Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.     

Glutathione protects Lactococcus lactis against oxidative stress

Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to approximately 60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.     

Germination of penicillium paneum Conidia is regulated by 1-octen-3-ol, a volatile self-inhibitor

Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10(9) conidia ml(-1). By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25 degrees C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.     

Flow cytometric assessment of membrane integrity of ethanol-stressed Oenococcus oeni cells

The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol on O. oeni cells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation of O. oeni cells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations.     

Multiple deletions of the osmolyte transporters BetL,Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (DeltabetL DeltaopuC Deltagbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 micro mol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (DeltabetL Deltagbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 micro mol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.     

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry.

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efflux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

Linkage disequilibrium inflates type I error rates in multipoint linkage analysis when parental genotypes are missing

OBJECTIVES: Describe the inflation in nonparametric multipoint LOD scores due to inter-marker linkage disequilibrium (LD) across many markers with varied allele frequencies. METHOD: Using simulated two-generation families with and without parents, we conducted nonparametric multipoint linkage analysis with 2 to 10 markers with minor allele frequencies (MAF) of 0.5 and 0.1. RESULTS: Misspecification of population haplotype frequencies by assuming linkage equilibrium caused inflated multipoint LOD scores due to inter-marker LD when parental genotypes were not included. Inflation increased as more markers in LD were included and decreased as markers in equilibrium were added. When marker allele frequencies were unequal, the r2 measure of LD was a better predictor of inflation than D'. CONCLUSION: This observation strongly supports the evaluation of LD in multipoint linkage analyses, and further suggests that unaccounted for LD may be suspected when two-point and multipoint linkage analyses show a marked disparity in regions with elevated r2 measures of LD. Given the increasing popularity of high-density genome-wide SNP screens, inter-marker LD should be a concern in future linkage studies.