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