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PubMed2006

Stabilizing behavior of soy soluble polysaccharide or high methoxyl pectin in soy protein isolate emulsions at low pH.

The stability of emulsions prepared with soy protein isolates was investigated as a function of pH in the presence of two negatively charged polysaccharides: high methoxyl pectin (HMP) and soy soluble polysaccharide (SSPS). Both polysaccharides are composed of a backbone which contains galacturonic acid but, when added to soy protein isolate-stabilized emulsions, SSPS showed a different behavior than that of HMP. At neutral pH and above a critical concentration of stabilizer (0.05%), HMP caused flocculation of the emulsion droplets via a depletion mechanism. On the other hand, the emulsions containing a similar amount of SSPS did not show creaming or flocculation. At acidic pH (<4.0) the addition of pectin caused extensive droplet aggregation, while no aggregation was observed with the addition of SSPS. The differences in the stabilization behavior between the two polysaccharides can be attributed to their differences in charge, neutral sugars side chains, and molecular weight.

Drug Stability↗

PubMed2006

Stability of acidic egg white protein emulsions containing xanthan gum.

The influence of xanthan gum concentration on the physicochemical stability of model oil-in-water emulsions prepared with egg white protein at pH 3.8 and containing 150 mM NaCl was investigated by following droplet aggregate formation, rheological changes, and serum separation with storage time. Egg white emulsions were more strongly flocculated and exhibited higher stability against creaming than those of yolk, irrespective of the presence or absence of xanthan. Depletion effects, originating from the presence in the continuous phase of the emulsions of nonadsorbing xanthan molecules, intensified droplet-droplet flocculation effects and resulted in large droplet flocs. At relatively low xanthan contents, the emulsions exhibited higher stability against creaming compared to the respective control emulsions probably due to the formation of a continuous droplet aggregate network structure. At higher xanthan contents, less extensive droplet interactions, due to slowly evolving microstructure of phase-separated xanthan-rich and xanthan-depleted regions, resulted in emulsions exhibiting increased stability against creaming. The role of interactions between protein molecules adsorbed on neighboring droplets in these changes and their effect on emulsion aging are discussed.

Chemical Phenomena↗

PubMed2007

Impact of electrostatic interactions on formation and stability of emulsions containing oil droplets coated by beta-lactoglobulin-pectin complexes.

Interfacial protein-polysaccharide complexes can be used to improve the physical stability of oil-in-water emulsions. The purpose of this study was to examine the impact of ionic strength on the formation and stability of oil-in-water emulsions containing polysaccharide-protein-coated droplets. Emulsions were prepared that contained 0.1 wt % corn oil, 0.05 wt % beta-lactoglobulin, and 0.02 wt % pectin at pH 7. The emulsions were then adjusted to pH 4 to promote electrostatic deposition of the pectin molecules onto the surfaces of the protein-coated droplets. The salt concentration of the aqueous phase (0 or 50 mM NaCl) was adjusted either before or after deposition of the pectin molecules onto the droplet surfaces. We found that stable emulsions containing polysaccharide-protein-coated droplets could be formed when the salt was added after pectin adsorption but not when it was added before pectin adsorption. This phenomenon was attributed to the ability of NaCl to promote droplet flocculation in the protein-coated droplets so that the pectin molecules adsorbed onto the surfaces of flocs rather than individual droplets when salt was added before pectin adsorption. We also found that polysaccharide-protein-coated droplets had a much improved stability to salt-induced flocculation than protein-coated droplets with the same droplet charge (zeta-potential). Theoretical predictions indicated that this was due to the ability of the adsorbed polysaccharide layer to strongly diminish the van der Waals attraction between the droplets.

Adsorption↗

PubMed1998

Influence of Protein Interfacial Composition on Salt Stability of Mixed Casein Emulsions.

The influence of casein surface composition on the stability toward flocculation by sodium chloride has been investigated for oil-in-water emulsions (20 vol % oil, 1 wt % total protein) prepared with a mixture of alpha(s1)-casein + beta-casein. It has been observed that the poor salt stability of emulsions containing 100% alpha(s1)-casein is greatly enhanced by the replacement of about one-third of the alpha(s1)-casein by beta-casein. With around half of the adsorbed protein layer consisting of beta-casein, emulsions remain stable at ionic strengths of 2 M NaCl in the presence or absence of calcium ions (5 mM). At the point of droplet flocculation in these emulsions, most of the casein present is associated with the surface of the droplets. These results provide definitive evidence for the important influence of the compositional balance in sodium caseinate on the colloidal stability behavior of casein-based emulsions.

