under the same conditions, increasing {KCl} to 100 mM nearly completely blocked IF1-mediated ATPase inhibition . While the species-endogenous level of rat heart IF1 (i.e., 1x IF1) inhibited IF1-depleted rat heart SMP virtually not at all at any {KCl} examined, the 8x rat heart IF1 was nearly as inhibitory as the 1x rabbit heart IF1 at varying ionic strengths . When rabbit, pigeon, or rat heart IF1 was bound to rabbit versus pigeon IF1-depleted SMP, the effect of varying ionic strength on IF1-mediated ATPase inhibition was related to the species source of the IF1, not to the species source of the enzyme; 1x bovine heart IF1 purified to homogeneity behaved much like 1x crude rabbit heart IF1 when binding to either the rabbit or the pigeon heart enzyme . This suggests that an IF1-ATPase complex stabilizing factor such as has been isolated from baker's yeast cells in neither lacking in the pigeon heart system nor required for the more ionic-strength-resistant binding of IF1 observed in slow heart-rate mammalian heart mitochondria.

Appl Biochem Biotechnol, 1993 Jun, 41(3), 145 - 55
The principle of parabolic feed for a fed-batch culture of baker's yeast; He RQ et al.; A parabolic nutrient supplementation has been derived by the specific growth rate, sugar conversion rate, and Pasteur effect, followed by high qualitative and quantitative biomass production with the lowest sugar consumption . The method produced about 95% cells in the G1 phase that were more resistant to drying and aging . These features are particularly important in the process of making dry yeast . It appears that the parabolic feed method may be used to species culture that show the Pasteur effect or produce byproducts from sugar . This may be because the supplementation is in conformity with reproducible kinetic growth during the fed-batch culture.

J Bacteriol, 1993 May, 175(9), 2607 - 12
The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli; Gibbs PE et al.; We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site . The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum . In SOS-induced E . coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations . In S . cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations) . Each of these yeast mutations was a single unique event, and none were like those in E . coli, suggesting that in fact the difference in error rate is much greater . Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S . cerevisiae (21 to 36% targeted mutations) than in E . coli (13%) . However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms . We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template . Unlike E . coli, bypass in S . cerevisiae did not require UV-induced functions.

J Immunol, 1993 May 1, 150(9), 3932 - 40
Pneumocystis carinii stimulates tumor necrosis factor-alpha release from alveolar macrophages through a beta-glucan-mediated mechanism; Hoffman OA et al.; Recent studies suggest that TNF-alpha plays a central role in host defenses during Pneumocystis carinii pneumonia . To determine whether P . carinii directly stimulates TNF-alpha secretion, rat alveolar macrophages were cultured in the presence of purified P . carinii . Whereas unstimulated alveolar macrophages released only 13.0 +/- 2.7 pg/ml of TNF-alpha into the medium, macrophages stimulated with P . carinii released 108.2 +/- 20.4 pg/ml of TNF-alpha after overnight culture (p = 0.0001) . Maximal TNF-alpha release was observed after 8 h of incubation and required a P . carinii: macrophage ratio of at least 2.5:1 . Autoclaved P . carinii were also able to trigger TNF-alpha release from macrophages albeit at a reduced level . In view of recent evidence that P . carinii is phylogenetically related to the fungi and contains a beta-glucan-rich cell wall, we hypothesized that TNF-alpha release might in part be mediated by this cell wall component . Preincubation of macrophages with particulate beta-glucan derived from Baker's yeast resulted in complete inhibition of TNF-alpha release in response to P . carinii . In addition, digestion of P . carinii with zymolyase, a preparation containing predominantly beta-glucanase activity, substantially reduced the ability of P . carinii to cause TNF-alpha release from macrophages . In a similar manner, macrophages incubated with P . carinii in the presence of laminariheptaose, an oligosaccharide that binds to macrophage beta-glucan receptors, also displayed decreased TNF-alpha release . Interestingly, TNF-alpha release may not be completely linked to the adherence of the organism to macrophages . Particulate beta-glucan significantly reduced P . carinii adherence to macrophages and also impaired TNF-alpha release . However, yeast mannan also significantly reduced P . carinii adherence to macrophages and also impaired TNF-alpha release . However, yeast mannan also significantly reduced P . carinii adherence but had no effect on TNF-alpha release . These data demonstrate that P . carinii can directly stimulate the secretion of TNF-alpha from alveolar macrophages and that this effect is largely mediated by beta-glucan components of the P . carinii cell wall.

Appl Environ Microbiol, 1993 Apr, 59(4), 1065 - 71
Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae; Lewis JG et al.; The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture . Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose . However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose . The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion . Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants . These results imply a direct link with respiration, rather than exhaustion of glucose . The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect . Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations . Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability . Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.

