Volatile metabolites produced by different flor yeast strains during wine biological ageing.

Sherry white wine called Fino is produced by dynamic biological ageing under the action of flor yeasts using traditional practices aimed at ensuring uniform quality and characteristics over time. These kinds of yeasts provide typical sensory properties to Fino wines. Although there are studies of the volatile composition of these wines submitted to biological ageing in wood barrels, there is a lack of knowledge on the particular volatile profile produced by different flor yeast strains from Sherry zone wineries. For this reason, the aim of this study was to analyse the volatile profiles produced by 15 pure culture flor velum yeasts, with the goal of observing their suitability for obtaining high quality Fino sherry wines. Volatile composition was determined by dual sequential stir bar sorptive extraction, followed by GC-MS analysis. All yeast strains studied produced the increase of most acetals, highlighting acetaldehyde diethylacetal which was the compound that most increased. Among terpenes, nerolidol and farnesol underwent remarkable increases. However, results showed that in a month of biological ageing, significant differences were observed among the volatile metabolites produced by flor yeast strains studied. Only some of them stood out for their high production of volatile compounds characteristic of Sherry Fino wines, which are good candidates for producing starter cultures.

[Advance directives in chronic dialysis patients]. / Documento de voluntades anticipadas de pacientes con insuficiencia renal crónica terminal en tratamiento sustitutivo mediante diálisis.

BACKGROUND AND OBJECTIVE: Knowledge of the life-sustaining treatment preferences of the dialysis patients would be extremely helpful to substitute decision-makers and nephrologists in deciding whether to continue or stop a treatment. The population of the Mediterranean countries show this opinion with less frequency. The objective of this study is: 1) the knowledge of the patient's view for the advance directives; it may increase the likelihood to get the correct decisions of the staff when complications break the normal course of chronic dialysis, and 2) the statement of the advance directives. MATERIAL AND METHOD: We distributed 135 questionnaires to patients with chronic renal failure in dialysis treatment of the Sabadell's Hospital to explore demographic information about responders and not-responders and explore the rate of questionnaires was completed about the cardiopulmonary resuscitation, respirator, tube feeding and dialysis in case of coma, persistent vegetative state, severe dementia and terminal illness. We explore about the representative of patients and in case of not-responders about the cause to not answer. RESULTS: Sixty-four of 135 patients (47,8%) did not want cardiopulmonary resuscitation, respirator, tube feeding or dialysis in case of coma, persistent vegetative state, severe dementia or terminal illness. Compared with patients who wanted the treatments, those who did not were older (71,2 versus 62,2 years; p = 0.002). There was no difference in the other demographic questions, including sex (p=0.674), cause of kidney failure (p=0.815), comorbid conditions (p=0.824), and social status (language of questionnaire -0.155- and standard of education -0.288-). Advance care planning does not occur solely within the context of the physician-patient relationship; the respondents reported the representative in the family, essentially. The patients not-responders doesn t want to think in those situations and also they show doubt about the interpretation of their answers. CONCLUSIONS: near 50% patients in chronic dialysis want to stop certain treatments in case of resuscitation cardiopulmonary, coma, persistent vegetative state, severe dementia or terminal illness. The older patients want the limitation of treatments more frequently.

[Choosing not to dialysis in chronic renal failure in stage V (Renal Failure). Evolution of the characteristics of patients between 1992-1995 and 2000-2003]. / Elección de no diálisis en insuficiencia renal crónica en estadio V (fallo renal). Evolución de las características de los pacientes entre 1992-1995 y 2000-2003.