Journal Article↗

PubMed1999

Involvement of proteins in cloud instability of shamouti orange [Citrus sinensis (L.) Osbeck] juice.

The present study provides evidence for the involvement of protein in cloud instability of natural orange juice. No heat-coagulable proteins were found in the serum. Insoluble cloud matter (ICM) was heat-flocculated following enzymatic pectin degradation (EPD). The degree of flocculation depended on temperature (from approximately 50 to 75 degrees C) and was highest at pH 3.5. The fresh juice contained about 6.5 and 1.8 mg mL(-1) of ICM and alcohol-insoluble solids of the serum (AISS), respectively. The ICM and the AISS contained, respectively, proteins (182+/-14 and 119+/-3 microg mg(-1)), galacturonic acid (37+/-6.6 and 175+/-1 microg mg(-1)), and neutral sugars (350+/-44 and 338+/-22 microg mg(-1)). EPD resulted in removal of a marked portion of the pectin and was accompanied by partial removal of neutral sugars (mainly glucose and galactose) and some proteins from the pectic polymer in both AISS and ICM. Under electrophoresis, proteins of the AISS included bands in the range of 20-52 kDa and 10-14 kDa and those of the ICM at 22 and 50 kDa.

Beverages↗

PubMed1999

Surface functional properties of native, acid-treated, and reduced soy glycinin. 2. Emulsifying properties.

Emulsifying properties of native and chemically modified soy glycinins were studied. The influence of ionic strength, protein sample composition and concentration, and assay conditions on the flocculation-creaming process and coalescence resistance was analyzed. Differences in these emulsifying properties were exhibited by native glycinins, which have a variable content of 4S, 11S, and 15S forms. Structure and functionality of native glycinin were modified by means of combined treatments: mild acidic treatments without heating or with heating at variable time and with or without disulfide bonds reduction. Modified glycinins presented different degrees of deamidation, surface hydrophobicity, and molecular mass. A slight enhancement of emulsifying stability at moderated deamidation degrees was observed. In different protein samples, a positive relationship between the flocculation-creaming rate constant and equilibrium oil volume fraction of emulsions with surface hydrophobicity was detected. A remarkable difference was observed between reduced and nonreduced samples, mainly with respect to behavior at low or high ionic strength.

Excipients↗

PubMed2006

Stabilization of gold nanoparticles by 6-mercaptopurine monolayers. Effects of the solvent properties.

6-Mercaptopurine-coated gold nanoparticles (6MP-AuNPs) have been prepared by modification of the nanoparticle surface with 6MP upon displacement of the protective layer of citrate anions. The modification has been studied by UV-vis and FTIR spectroscopies. A study of the stability of these 6MP-AuNPs in aqueous solutions as a function of ionic strength and pH has shown the importance of the charges on the stabilization. The protonation of N9 of the 6MP molecules brings about a sudden flocculation phenomenon. However, the flocculation is reversible upon changing the pH to values where the molecules become newly charged. Evidence of the competence between the interaction of capping solvent molecules and the attractive forces between particles is also shown in this paper.

Journal Article↗

PubMed2005

Colloid stability of sodium dihexadecyl phosphate/poly(diallyldimethylammonium chloride) decorated latex.