Biochem Mol Biol Int, 1993 Apr, 29(5), 907 - 12
Functional analysis of apf1 mutation causing defective amino acid transport in Saccharomyces cerevisiae; Horak J et al.; Mutation in the Apf1 locus causes a pleiotropic effect of H(+)-driven active amino acid transport in baker's yeast Saccharomyces cerevisiae . The uptake of other, presumably H(+)-driven, substances, e.g . of purine and pyrimidine bases, maltose and phosphate ions, is not significantly influenced by this mutation . The apf1 mutation decreases not only the initial rates of amino acid uptake but also the accumulation ratios of amino acids taken up but has virtually no effect on the membrane potential or on the delta pH which constitute the thermodynamically relevant source of energy for their transport . Similarly, no changes in intracellular ATP content, in ATP-hydrolyzing and H(+)-extruding H(+)-ATPase activities, in the efflux of intracellularly accumulated amino acids, or in rates of endogenous respiration, were observed in the apf1 mutant phenotype . Hence, all these data are in accordance with the experiments showing that the Apf1 protein, an integral protein of the endoplasmic reticulum, is required exclusively for efficient processing and translocation of transport proteins specific for amino acids from the endoplasmic reticulum to their final destination, the plasma membrane.

Clin Exp Allergy, 1993 Mar, 23(3), 179 - 84
IgE-binding components of baker's yeast (Saccharomyces cerevisiae) recognized by immunoblotting analysis . Simultaneous IgE binding to mannan and 46-48 kD allergens of Saccharomyces cerevisiae and Candida albicans; Kortekangas-Savolainen O et al.; The Saccharomyces cerevisiae allergens were characterized by IgE-immunoblotting with serum samples of 83 patients; 63 represented patients with atopic dermatitis with previous positive skin prick test or RAST for S . cerevisiae, seven patients with AD but negative test results and 13 were non-atopic controls . Disrupted whole body extract of S . cerevisiae was used in the assays . From the patients tested 41 patients with atopic dermatitis appeared positive in IgE immunoblotting revealing 22 IgE stained bands . From these bands 10 represented intermediate allergens, and 12 minor allergens . The most frequent staining was obtained with the 48 kD band (39%) . When the staining pattern of 45 kD and 48 kD bands and mannan was compared with Candida albicans allergens or purified baker's yeast enolase a simultaneous binding was seen with the 48 kD band of S . cerevisiae and the 46 kD band of C . albicans and enolase whereas the 45 kD band was neither associated with the 46 kD band of C . albicans nor purified enolase . High molecular weight staining was found in five samples . The staining pattern was associated with the mannose containing structures in parallel with C . albicans.

Cell Immunol, 1993 Mar, 147(1), 203 - 14
Immunization with recombinant Escherichia coli expressing retinal S-antigen-induced experimental autoimmune uveitis (EAU) in Lewis rats; Eto K et al.; Previously we have reported that microbial proteins having sequence homology with uveito-pathogenic peptide (peptide M) of retinal S-antigen (S-Ag) induced experimental autoimmune uveitis (EAU) in Lewis rats and subhuman primates . In order to evaluate the role of natural microbial infections in causing autoimmune diseases we have constructed a recombinant Escherichia coli expressing retinal S-Ag when injected it into Lewis rats which developed EAU . Control animals immunized with E . coli JM105 transfected with only plasmid DNA did not induce EAU . Similarly, baker's yeast (Saccharomyces cerevisiae), which has six amino acid residues in histone H3 identical with peptide M of S-Ag, induced EAU when injected into rats . Lymph node cells of rats immunized with recombinant E . coli or with the yeast show significant proliferative responses against peptide G and peptide M of S-Ag, respectively . The animals immunized with recombinant E . coli also produced antibody to S-Ag.

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 279 - 84
Chemical modification studies of the active site of glucosamine-6-phosphate synthase from baker's yeast; Milewski S; Glucosamine-6-phosphate synthase from baker's yeast has been purified 100-fold with a final recovery of 70% . The purification procedure involved thiol-affinity chromatography . Chemical modification studies of the enzyme revealed the presence of cysteine, Glu/Asp-carboxyl and probably histidine at the glutamine binding site and, on the other hand, arginine and probably another histidine at the D-fructose 6-phosphate binding site . A few glutamine analogs, including 6-diazo-5-oxo-L-norleucine (DON), anticapsin and N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), were shown to inactivate the enzyme in a time- and concentration-dependent manner . Anticapsin, the most active in the series, exhibited an inactivation constant, Kinact, of 9.5.10(-6) M.

Biochem J, 1993 Jan 15, 289 ( Pt 2), 453 - 61
Purification and properties of three (1-->3)-beta-D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare); Hrmova M et al.; Three (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography . The three (1-->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3 . N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes . Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme . The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1-->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx . 25 . The three enzymes are classified as endohydrolases, because they yield (1-->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin . Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat . constants of 36-155 s-1 and pH optima of 4.8 . Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1-->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1-->3;1-->6)-beta-D-glucans . However, the isoenzymes differ in their rates of hydrolysis of a (1-->3;1-->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly . The enzymes do not hydrolyse (1-->3;1-->4)-beta-D-glucans, (1-->6)-beta-D-glucan, CM-cellulose, insoluble (1-->3)-beta-D-glucans or aryl beta-D-glycosides.