BACKGROUND: The incidence of chronic renal failure increase with the age. The selection of patient to dialysis has been increasing in spite of the high comorbidity. Moreover, in our clinical practice the aged patient is not contraindicated to dialysis. However, in the nephrology clinical practice not all the patients start the treatment with dialysis. OBJECTIVE The aim of our study has been to compare the characteristics of the patients who had not been dialyzed between the periods 1992-1995 and 2000-2003 to analyze the trend of the nephrology clinical practice. MATERIAL AND METHODS: Comparative study of the characteristics and the evolution of patients with chronic renal failure in stage V, (renal failure) not incorporated to dialysis in one hospital during four years between the periods the 1992-1995 ( period A) and 2000-2003 (period B). RESULTS: Start dialysis (period A versus period B): 116 patients, age 59.9+15.5 years vs. 229 patients, age 64.0+15.8 years (p<0.05). Non-dialysis (period A versus period B): 38 patients, age 77.5+9.3 years vs. 37 patients, age 81.7+6.2 years (p<0.01). Renal function: serum creatinina 7.4+2.4 mg/dl vs. 5.3+1.2 mg/dl (p<0.001), MDRD estimate glomerular filtration 6.9+2.4 mg/dl ml/min/1.73 m2 vs. 10.0+2.3 ml/min/1.73 m2 (p<0.001). Primary renal disease: unknown etiology 31.5 % vs. 24.3 %, nephroangiosclerosis 23.6 % vs. 32.4 %, diabetes 28.9 % vs. 21.6 %. Functional status: dependent patients 34.2 % vs 83.8 % (p<0.001). The principal reason for non-dialysis were: personal decision: 26.3 % vs. 35.1 %, dementia 15.8 % vs. 29.7 %, brief life expectancy because of serious co-existing diseases 13.1 % vs. 21.7 % and serious chronic illness with inability for themselves care 44.7 % vs. 13.1 %. Comorbid conditions: 2.3+1.0 vs. 3.0+1.5 (p<0.05). Survival: 55+168 days vs. 168+236 days (p<0.001). CONCLUSION: Most of the patients that don't begin dialysis are elderly together with a poor functional capacity and with more autonomy in their decisions. The identification of patients with renal failure (stage V) was detected early in the last period than in the following one. The conservative management of non-dialyzed uremic patients is a significative nephrology clinical practice due to more survival of those persons.

[Secondary amyloidosis (AA-type) due to localized cutaneous vasculitis]. / Amiloidosis secundaria asociada a vasculitis cutánea localizada.

We report a case of a 49 year old man, diagnosed soon after the outcome of casual proteinuria, of AA-type amyloidosis in relation to small and medium vessel cutaneous vasculitis without systemic involvement. This combination is a rare entity and only two cases of cutaneous hypersensibility vasculitis complicated with AA-type amyloidosis had been reported. We describe the results of the use of several immunosuppressive drugs during four years follow up with temporally total remission of the disease.

[Diagnosis and prognosis of atheroembolic disease]. / Diagnóstico y pronóstico de la enfermedad ateroembólica.

Atheroembolic disease is recognized as an iatrogenic complication from an invasive vascular procedure, such as manipulation of the aorta during angiography or vascular surgery, and after anticoagulant and fibrinolytic therapy. Cholesterol crystal embolism is caused by showers of cholesterol crystals from an atherosclerotic aorta that occlude small arteries. The kidney is a frequent target organ for cholesterol emboli because of proximity of the renal arteries to abdominal aorta and it receive an enormous amount of blood flows. We describe the epidemiologic agents of 19 cases that were diagnosed by histologic sections of the affected tissues; the eosinophilia and the renal failure are the clinical features that guide to the diagnosis, in patients with ischemic nephropathy and general atherosclerosis. 53% among patients had a previous invasive procedure and 26% occurred spontaneously. We remark the importance of the kidney's biopsy in diagnosis of the atheroembolic disease and their bad prognosis with 63% of death rate in 18 months of average follow-up. We report patients with the multiple cholesterol emboli syndrome mimicking systemic vasculitis: they died by multivisceral acute failure. The subacute presentation of atheroembolic disease with progressive renal failure treated with hemodialysis is a sign of bad prognosis. The knowledge of the disease and their prevention are the better treatment.

[Ischemic renal disease: revascularization or conservative treatment?]. / Nefropatía isquémica: revascularización o tratamiento médico conservador?

Ischemic nephropathy is recognized as a distinct cause of renal insufficiency and it is defined as a significant reduction in glomerular filtration rate in patients with hemodynamically significant renovascular occlusive disease. We argue the epidemiologic and clinical manifestations of atherosclerotic renovascular disease, and we evaluate the pronostic agents. Published studies of the outcome of revascularization for renal-artery stenosis have been excellent, offering a durable patency and functional improvement but they have had numerous limitations. The atherosclerosis is a systemic disease and it provides the general prognosis of patients. We conclude that ischemic renal disease is a nephropathy of smoker men, with proteinuria excretion similar to nephropathy with unilateral stenosis. The age of patients is the clinical feature that decide the treatment: surgery, angioplasty/stent or medical management. Comparative analysis of percutaneous transluminal angioplasty and operation for renal revascularization and medically treated patients have proved that the advanced chronic renal insufficiency is associated with an unfavourable response of treatment of the ischemic nephropathy. But, in this nephropathy the revascularization can be the better therapy for selected patients. The revascularization with angioplasty/stent for patients with unilateral renal stenosis and chronic renal insufficiency has a doubtful effectiveness, as the chronic renal failure is result of nephroangiosclerosis.