The colloid stability of supramolecular assemblies composed of the synthetic anionic lipid sodium dihexadecyl phosphate (DHP) on cationic poly(diallyldimethylammonium chloride) (PDDA) supported on polystyrene sulfate (PSS) microspheres was evaluated via turbidimetry kinetics, dynamic light scattering for particle sizing, zeta-potential analysis, and determination of DHP adsorption on PDDA-covered particles. At 0.05 g/L PDDA and 5 x 10(9) PSS particles/mL, PDDA did not induce significant particle flocculation and a vast majority of PDDA covered single particles were present in the dispersion so that this was the condition chosen for determining DHP concentration (C) effects on particle size and zeta-potentials. At 0.8 mM DHP, charge neutralization, maximal size, and visible precipitation indicated extensive flocculation and minimal colloid stability for the DHP/PDDA/PSS assembly. At 0.05 g L(-1) PDDA, isotherms of high affinity for DHP adsorption on PDDA-covered particles presented a plateau at a limiting adsorption of 135 x 10(19) DHP molecules adsorbed per square meter PSS which was well above bilayer deposition on a smooth particle surface. The polyelectrolyte layer on hydrophobic particles was swelled and fluffy yielding ca. 6 +/- 1.5 nm hydrodynamic thickness. Maximal and massive adsorption of DHP lipid onto this layer produced polydisperse DHP/PDDA/PSS colloidal particles with low colloid stability and which, at best, remained aggregated as doublets over a range of large lipid concentrations so that it was not possible to evaluate the mean total thickness for the deposited film. The assembly anionic lipid/cationic PDDA layer/polymeric particle was relatively stable as particle doublets only well above charge neutralization of the polyelectrolyte by the anionic lipid, at relatively large lipid concentrations (above 1 mM DHP) with charge neutralization leading to extensive particle aggregation.

Journal Article↗

PubMed2006

pH-Dependent aggregation properties of mixtures of sugar-based gemini surfactants with phospholipids and single-tailed surfactants.

Sugar-based gemini surfactants (GSs) display rich pH-dependent phase diagrams and are considered to be promising candidates as gene- and drug-delivery vehicles for biomedical applications. Several sugar-based GSs form vesicles around neutral pH. The vesicular dispersions undergo transitions toward wormlike micelles and spherical micelles at acidic pH, whereas flocculation followed by redispersion upon charge reversal is observed at basic pH. The influence of various amounts of the double-tailed phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and of the single-tailed surfactants lyso-PC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) and OTAC (octadecyltrimethylammonium chloride) on the phase behavior of GS1 (1,8-bis(N-octadec-9-yl-1-deoxy-D-glucitol-1-ylamino)3,6-dioxaoctane) was determined as a function of pH, in water and in water at physiological ionic strength. The pH corresponding to the phase transitions and the characteristics of the aggregates were determined by means of a combination of physical techniques: static and dynamic light scattering (SLS and DLS), fluorescence spectroscopy, cryo-TEM and diffusion- and (31)P NMR. The results show that the additives affect the phase behavior of the GS1 dispersions in a pH-dependent fashion. In the presence of double-tailed phospholipids, a higher degree of protonation of GS1 must be reached to observe micelle formation, whereas single-tailed surfactants affect these transitions only slightly. In the presence of increasing amounts of lyso-PC, the pH range of flocculation becomes more narrow, indicating the increased hydration of the vesicles. The pH of redispersion after charge reversal is particularly sensitive to the presence of positively charged additives. It is suggested that the cationic headgroups disturb the hydrogen-bond structure of water at the vesicular surface, hampering OH(-) binding. The effect of an increase in ionic strength to physiological values is found to be modest, except for the dispersions containing the positively charged additives.

Journal Article↗

PubMed2006

Heteroflocculation of particle mixtures by a coacervation mechanism. A rheological study.

In most of the classical studies of heteroflocculation two sets of oppositely charged particles are mixed. In this current study, a somewhat different mechanism of heteroflocculation is described. Two sets of concentrated dispersions of polyacrylate latex particles (having the same surface-charge sign) have been mixed, where the surface of one set had been functionalized with methacrylic acid (MAAc) groups and the second set with poly(ethylene oxide) (PEO) chains. The resultant heteroflocculation has been investigated as a function of the number fraction (F) of MAAc-functionalized particles, the size ratio (R) of the two sets of particles (R = d(MMAc)/d(PEO)), the background electrolyte concentration (0-0.2 M KCl), the pH (3 or 9), and the order of mixing of the particles. The relative extent and strength of flocculation were assessed using two basic rheological techniques: (i) the plastic viscosity (eta(pl)) and the Bingham yield stress (sigmaB) were determined from steady-state shear experiments, (ii) the modulus (G) and the viscosity (eta) (both at a given applied stress) and the actual yield stress (sigmaY) were determined from creep-recovery experiments. Heteroflocculation was observed at a pH value of 3, where the carboxylic groups at the surface of the MAAc-functionalized particles remain largely undissociated. However, no flocculation was observed at pH 9, where the COOH groups dissociate to become COO-. The aggregation mechanism is, therefore, believed to be due to hydrogen-bonding between the hydrogens of the carboxylic acid groups and the ether oxygens present on the surface of the PEO-functionalized particles. To this extent, this mechanism of heteroflocculation resembles the coacervation of mixtures of solutions of two H-bonding polymers, for which aqueous mixtures of PMAAc and PEO (at low pH) are a well-known example. Because both sets of particles carried negative surface charge groups, arising from the polymerization initiator used in their preparation, a minimum concentration of added electrolyte (KCl) was needed before any heteroflocculation between the two sets of particles was observed. However, this minimum KCl concentration for the onset of heteroflocculation was significantly lower than the concentration of KCl required to induce homoflocculation of either set of latex particles separately. At an R value of 1.3, all the rheological parameters passed through a maximum value at F = 0.5, whereas when R was 6.2 the maximum occurred at a value of F = 0.018 or F = 0.025, depending on the order of mixing.