Life Sci, 1993, 53(17), 1383 - 9
Antimicrobial and antioxidant activities of unripe papaya; Osato JA et al.; The meat, seed and pulp of Carica papaya Linn., a popular traditional medicinal herb grown in the tropics, was shown by the agar-cup method to be bacteriostatic against several enteropathogens such as Bacillus subtilis, Enterobacter cloacae, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Klebsiella pneumoniae . The same parts of papaya were unequivocably demonstrated by electron spin resonance spectrometry to scavenge 1,1-diphenyl-2-picrylhydrazyl (5.8 x 10(14) spins/ml), hydroxyl (5.1 x 10(14) spins/ml) and superoxide (1.2 x 10(14) spins/ml) radicals with the seed giving the highest activity at concentrations (IC50) of 2.1, 10.0 and 8.7 mg/ml, respectively . The superoxide dismutase (SOD)-like activity in the meat, seed and pulp amounts to about 32, 98 and 33 units/ml; comparable to those of soybean paste miso, rice bran and baker's yeast . Vitamin C, malic acid, citric acid and glucose are some of the possible antioxidative components in papaya . Our study correlates the bacteriostatic activity of papaya with its scavenging action on superoxide and hydroxyl radicals which could be part of the cellular metabolism of such enteropathogens . This is indicative of the pathophysiological role of these reactive oxygen species in gastrointestinal diseases and papaya's ability to counteract the oxidative stress.

Agents Actions Suppl, 1993, 44, 31 - 6
R-flurbiprofen: isomeric ballast or active entity of the racemic compound?
Geisslinger G, Menzel-Soglowek S, Beck WS, Brune K.
The enantioselective pharmacokinetic behaviour of the flurbiprofen enantiomers was investigated following administration of optically pure R- or S-flurbiprofen to various species . Only negligible inversion (< 5%) of flurbiprofen occurred in the rat and in man . Consequently, pharmacodynamic experiments evaluating pain and inflammation as parameters have been carried out enantioselectively for both flurbiprofen enantiomers in the rat . R-flurbiprofen, which is not an inhibitor of prostaglandin synthesis in vitro, had only marginal anti-inflammatory effects as defined by the carrageenan edema of the rat paw in contrast to the S-enantiomer . In contrast, to S-flurbiprofen, R-flurbiprofen caused only marginal mucosal damage in the GI-tract . Both enantiomers, however, were of similar potency as antinociceptive drugs in the rat Randall-Selitto assay following the injection of interleukin-1 or baker's yeast . Using the pure enantiomers of flurbiprofen it appears possible to establish a more specific drug treatment: the R-enantiomer in occasional pain, the S-enantiomer in rheumatic disorders.

Chin J Biotechnol, 1993, 9(3), 189 - 96
Profit optimization for baker's yeast fed-batch fermentation; Yuan J et al.; The model-based profit optimization for baker's yeast fed-batch fermentation was done in this paper . The model system used for simulation was a combined metabolic and cell cyclic model for baker's yeast Saccharomyces cerevisiae . The objective function was built according to cost-effect balances, while the required data came from a baker's yeast factory . The simulation of profit optimization revealed that in order to obtain the highest profit, there exist optimal operation regions for major manipulating variables, such as substrate feeding rate, sugar concentration in feed, aeration rate and fermentation period.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 871 - 9
Characterization and purification of a novel dATP-binding protein in eukaryotes; Dalton A et al.; We characterized and purified an acidic dATP-binding protein, which, in its active form, resides in the nuclear fraction of a range of cells from mammals (including pig liver) and baker's yeast (Saccharomyces cerevisiae) . This protein exhibits a high degree of specificity for the deoxy form of the naturally occurring nucleoside triphosphates and shows a marked preference for the purine deoxynucleoside triphosphates dATP and dGTP . The protein cleaves the terminal phosphate of dATP and appears to retain the dADP moiety of the nucleotide in a reaction that is resistant to both SDS and 8 M-urea . Fractionation of the nuclear preparation followed by non-denaturing PAGE and SDS/PAGE electrophoresis was sufficient to produce pure protein . The occurrence of this activity in all nuclei tested suggests that it plays an important role in nuclear metabolism . The specificity of the enzyme for deoxynucleoside triphosphates further suggests a role for this enzyme in DNA replication or repair, but the acidity of the protein argues against a direct interaction with DNA, and, indeed, the catalytic activity is not modulated by the inclusion of DNA in a variety of physical forms.

Mol Cell Biol, 1992 Sep, 12(9), 3766 - 75
Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor; Zhou P et al.; The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium . On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C . glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1 . AMT1 forms multiple specific DNA-protein complexes with both C . glabrata MT-I and MT-IIa promoter DNA fragments . In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding . Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C . glabrata . Although AMT1-mediated trans activation of the C . glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance . The distinct function that metallothionein genes have in copper but not cadmium detoxification in C . glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.

