Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.     

Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex

Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps-40 ns time range.     

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P < 0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P < 0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P < 0.0001). The DGS produced significantly more (P = 0.003) intact cells than CB and LVF. Distorted cells were significantly (P = 0.001) more frequent in smears by LVF. The CB harvested significantly (P = 0.009) more fragmented cells. CB and LVF produced significantly (P < 0.0001; P = 0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P = 0.0062; P = 0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P = 0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k ≤ 0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.     

Stability in by-product formation as a strain selection tool of Saccharomyces cerevisiae wine yeasts

A strain of Saccharomyces cerevisiae homozygous for different physiological and metabolic characters was inoculated into two grape musts and the stability of the characters was tested by isolating clones at different fermentation stages. A total of 60 cell-clones were collected and asci dissected from each, yielding a total of 1200 single spore cultures, which were then tested for the segregation of several genetically controlled traits. From the parental strain, 10 asci were dissected and the 40 single spore cultures obtained were used as controls. Micro-fermentations were performed with the 200 single spore cultures obtained from clones isolated at the end of Trebbiano and Aglianico must fermentations. The majority of these spore cultures produced amounts of the secondary compounds at the same level as the parental strain. The progeny of three clones from the Trebbiano fermentation exhibited a significant increase in the production of isoamyl alcohol, whereas the progeny of one clone from the Aglianico fermentation differed in the production of acetoin and amyl alcohols. The variability found in the levels of by-products can also affect the organoleptic properties of the final product. The introduction of the 'metabolic characteristics stability' as a selective index for industrial strains is advised.     

Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa

New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H₂O₂ and O₂ production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24h, 48h and 72h.Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24h, 48h and 72h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.     

Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.     

Genetic segregation of natural Saccharomyces cerevisiae strains derived from spontaneous fermentation of Aglianico wine

AIMS: Investigation of the meiotic segregation of karyotypes and physiological traits in indigenous Saccharomyces strains isolated from Aglianico (South Italy) red wine. METHODS AND RESULTS: Segregation was studied in F1 and F2 descendants. Tetrads were isolated from sporulating cultures by micromanipulation. The spore clones were subjected to karyotype analysis by pulse-field gel electrophoresis (Bio-Rad model CHEF-DR II) and to various physiological tests. Certain chromosomes of the isolates showed 2:2 segregation patterns in F1 but proved to be stable in F2. The ability of cells to utilize maltose also segregated in a 2 : 2 manner in F1 and did not segregate in F2. Resistance to CuSO4, SO2 tolerance, the fermentative power and the production of certain metabolites segregated in both F1 and F2 generations and showed patterns indicating the involvement of polygenic regulation. CONCLUSIONS: The analysis revealed a high degree of genetic instability and demonstrated that meiosis can improve chromosomal and genetic stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Winemaking is critically dependent on the physiological properties and genetic stability of the fermenting Saccharomyces yeasts. Selection of clones from F2 or later generations can be a method of reduction of genetic instability.