Journal Article↗

PubMed2006

Poly(DEAEMa-co-PEGMa): a new pH-responsive comb copolymer stabilizer for emulsions and dispersions.

Stimulus responsive copolymers are an important class of surfactants that are attracting growing attention in the literature. When used to stabilize colloids, they confer responsiveness to an otherwise nonresponsive system. In this work, a new pH-responsive comb copolymer surfactant, poly(DEAEMa-co-PEGMa), where DEAEMa and PEGMa are diethylaminoethyl methacrylate and poly(ethylene glycol) methacrylate, is introduced and used to stabilize emulsions and particulate dispersions. The copolymer contained 70 mol % of DEAEMa. Turbidity versus pH measurements and photon correlation spectroscopy of the copolymer solutions revealed pH-triggered collapse of the chains above the pK(a). The surface activity of the copolymer increased with pH. The minimum surface tension measured was 33.6 mN/m at pH = 10. These data enabled identification of the pK(a) for poly(DEAEMa-co-PEGMa) as 6.8. The emulsions consisted of tetradecane-in-water and had a droplet size in the range 5-11 mum. They were slightly flocculated when the pH value was close to the isoelectric point. The emulsions phase separated at low pH values. The particulate dispersions were based on carbendazim, which is a fungacide, and had an average size of 1.8 mum. The data for the emulsions and carbendazim dispersions show that the extent of flocculation decreases with decreasing size of the dispersed phase. Analysis of the data suggest that optimum emulsion stability occurs in the pH region of 5.3-6.8 as judged by turbidity measurements. Electrophoretic mobility measurements as a function of pH for the emulsions and carbendazim dispersions reveal a similar isoelectric point in the range of 8.5-9.0, which is about two pH units higher than the pK(a) of the copolymer. A mechanism that explains the pH-responsive stability of the emulsions and dispersions is presented and discussed.

Benzimidazoles↗

PubMed2002

Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway.

Filamentation and adherence to host cells are critical virulence factors of Candida albicans. Multiple filamentation regulatory pathways have been discovered in C. albicans using Saccharomyces cerevisiae as a model. In S. cerevisiae, these pathways converge on Flo11p, which functions as a downstream effector of filamentation and also mediates cell-cell adherence (flocculation). In C. albicans, such effector(s) have not yet been identified. Here, we demonstrate that the cell surface protein Als1p is an effector of filamentation in C. albicans. We show that Als1p expression is controlled by the transcription factor Efg1p, which is known to be a key regulator of filamentation in C. albicans. Further, disruption of ALS1 inhibited filamentation, and autonomous expression of Als1p restored filamentation in an efg1 homozygous null mutant. Thus, Als1p functions as a downstream effector of the EFG1 filamentation pathway. In addition, we found that Als1p mediates both flocculation and adherence of C. albicans to endothelial cells in vitro. As a cell surface glycoprotein that mediates filamentation and adherence, Als1p has both structural and functional similarity to S. cerevisiae Flo11p. Consistent with our in vitro results, Als1p was required for both normal filamentation and virulence in the mouse model of haematogenously disseminated candidiasis.

Candida albicans↗

PubMed2005

Bi-modal hetero-aggregation rate response to particle dosage.