Appl Biochem Biotechnol, 1992 Sep, 36(3), 153 - 61
New alcohol resistant strains of Saccharomyces cerevisiae species for potable alcohol production using molasse; Argiriou T et al.; Two alcohol resistant strains of Saccharomyces cerevisiae species were isolated from a Greek vineyard plantation . The strain AXAZ-1 gave a concentration of 17.6% v/v alcohol and AXAZ-2 16.5%, when musts from raisin and sultana grapes, respectively, were employed in alcoholic fermentations . They were found to be more alcohol tolerant and fermentative in the fermentation of molasse than the traditional baker's yeast . Specifically, using an initial {symbol: see text} Be density of 16 {symbol: see text} Be at the repeated batch fermentation process, in the first as well as fourth batch, the better AXAZ-1 gave final {symbol: see text} Be densities of 6.0 and 10.5 respectively, and the baker's yeast 11.6 and 14.5.

Antonie Van Leeuwenhoek, 1992 Aug, 62(1-2), 79 - 93
Heterologous protein production in yeast; Gellissen G et al.; The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade . Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production . Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology . Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application . In the following review a selection from the different yeast systems is described and compared.

Gut, 1992 Aug, 33(8), 1071 - 5
Antibodies to Saccharomyces cerevisiae in patients with Crohn's disease and their possible pathogenic importance; Giaffer MH et al.; Saccharomyces cerevisiae (baker's yeast) may play an important part in the pathogenesis of Crohn's disease . Because of this the levels of IgG and IgA antibodies against three S cerevisiae strains (NCYC 77, NCYC 79, and NCYC 1108) were assayed in 49 patients with Crohn's disease, 43 with ulcerative colitis, 14 with coeliac disease, and 21 healthy controls . Coded serum samples were tested by ELISA . Similar antibody patterns to all three strains were found . IgG and IgA antibody levels were significantly raised in patients with Crohn's disease compared with healthy controls (p < 0.001 and p < 0.0001 respectively) and with ulcerative colitis patients (p < 0.0001 and p < 0.0006 respectively) . Raised IgA, but not IgG, yeast antibody levels were found in two patients with Crohn's disease who were intolerant to yeast, but these values were similar to those in other patients without yeast intolerance . In ulcerative colitis, both IgG and IgA levels were similar to normal controls . Patients with small bowel Crohn's disease had significantly higher IgG antibody levels than those with colonic disease (p < 0.01) . High levels of IgG, but not IgA, antibody were present in patients with coeliac disease, the antibody responses being indistinguishable from those found in Crohn's disease . It is concluded that the presence of IgG antibody to S cerevisiae is characteristic but not specific to Crohn's disease . Although raised IgA antibody levels are more frequently found in Crohn's disease, their pathogenic importance remains to be established.

Gut, 1992 Jul, 33(7), 909 - 13
Antibody (IgG, IgA, and IgM) to baker's yeast (Saccharomyces cerevisiae), yeast mannan, gliadin, ovalbumin and betalactoglobulin in monozygotic twins with inflammatory bowel disease; Lindberg E et al.; To assess whether dietary antigens play a role in inflammatory bowel disease, 26 monozygotic twin pairs with inflammatory bowel disease and 52 healthy controls were investigated for serum antibodies (IgA, IgG, IgM) against ovalbumin, betalactoglobulin, gliadin, whole yeast (Saccharomyces cerevisiae) and yeast cell wall mannan . The twins were made up of five pairs concordant and nine pairs discordant for Crohn's disease, and two pairs concordant and 10 pairs discordant for ulcerative colitis . Two patients with Crohn's disease had a slight increase in disease activity, the others were in clinical remission . Two striking observations were made: first, individuals with ulcerative colitis were indistinguishable from healthy twins, and controls except for the response to gliadin . Both healthy and diseased twins had higher IgA levels to gliadin than controls . Second, twins who had developed Crohn's disease displayed higher antibody titres towards yeast cell wall mannan in particular, but also to whole yeast (Saccharomyces cerevisiae) of all antibody types (IgA, IgG, and IgM) . In contrast, the response to gliadin, ovalbumin, and betalactoglobulin did not differ from healthy twins and was even lower than in the controls . The results argue against an increased systemic antigen presentation caused by an impaired mucosal barrier in the inflammatory bowel disease . Rather, they suggest that yeast cell wall material--that is, mannan, or some antigen rich in mannose and cross reacting with mannan, may play an aetiological role in Crohn's disease, but not in ulcerative colitis . The increases in IgA and IgM, as well as IgG suggest that local and systemic immune systems are selectively activated by antigen(s) present in the cell wall of baker's yeast.

Appl Environ Microbiol, 1992 Jul, 58(7), 2116 - 22
Microbial oxidation of oleic acid; el-Sharkawy SH et al.; Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield . Nocardia aurantia (ATCC 12674), Nocardia sp . (NRRL 5646), and Mycobacterium fortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively . Structures of all metabolites were suggested by 1H and 13C nuclear magnetic resonance and by infrared and mass spectrometry . Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison . We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones . We further introduce a simple method based on 1H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.