Monitoring Cole-Cole parameters during haemodialysis (HD).

UNLABELLED: The investigation of the hydration process during the haemodialysis treatment sessions is very important for the development of methods for predicting the unbalanced fluid shifts and hypotension crisis hence improving the quality of the haemodialysis procedure. Bioimpedance measurements can give valuable information about the tissue under measurement, therefore characterizing the tissue. In this work we propose a non-invasive method based on local multifrequency bioimpedance measurements that allow us to determine the fluid distribution and variations during haemodialysis. METHODS: Clinical measurements were done using 10 HD patients during 60 HD sessions. Bioimpedance data, ultrafiltration volume, blood volume and blood heamatocrit variations were recorded continuously during the HD sessions. Bioimpedance of the local tissue was measured with a 4-elctrode impedance system using surface electrodes with sampling rate of 1meas./4min. at 6 different frequencies. The measured impedances were fitted into Cole-Cole model and the Cole-Cole parameters were continuously determined for each measurement point during the HD session. ANALYSIS: The 4 Cole-Cole parameters (R 00, R 0, Fc,alpha) and their variations were evaluated. Impedance values at infinite and zero (R 00, R 0) frequencies were extrapolated from Cole-Cole mathematical model. These values are assumed to represent the impedance of total tissue fluid and the impedance of the extracellular space respectively.

Genomic complexity and chromosomal rearrangements in wine-laboratory yeast hybrids.

In this report we describe the genomic complexity of a number of Saccharomyces yeast strains isolated from sherry wine (flor yeasts), and the genomic stability of a yeast hybrid derived from one of these and a laboratory strain. Flor yeast strains largely differed in their DNA content, but showed very few variations their molecular karyotype. These strains contained a large number of Ty2 sequences, but lacking the Ty1 elements commonly found in laboratory strains. The genetic analysis of a flor-laboratory hybrid indicated that flor yeasts were aneuploid. Hybridization patterns obtained with Ty1 and Ty2 probes in the meiotic progeny of this hybrid suggested that recombination may occur not only among homologous chromosomes of similar length, but also among polymorphic partners with different sizes. New chromosomal variants were frequently observed in the meiotic products, suggesting that polymorphism in chromosome length may itself be a major source of karyotypic variation. The genetic analysis of such variants indicated that recombinational events leading to new chromosomal forms may occur both mitotically and meiotically.

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.

Detection of Dekkera-Brettanomyces strains in sherry by a nested PCR method.

Brettanomyces sp. and its ascosporogenous sexual state, Dekkera sp., have been well documented as spoilage microorganisms, usually associated with barrel-aged red wines. In this report, we describe the genetic characterization, on the basis of DNA content per cell, electrophoretic karyotyping, and mitochondrial DNA restriction patterns, of a Dekkera yeast strain isolated from sherries and of a number of other Brettanomyces and Dekkera strains. By using a genomic DNA fragment of the isolated Dekkera strain, we developed a two-step PCR method which directs the specific amplification of target DNA from this strain and from other Brettanomyces-Dekkera strains. The method efficiently amplified the target DNA from intact cells, obviating DNA isolation, and yielded a detection limit of fewer than 10 yeast cells in contaminated samples of sherry.

Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein.

Osmotin is a plant PR-5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an alpha-1, 2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an alpha-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins.

Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility.

The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

A plant defense response effector induces microbial apoptosis.

Osmotin is a tobacco PR-5 protein that has antifungal activity and is implicated in host-plant defense. We show here that osmotin induces apoptosis in Saccharomyces cerevisiae. Induction of apoptosis was correlated with intracellular accumulation of reactive oxygen species and was mediated by RAS2, but not RAS1. Osmotin treatment resulted in suppression of transcription of stress-responsive genes via the RAS2/cAMP pathway. It was therefore concluded that osmotin induced proapoptotic signaling in yeast. The results indicate that the ability of antimicrobial proteins to induce microbial apoptosis could be an important factor in determining a pathogen's virulence and could therefore be targeted for the design of new antifungal drugs.

Resistance to the plant PR-5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition.

The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1-v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Delta mutation displayed high sensitivity to osmotin. The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner. Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression. The sensitivity of ssd1Delta cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans. Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.