A 'fragile cell' sub-population revealed during cytometric assessment of Saccharomyces cerevisiae viability in lipid-limited alcoholic fermentation.

AIMS: To show that in anaerobic fermentation with limiting lipid nutrients, cell preparation impacts the viability assessment of yeast cells, and to identify the factors involved. METHODS AND RESULTS: Saccharomyces cerevisiae viability was determined using propidium iodide staining and the flow cytometry. Analyses identified intact cells, dead cells and, under certain conditions, the presence of a third subpopulation of apparently damaged cells. This intermediate population could account for up to 40% of the entire cell population. We describe, analyse and discuss the effects of different solutions for cell resuspension on the respective proportion of these three populations, in particular that of the intermediate population. We show that this intermediate cell population forms in the absence of Ca(2+)/Mg(2+). CONCLUSIONS: Cell preparation significantly impacts population viability assessment by FCM. The intermediate population, revealed under certain conditions, could be renamed as 'fragile cells'. For these cells, Ca(2+) and Mg(2+) reduce cell membrane permeability to PI. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that analyses and discusses the factors influencing the formation of an intermediate population when studying viability in yeast alcoholic fermentation. With a wider application in biological research, this study provides important support to the relatively new questioning of propidium iodide staining as a universal cell death indicator.

Carbonic maceration wines: characteristics and winemaking process.

Invented by Michel Flanzy in 1934, carbonic maceration involves placing the intact grape clusters into a closed tank with a carbon dioxide-rich atmosphere. The berries subsequently undergo an intracellular fermentation without yeast intervention. Complex changes occur during this process which entail the transformation of a small amount of sugar into alcohol (1.5-2% alcohol), the reduction of malic acid content by about half, and the generation of secondary products. Compared with wines produced by conventional techniques, carbonic maceration produces wines of distinctive character of superior quality possessing a harmonious balance. It can be used to generate a wide range of wines (red as well as rosé), to be drunk young or aged. The process is composed of four steps: vatting of intact berries, "maceration-fermentation," pumping off, and pressing, followed by a second fermentation phase. Exchanges and interactions occur between grape berries, the gaseous atmosphere, and the must present at the bottom of the tank during the first step of the carbonic maceration winemaking process. Yeast fermentation starts at this stage, in the liquid phase, and continues throughout the second step as well, with the malolactic fermentation. The specific conditions required for a good handling of carbonic maceration are presented.

[Access to medical records: who asks and why?]. / Accès au dossier médical: caractéristiques et motifs des demandeurs.

OBJECTIVE: The aim of the survey was to study the characteristics of patients who (or whose families) request access to their medical records, their satisfaction with the handling of their requests, and the reasons for them. METHODS: This prospective study analyzed all the requests for access to medical records sent to two hospitals and collected data from the hospitals at that time (characteristics of the requester, patient and hospitalization) and from the requesters afterwards (opinion survey). RESULTS: The study reviewed 94 requests for medical records. Delays in providing the records exceeded the statutory periods. Most requests were intended to allow continuity of care, to provide information the patient did not receive during hospitalization, or because the patient needed to transmit it to a third party (new doctor, insurance, attorney, worker compensation). The hospitals mailed records to patients by regular mail in 90% of cases. One third of the patients were not satisfied by the handling of their request, complaining about the following difficulties: length of wait for records, complexity of the request procedure, its expense, absence of some documents, and difficulty in understanding the files without medical assistance. CONCLUSION: Improvements are needed, both to decrease the number of requests and to improve the requesters' satisfaction. Specific improvements are suggested.

Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development.

Three full-length cDNAs (VvAdh1, VvAdh2, and VvAdh3) encoding alcohol dehydrogenases (EC 1.1.1.1) were obtained from grape berries (Vitis vinifera L.) by means of PCR and RACE. Pairwise comparisons at the nucleotide level showed that the three cDNAs displayed strong homology in the coding region, but were highly divergent in the 5' and 3' untranslated regions. VvAdh1 and VvAdh2 corresponded to the two previously characterised Adh genes from grapevine, but VvAdh3 was unrelated to known grapevine Adh sequences. The two first cDNAs presented a single ORF of 380 amino acids, whereas the last one has two additional residues. Moreover, the three encoded polypeptides possessed the 22 residues strictly conserved between Adh from different kingdoms. Expression pattern of the individual isogenes was investigated during fruit development. Specific primers were designed, and quantitative RT-PCR experiments were performed to increase the sensitivity of detecting isogenes with a low expression level. Results presented here revealed different developmental regulation of the three Adh isogenes during fruit ripening. VvAdh1 and VvAdh3 transcripts were temporarily accumulated in young, developing berry, whereas VvAdh2 was overexpressed later in fruit development, from the onset of ripening (véraison). Expression analysis also indicated that VvAdh2 accounted for most of the Adh mRNAs present in berries during development. The increased ADH activity detected in berries correlated with the expression pattern of VvAdh2 transcripts. The VvAdh2 and VvAdh3 encoded enzymes were purified from overexpressing E. coli cells. Comparison of kinetic properties of the two ADH enzymes showed a difference in affinity with either ethanol or acetaldehyde as substrates. Significance of multiple Adh expressed in berries is discussed.

Cloning and characterization of Vine-1, a LTR-retrotransposon-like element in Vitis vinifera L., and other Vitis species.

We report the organization of a grapevine chimeric gene Adhr-Vine-1, composed by an Adhr gene, into which a retroelement, Vine-1, was inserted. Sequence analysis revealed that Adhr is a member of the Adh multigene family, but does not correspond to any other grapevine Adh described to date. Vine-1, albeit defective, is the most complete LTR (long terminal repeat)-retrotransposon-like element described in Vitis vinifera L. It is 2392 bp long, with two almost identical LTRs (287 bp) in the same orientation, and flanked by direct repeats of a 5 bp host DNA. This element presents other features, characteristic of retroviruses and retrotransposons including inverted repeats, a primer binding site, and a polypurine tract. It has a single open reading frame (ORF) of 581 amino acids, potentially encoding for a gag protein and parts of the protease and integrase proteins. Vine-1 is most likely related to the copia-like type family, but with no significant similarity to any previously described plant retrotransposon or inserted element, nor to any eukaryotic element described to date. Vine-1 element has been found in Adhr at the same location in different V. vinifera cultivars, but not in some other analyzed Vitis species. These data suggest that Vine-1 insertion in Adhr is specific to V. vinifera, and has occurred after the Adh isogene separation, but prior to cultivar development. Sequences related to Vine-1 were revealed in multiple copies in the V. vinifera genome and, to a lesser extent, in other analyzed Vitis species. The polymorphism observed prompts us to question the role played by transposition in the evolution of the Vitis genus.

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.