Pharmazie, 1992 Jul, 47(7), 531 - 7
{The yeast test: an alternative method for determination of acute toxicity of drugs and environmental chemicals}; Koch HP; Starting point for this study was the urgent need for replacement of the contemporary mode of acute toxicity testing of chemicals in vertebrates (LD50-test) by an experimental model with equal power that can be performed on non-pain sensitive matter . For this purpose, a testing procedure ought to be developed using a non-pathogenic microorganism as testing object that is available at any time, easy to cultivate, and in its indicative power equivalent to the LD50 test in mice, rats, and other laboratory animals . Such an organism has been found with ordinary yeast (baker's yeast, brewer's yeast, Saccharomyces cerevisiae) which can always be obtained in good quality . This procedure (shortly denominated as 'yeast test' offers several advantages: It is rather simple, inexpensive, outstandingly well reproducible, and it can be carried out conveniently also in routine experiments . The results correlate well with those furnished by the customary methods of acute toxicity testing.

Mol Endocrinol, 1992 Jul, 6(7), 1043 - 50
Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast; Pham TA et al.; The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes . The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function . However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast . In this report, we have characterized the activity of the transactivation functions of the ER in yeast . Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity . These results are consistent with those obtained in mammalian cells . Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone . Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast . Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.

Eur J Biochem, 1992 Jun 1, 206(2), 463 - 70
Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase; Baykov AA et al.; Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme-bound and free pyrophosphate were measured over wide ranges of Mg2+ and respective substrate concentrations . Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange {Kasho, V . N . & Baykov, A . A . (1989) Biochem . Biophys . Res . Comm . 161, 475-480}, yielded values of the equilibrium constants for Mg2+ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps . All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi bound . Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four-metal pathway . In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four-metal pathway, dissociation of the second Pi is faster for the three-metal pathway, while PPi hydrolysis and the release of the first Pi may proceed with similar rates . Release of pyrophosphate formed on the enzyme is faster for the three-metal pathway . Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg2+ concentration, thus providing a means for pyrophosphatase-activity regulation.

Photodermatol Photoimmunol Photomed, 1992 Jun, 9(3), 113 - 20
Photoprotective effects of sunscreens in cosmetics on sunburn and Langerhans cell photodamage; Elmets CA et al.; It has become common practice to add sunscreening agents of variable potency to cosmetics to protect against the adverse effects of ultraviolet (UV) radiation exposure . The purpose of this study was to determine whether cosmetic preparations containing sunscreening agents protected against the adverse effects of acute UV radiation exposure and, if so, to identify the components responsible for the photoprotective effects . Pretreatment of skin with one such cosmetic product provided complete protection against UV-induced erythema, sunburn cell formation and Langerhans cell damage in volunteers, skin types II and III, whose skin was exposed to a 1.5 minimal erythema dose daily for 4 consecutive days . When individual components of the cosmetic preparation were analyzed for their photoprotective activities, it was found that both the cinnamate and benzophenone sunscreen combination and an extract of baker's yeast present in the preparation had photoprotective properties . These studies indicate that incorporation of photoprotective agents into cosmetic preparations provides a beneficial function and should therefore be encouraged.

Appl Environ Microbiol, 1992 May, 58(5), 1661 - 9
Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant; Linden T et al.; From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens . One of the isolates of S . cerevisiae, no . 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast . The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation . Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially . Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6 . Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5 . The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S . cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054 . The S . cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P . stipitis value of 890 nmol/min/mg of protein . The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P . stipitis (330 nmol/min/mg of protein).

Appl Microbiol Biotechnol, 1992 May, 37(2), 230 - 4
Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer, using a kar1 mutant as vector; Spencer JF et al.; A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector . The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S . cerevisiae (S . diastaticus) and the melibiose-metabolism (mel) gene(s), from S . kluyveri, to the kar1-1 mutant {K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion . From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating . Strains that utilized starch or melibiose were obtained by all three methods . Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids . The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S . cerevisiae . The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.

J Chromatogr, 1992 Apr 15, 576(1), 63 - 70
Determination of free N-acetylamino acids in biological samples and N-terminal acetylamino acids of proteins; Kawakami Y et al.; N-Acetylamino acids were derivatized with 9-anthryldiazomethane to the corresponding esters . The anthryl esters were separated by high-performance liquid chromatography and detected fluorimetrically (excitation at 365 nm; fluorescence emission measured at 412 nm) . N-Acetyl derivatives of Asn, Gln, Ser, Thr, Gly, Ala, Tyr, Pro, Met, Val, Ile and Leu as well as N-formyl-Met could be separated and identified in the same chromatographic run . The detection limit was from 0.10 pmol for AcGln to 5.5 pmol for AcIle and AcLeu . When the acetylamino acids listed above were added to the 700 g supernatant of a rat liver homogenate, the mean recovery was 72% . AcAla and AcTyr were found in free form in baker's yeast . Proteins with an acetylated N-terminus were digested by a protease, and the peptides formed were treated with an N-acylamino acid-releasing enzyme . This method was applied to end-group determination of four proteins (each 0.5 nmol).

Protein Sci, 1992 Apr, 1(4), 540 - 8
Extreme pKa displacements at the active sites of FMN-dependent alpha-hydroxy acid-oxidizing enzymes; Lederer F; Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases . Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation . A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction . Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free . The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion . Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2 . The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.