The rate of flocculation of cationic polystyrene latex (PSL) particles by smaller, anionic PSL particles has been measured using a low-angle static light scattering technique. The rate of aggregate growth has been investigated as a function of particle size ratio and relative concentration of each particle species (for a constant dose of cationic particles). Contrary to many previous reports, two peaks in the flocculation rate were observed as a function of dose. It is speculated that the peak observed at the lower particle concentration coincides with the dose yielding maximum constant collision efficiency in the steady-state regime, a condition which is attained only after complete adsorption of the smaller particles onto the larger particle species. The peak at the higher particle concentration is believed to be related to the maximum collision rate constant upon reaching the steady-state regime, the value of which corresponds to maximum degree of aggregation and therefore the maximum mean collision efficiency prior to reaching this condition. From classical collision kinetics, the rate of aggregate growth may be represented as being proportional to the product of the collision rate constant and collision efficiency at any given time. Given then that the maximum value of these two variables coincides with different particle concentrations, the product of the response of each to particle dosage can in some cases yield a net bi-modal aggregation rate response to particle dosage.

Journal Article↗

PubMed2002

Rubritepida flocculans gen. nov., sp. nov., a new slightly thermophilic member of the alpha-1 subclass of the Proteobacteria.

A bacterial isolate, with an optimum growth temperature of about 50 degrees C, was recovered from the hot spring at Egerszalók in Hungary. Phylogenetic analyses using the 16S rRNA gene sequence of strain H-8T indicated that the new organism represented a new genus and species of alpha-1 subclass of the Proteobacteria. The major fatty acids of strain H-8T are 16:0, 18:1 omega7c; the rare fatty acid 19:0 20H cyclo 11,12 is also present. Ubiquinone 9 is the major respiratory quinone, the polar lipids are phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol in addition to two unidentified aminolipids. The new isolate forms red-colored colonies, flocculates in liquid media, is heterotrophic and strictly aerobic. Thiosulfate is oxidized to sulfate, but an increase in biomass could not be measured because of the flocculating behavior. Bacteriochloropyll a was detected by direct spectrophotometric analysis when the organism was grown at 30 degrees C, but could not be detected after growth at 50 degrees C. pufL and pufM genes were present. Heterotrophic growth of strain H-8T occurs on a few carbohydrates, amino acids and organic acids. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strain H-8T represents a new genus and a new species most closely related to Roseococcus thiosulfatophilus for which we propose the name Rubritepida flocculans.

Alphaproteobacteria↗

PubMed2005

Pharmacokinetics and tissue distribution of etoposide delivered in long circulating parenteral emulsion.

PURPOSE: The aim of the study is to ascertain the influence of pegylation of parenteral emulsion (PE) on their long circulating property. METHODS: Etoposide encapsulated parenteral emulsion (EPE) was prepared using soybean oil, egg lecithin and cholesterol. Etoposide encapsulated long circulating parenteral emulsion (PEG-EPE) was prepared using PEG (2000)-DSPE as a stealth agent. The effect of monovalent and divalent electrolytes on the stability of EP was assessed by measuring the fixed aqueous layer thickness (FALT) and flocculation rate. Pharmacokinetics and tissue distribution pattern of PE following i.v. (bolus) were assessed in Wistar rats and Swiss albino mice. RESULTS: FALT of PEG-EPE was larger than that of EPE. In case of PEG-EPE, as the concentration of pegylated lipid (PEG) increased from 0.15 to 0.45% w/v the flocculation rate decreased asymptomatically in the presence of monovalent and divalent electrolytes. The increased circulation time of PEG-EPE (0.3%) after intravenous injection to rats confirms the presence of FALT around globules. PEG-EPE showed improved pharmacokinetic parameters with 5.5 times higher AUC than etoposide commercial formulation (ETP). Tissue distribution results show that etoposide levels in all tissues except in brain and heart were lower in case of PEG-EPE than ETP. The percentage of tumor growth suppression rate (%T/C) in Lewis lung carcinoma bearing mice was 63.23, 62.83 and 33.78% in EPE, PEG-EPE and ETP treated mice, respectively. The improved activity of PEG-EPE is due to enhanced permeability and retention effect (EPR). CONCLUSION: Encapsulation of etoposide in PEG-coated PE produced improved pharmacokinetic profile than that of EPE and ETP.

Animals↗

PubMed1976

Coated-vesicle shells, particle/chain material, and tubulin in brain synaptosomes. An electron microscope and biochemical study.