Eur J Biochem, 1992 Feb 1, 203(3), 557 - 62
New site-directed reversible inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase; Sixt B et al.; 1 . CoA-thioether analogues of 3-hydroxy-3-methylglutaryl-CoA containing an additional methyl group at positions 2, 6(methyl at C3) or 4 of the acyl residue were prepared . To probe for hydrophobic interaction, their inhibitory properties were determined with 3-hydroxy-3-methylglutaryl-CoA reductase purified from baker's yeast . The CoA-thioethers were purely competitive inhibitors whose affinity to the reductase was near to that of the physiological substrate . 2 . CoA-sulfoxides derived from the CoA-thioethers displayed affinities to the reductase superior to that of the physiological substrate (Km = 7 microM) . Depending on the degree of recognition of diastereomers by the enzyme, the inhibitor constants of the two best inhibitors vary from Ki = 200 nM and Ki = 80 nM (diastereomeric mixtures) to 25 nM and 20 nM, respectively (if only one diastereomer would interact with the enzyme).

Cell Immunol, 1992 Jan, 139(1), 81 - 90
Suppression of experimental autoimmune uveitis in rats by the oral administration of the uveitopathogenic S-antigen fragment or a cross-reactive homologous peptide; Singh VK et al.; The oral administration of S-antigen fragment (a synthetic peptide designated as peptide M and known to be uveitopathogenic for rat, guinea pig, and monkey) to Lewis rats prior to challenge with an emulsion of peptide M and CFA resulted in either a total or partial suppression of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease studied as a model for human uveitis and experimental autoimmune pinealitis (EPA) . Both the clinical and histopathologic manifestations of the disease were suppressed in a dose-dependent manner . Pinealitis associated with EAU was also suppressed by the oral administration of peptide M . Additionally, ingestion of a fragment of baker's yeast (Saccharomyces cerevisiae) histone H3, which has five consecutive amino acids identical to peptide M and which has been found to be uveitopathogenic in Lewis rats, induced tolerance to either peptide M or synthetic histone H3 peptide . In addition, the proliferative response to peptide M was inhibited in peptide M-fed rats . The suppression of EAU and in vitro lymphocyte proliferative responses to peptide M were observed to be antigen specific, since oral feeding of a control protein (BSA) exerted no suppressive effect . Furthermore, the T cells isolated from the spleen and lymph nodes of animals rendered tolerant by oral administration of peptide M can transfer protection against EAU adoptively . These results demonstrate that the oral administration of an autoantigen or its homologous peptide initiates an antigen-specific cellular mechanism which may ameliorate EAU.

Yeast, 1992 Jan, 8(1), 47 - 55
Effects of phosphoglycerate kinase overproduction in Saccharomyces cerevisiae on the physiology and plasmid stability; van der Aar PC et al.; In this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyces cerevisiae containing the PGK1 gene on an episomal plasmid are described . This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10-15% of the total soluble protein . Strains containing the plasmid and the host strain were grown in non-selective batch cultures and continuous culture, under different growth conditions . Plasmid-containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12% . This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose-limited conditions . During respiro-fermentative growth, PGK-overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain . The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose . The results are consistent with previously reported data on the physiology and gene stability of PGK-overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.

Scand J Gastroenterol, 1992, 27(3), 196 - 200
The effect of dietary yeast on the activity of stable chronic Crohn's disease; Barclay GR et al.; The effect of dietary yeast on the activity of stable Crohn's disease was assessed in 19 patients . During the 1st month patients continued their usual diet (base-line period), but during the next 2 months dietary yeast was excluded except that during 1 month patients took baker's yeast capsules while for the other month they took placebo capsules . The patients' mean Pettit Crohn's disease activity index (CDAI) while taking baker's yeast (mean, 107.9; SE, 6.1) was significantly greater than during yeast exclusion (mean, 102.1; SE, 5.7; p less than 0.05) . The mean of each patient's maximum CDAI during yeast exclusion (mean, 107.1; SE, 5.7) was significantly lower than those during the base-line (mean, 115.2; SE, 6.1; p less than 0.05) and baker's yeast inclusion periods (mean, 113.9; SE, 6.7; p less than 0.05) . Patients with elevated yeast antibodies tended to develop a higher CDAI while receiving baker's yeast (13 of 15) . These results suggest that dietary yeast may affect the activity of Crohn's disease.

EMBO J, 1991 Dec, 10(12), 3923 - 9
Rolling circle replication of DNA in yeast mitochondria; Maleszka R et al.; The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy . The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units . A small proportion of mtDNA is present as supercoiled or relaxed circular molecules . Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats) . The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism . Replication of mtDNA in other yeasts is thought to occur by the same mechanism . For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length . Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus . The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.

J Bioenerg Biomembr, 1991 Dec, 23(6), 873 - 88
Regulation of the mitochondrial ATPase in situ in cardiac muscle: role of the inhibitor subunit; Rouslin W; The mitochondrial F1-ATPase inhibitor protein, IF1, binds to beta subunits of the F1-ATPase both in vitro and in situ under nonenergizing conditions, i.e., under conditions that allow a net hydrolysis of ATP by the mitochondrial ATPase to take place . This reversible IF1 binding occurs in a wide variety of cell types including (anaerobic) baker's yeast cells and (ischemic) mammalian cardiomyocytes under conditions that limit oxidative phosphorylation . The binding of inhibitor results in a marked slowing of ATP hydrolysis by the undriven mitochondrial ATP synthase . An apparent main function of this reversible IF1 binding, at least in cells that undergo aerobic-anaerobic switching, is the mitigation of a wasteful hydrolysis of ATP produced by glycolysis during anoxic or ischemic intervals, by the mitochondrial ATPase . While this apparent main function is probably of considerable importance in cells that normally either can or must undergo aerobic-anaerobic switching such as baker's yeast cells and skeletal myocytes, one wonders why a full complement of IF1 has been retained in certain cells that normally do not undergo such aerobic-anaerobic switching, cells such as adult mammalian cardiomyocytes of many species . While some mammalian species have, indeed, not retained a functional complement of IF1 in their cardiomyocytes, those that have can benefit significantly from its presence during intervals of myocardial ischemia.

Yeast, 1991 Dec, 7(9), 925 - 31
Participation of proteinase yscA in the in vitro formation of the smaller subunit of glycogen phosphorylase in extracts of Saccharomyces cerevisiae; Ziegler KJ et al.; Analyses by sodium dodecyl sulphate-polyacrylamide gradient-gel electrophoresis and Western blotting of the proteins from boiled cell samples from all growth phases of yeast cultures, and from all stages of extract preparation indicate that the smaller subunit (s-monomer), which is found in purified glycogen phosphorylase (EC 2.4.1.1) from baker's yeast, is not present in the living cell . It is observed in extracts of Saccharomyces carlsbergensis and S . cerevisiae after incubation at ambient temperatures or even after storage in the frozen state at -25 degrees C . Its formation is sensitive towards pepstatin A, and it is absent from extracts of several mutants of S . cerevisiae that do not contain active proteinase yscA (EC 3.4.33.6) . When purified proteinase yscA is added to the extracts of these mutants, the formation of s-monomer is restored . When the proteinase yscA-deficient strains are grown with a reduced amount of complex nitrogen compounds, the slightly smaller sc-monomer is formed in their extracts . This event must be attributed to a different proteinase, since it is sensitive towards p-hydroxymercuriphenylsulphonate, but not towards pepstatin A . The N-terminal amino acid of the sc-monomer was found to be blocked, as in the case of the native l-monomer, but not the s-monomer.

J Gen Microbiol, 1991 Dec, 137 ( Pt 12), 2879 - 83
Effects of alcohols on the respiration and fermentation of aerated suspensions of baker's yeast; Carlsen HN et al.; The immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker's yeast were studied by stopped-flow membrane inlet mass spectrometry . Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0-20%, v/v) . The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects . For five different alkanols (methanol, ethanol, 1-propanol, 2-propanol and 1-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu . This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane . Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.

Biotechnol Appl Biochem, 1991 Dec, 14(3), 284 - 95
Preparative isolation of polyphosphoinositides and other anionic phospholipids from natural sources using chromatography on adsorbents containing primary amino groups; Klyashchitsky BA et al.; A new method for the preparative isolation of anionic phospholipids with the use of chromatography on adsorbents containing primary amino groups has been developed . The method combines elements of bioaffinity and ion-exchange chromatography . Synthesis of adsorbents based on Spheron and silica supports with immobilized neomycin, L-lysine, or aminoalkyl groups was carried out . Optimal conditions for the separation of phospholipid mixtures on aminosorbents were determined . Separation of rat brain bis- and trisphosphoinositides and preparative isolation of bovine heart cardiolipin and baker's yeast phosphatidylinositol are described . The chromatographic behavior of anionic phospholipids on three types of adsorbent was studied . The contribution of biospecific interactions to the adsorption of polyphosphoinositides on aminosorbents is noted.

J Chromatogr, 1991 Nov 8, 586(1), 51 - 9
High-performance affinity chromatography of NADP+ dehydrogenases from cell-free extracts using a nucleotide analogue as general ligand; Alhama J et al.; An epoxy-activated silica column (50 cm x 0.45 cm I.D.) was derivatized with 8-{6-aminohexyl)amino}-2'-phosphoadenosine-5'-diphosphoribose; the bound ligand concentration was 11.4 mumol/g of dry silica, and the useful loading capacity was 2.3 mg of glutathione reductase . The new high-performance liquid chromatographic column specifically retained NADP(+)-dependent enzymes, which were quantitatively eluted specifically by NADP+ or, with better resolution, by potassium chloride . The new high-performance liquid chromatographic support was applied to the purification of glutathione reductase and glucose-6-phosphate dehydrogenase from cell-free extracts of baker's yeast, fish liver and rabbit hemolysates, with high recoveries and excellent purification factors.

Biol Chem Hoppe Seyler, 1991 Nov, 372(11), 1021 - 6
Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors; Jaenicke L et al.; Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L . and Siegmund H.-U., Chem . Phys . Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines . The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells . Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units . Km determinations confirmed the results . Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive . The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.