Coated vesicles (CVs), plain synaptic vesicles (PSVs), and nonvesicular flocculent material were isolated from synaptosomes and examined with goniometry and high-resolution electron microscopy after either negative staining or various biochemical procedures. The flocculent material (i.e. the presynaptic matrix material except CV shells) is largely composed of particulate or elongated (chainlike) structures; some of this material (here referred to as particle/chain material) is attached to PSVs. The results obtained were: (a) the proteinaceous properties of the CV coat (also referred to as CV shell) and the particle/chain material were demonstrated with chymotrypsin; (b) the CV shell, studied with various negative-staining techniques, differs from the particle/chain material since it has no 3-4-nm globular subunits and reacts differently to alkaline pH; (c) the particle/chain material consists of aggregates of 3-4-nm globular subunits, four of which yield 8-10-nm fine particles; and these particles can be further aggregated into chains 8-10 nm wide and up to 30-60 nm long showing a "hollow" core; (d) vinblastine sulfate induced ringlike or helical crystalloid precipitates closely resembling the vinblastine-induced microtubule crystals reported in the literature, but vinblastine had no effect on either the CV shell material or the particle/chain material.

Animals↗

PubMed1961

Immunologic studies of heart tissue. IV. Serologic reactions with human heart tissue as revealed by immunofluorescent methods: isoimmune, Wassermann, and autoimmune reactions.

Reactions of normal and pathologic sera with heart tissue have been investigated by the immunofluorescent method, with particular reference to presumptive autoantibodies to heart and their differentiation from blood group isoantibodies and Wassermann antibody. In the heart, blood group substances A and B were found distributed in capillary walls, vascular endothelium, and interstitial connective tissue. In surveys of randomly selected sera, isoimmune reactions against tissue blood group substances A and B were noted infrequently. This finding was considered related to the limited sensitivity of fluorescent antibody methods. Heart tissue from blood group O individuals was used for screening of pathologic sera for presence of tissue-reactive factors. Wassermann antibody was found reactive with constituents of myocardial sarcoplasm, of which the major reactant was cardiolipin. Wassermann-positive sera absorbed with beef cardiolipin gave evidence of reaction with other constituents of myofiber sarcoplasm. Sera of patients with rheumatic fever, rheumatic heart disease, rheumatoid arthritis, disseminated lupus, and liver disease frequently showed a marked reactivity with constituents of myofiber sarcoplasm. These serum factors were differentiated from Wassermann antibody. At least three patterns of immunofluorescent staining could be differentiated by the distribution of reactants in the myofiber sarcoplasm. These reactants were extractable with ethanol and methanol but not by acetone. Sera found reactive by immunofluorescence frequently gave positive flocculation and complement-fixation tests with alcohol extracts of human heart. Immunofluorescent tests were best correlated with flocculation reactions. Sarcoplasmic-reactive factors were associated in some sera with 19S gamma globulin as demonstrated by the use of fluorescent anti-19S gamma globulin. Serologic reactions with homologous or autologous heart were observed particularly frequently with sera from rheumatic patients approximately 2 weeks following cardiac surgery, as well as in some non-rheumatic patients following cardiac or thoracic surgery or acute myocardial infarction. The pathogenetic significance of these presumptive autoantibodies to heart is unknown. As yet, no definite conclusions may be drawn regarding their relationship to bound gamma globulin in rheumatic hearts or to the postcardiotomy and post-infarction syndromes.

Antibodies↗

PubMed1998

Mutational analysis of the Tup1 general repressor of yeast.

The Tup1 and Ssn6 proteins of Saccharomyces cerevisiae form a general transcriptional repression complex that regulates the expression of a diverse set of genes including aerobically repressed hypoxic genes, a-mating type genes, glucose repressed genes, and genes controlling cell flocculence. To identify amino acid residues in the Tup1 protein that are required for repression function, we selected for mutations that derepressed the hypoxic genes. Three missense mutations that accumulated stable protein were isolated, and an additional three were generated by site-directed mutagenesis. The mutant protein L62R was unable to complex with Ssn6 or repress expression of reporter genes for the hypoxic and glucose repressed regulons or the flocculence phenotype, however, expression of the a-mating type reporter gene was still repressed. The remaining mutations fell within the WD repeat region of Tup1. These mutations had different effects on the expression of the four Tup1 repressed regulons assayed, indicating that the WD repeats serve different roles for repression of different regulons.

Amino Acid Sequence↗