Biotechnology (N Y), 1991 Nov, 9(11), 1067 - 72
Yeast systems for the commercial production of heterologous proteins; Buckholz RG et al.; Yeasts are attractive hosts for the production of heterologous proteins . Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins . In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation . The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae . However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production . These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica . The performance of these alternative yeast expression systems is reviewed here relative to S . cerevisiae, and the advantages and limitations of these systems are discussed.

J Neurosci Methods, 1991 Oct, 39(3), 225 - 30
A triple staining procedure to evaluate phagocytic role of differentiated astrocytes; Iacono RF et al.; To determine whether phagocytic activity is affected by a viral infection known to induce astrocyte differentiation, a triple procedure (PAP labeling for GFAP, PAS reaction for added baker's yeast cells and hematoxylin for nuclear staining of the whole monolayer) was applied to Junin virus-inoculated cultures, as well as matched controls . The three-step staining simplified yeast cell count for subsequent statistical analysis, which discerned preferential uptake by differentiated rather than immature astrocytes . Accordingly, greater cell maturation induced by Junin virus was concomitant with early enhancement of phagocytic activity.

Clin Ther, 1991 Sep-Oct, 13(5), 569 - 78
The use of human insulin derived from baker's yeast by recombinant DNA technology; Raskin P et al.; Human insulin has gradually replaced animal insulin as the therapeutic agent of first choice among insulin-dependent and insulin-requiring patients with diabetes . Like animal insulin, human insulin manufactured by several different methods is available in Regular, NPH, Lente, and 70/30 (NPH/Regular) formulations . The most recently developed method of manufacturing human insulin uses recombinant DNA technology with baker's yeast as the host cell {rDNA HI (BY)}, offering potentially limitless supplies of insulin structurally identical to that made by the human pancreas . Clinical studies have demonstrated that the extent of insulin absorption, the glucose-lowering effects, and the clinical effects on glycemic control and on incidence of hypoglycemia with rDNA HI (BY) are similar to those observed when patients are treated with semisynthetic human insulin (ssHI) . Dose-for-dose transfer of patients from ssHI to rDNA HI (BY) is therefore appropriate . It is standard practice to recommend that any change in insulin be conducted under medical supervision.

Clin Ther, 1991 Sep-Oct, 13(5), 557 - 68
Transfer of patients with diabetes from semisynthetic human insulin to human insulin prepared by recombinant DNA technology using baker's yeast: a double-blind, randomized study; Davidson JA et al.; A multicenter, double-blind, randomized, parallel-group, 12-month study was conducted to determine if patients with diabetes could be transferred safely and effectively from semisynthetic human insulin (ssHI) to human insulin produced by recombinant DNA technology using baker's yeast as the host cell {rDNA HI (BY)} . Sixty-five patients who ranged in age from 19 to 67 years and who had been on a stable dose of NPH or Lente ssHI with or without Regular ssHI participated . Forty-three were transferred randomly to rDNA HI (BY) and 22 continued on ssHI . Evaluation at both six and 12 months of treatment indicated no statistically or clinically significant differences between the ssHI and rDNA HI (BY) groups with regard to the mean changes from baseline in glycosylated hemoglobin, total daily insulin dose, or body weight . In addition, the proportions of patients who had episodes of mild or moderate hypoglycemia, severe hypoglycemia, hyperglycemia, or other clinical experiences related to diabetes or treatment were similar in the ssHI and rDNA HI (BY) groups . No pattern of unexpected retinal changes occurred, nor were there any remarkable changes in human insulin antibody binding or in mean or individual yeast antibody values in either group . These results show that patients with diabetes can be safely and effectively transferred from semisynthetic human insulin to human insulin produced by rDNA technology from baker's yeast with no change expected in the insulin dose.

Plant Mol Biol, 1991 Sep, 17(3), 415 - 29
Stress responses in alfalfa (Medicago sativa L.) 12 . Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants; Gowri G et al.; An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa . A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region . The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865 . A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1 . Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa . PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast . PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings . These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

Biochim Biophys Acta, 1991 Aug 9, 1079(1), 87 - 95
Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae); Nalecz MJ et al.; Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite . Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nalecz K.A . and Azzi, A . (1989) J . Biol . Chem . 264 18024-18030) . The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa . The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes . The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate . In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein . Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate . Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

Anal Biochem, 1991 Aug 1, 196(2), 234 - 7
Preparation of NADH/NADPH using cetyltrimethylammonium bromide permeabilized baker's yeast cells; Naina NS et al.; Alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) activities of cetyltrimethylammonium bromide permeabilized baker's yeast whole cells were employed to prepare reduced nicotinamide nucleotides NADH and NADPH from their corresponding oxidised forms . Both NADH and NADPH were found to be stable in the presence of permeabilized cells under the conditions of preparation . No dephosphorylation of NADP+ to NAD+ or of NADPH to NADH was found . Reduction is complete and the prepared NADH and NADPH are chromatographically pure . Since readily available Baker's yeast cells were used instead of expensive isolated enzyme the method described here is simple, economical, and easy to scale up.

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