Improvement of the alcohol dehydrogenase from baker's yeast using polymers and salts for alcohol determinations in beverages

An alcohol dehydrogenase (ADH) was purified from dry baker’s yeast. This is a key enzyme of the primary short-chain alcohol metabolism in many organisms. In the present study, the obtained enzymatic preparation of baker’s yeast, containing 2.7 U/mg of ADH, was used in the reactions. The purified extract of the ADH obtained from Fermix commercial dry yeast, presented the highest activity and purification factor when ammonium sulfate was added in the precipitation of protein, in the range 35-60% (w/v). The enzymatic preparation was maintained for 2 months in the lyophilized form at 4ºC (retention of 96.2% of activity) in the presence of 1 mmol/L of sodium azide, and it maintained 47% of activity for 30 days at 30°C in the presence of 15% PEG. The assays of ethanol (detection range 5 mM -150 mM or 2.3 x 10-4 – 6.91 x 10-3g/L) in different samples in alcoholic beverages, presented a maximum deviation of only 2.1%. Assays of recovery of the substrate (99.25%) added in the wine showed that the methodology is viable for this sample type. The standard curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable analytical method.

CHEMICAL COMPOSITION OF SUGAR CANE SPIRITS FERMENTED BY DIFFERENT Saccharomyces cerevisiae YEAST STRAINS

CHEMICAL COMPOSITION OF SUGAR CANE SPIRITS FERMENTED BY DIFFERENT Saccharomyces cerevisiae YEAST STRAINS. The aim of this study was to evaluate the chemical composition of sugar cane spirits, fermented by different commercial Saccharomyces cerevisiae yeast strains and double distilled by pot still. Sugar cane juices were separately fermented by yeasts CA-11, Y-904, BG-1, PE-2, SA-1 and CAT-1 and distilled by pot still according to the methodology used for whisky production. The alcoholic liquids from first and second distillations were analyzed for concentrations of ethanol, volatile acidity, aldehydes, esters, furfural, higher alcohols and methanol. The sugar cane spirits derived from fermentation by the different yeast strains presented distinct chemical compositions.

Production and characterization of an alcoholic fermented beverage from grape must and beer wort

Mestrado Vinifera Euromaster - Instituto Superior de Agronomia - UL

Non-alcoholic fatty liver disease : can ultrasound assist in early diagnosis of prediabetes and delay progression to type2 diabetes mellitus

Non Alcoholic Fatty Liver Disease (NAFLD) is a condition that is frequently seen but seldom investigated. Until recently, NAFLD was considered benign, self-limiting and unworthy of further investigation. This opinion is based on retrospective studies with relatively small numbers and scant follow-up of histology data. (1) The prevalence for adults, in the USA is, 30%, and NAFLD is recognized as a common and increasing form of liver disease in the paediatric population (1). Australian data, from New South Wales, suggests the prevalence of NAFLD in “healthy” 15 year olds as being 10%.(2) Non-alcoholic fatty liver disease is a condition where fat progressively invades the liver parenchyma. The degree of infiltration ranges from simple steatosis (fat only) to steatohepatitis (fat and inflammation) steatohepatitis plus fibrosis (fat, inflammation and fibrosis) to cirrhosis (replacement of liver texture by scarred, fibrotic and non functioning tissue).Non-alcoholic fatty liver is diagnosed by exclusion rather than inclusion. None of the currently available diagnostic techniques -liver biopsy, liver function tests (LFT) or Imaging; ultrasound, Computerised tomography (CT) or Magnetic Resonance Imaging (MRI) are specific for non-alcoholic fatty liver. An association exists between NAFLD, Non Alcoholic Steatosis Hepatitis (NASH) and irreversible liver damage, cirrhosis and hepatoma. However, a more pervasive aspect of NAFLD is the association with Metabolic Syndrome. This Syndrome is categorised by increased insulin resistance (IR) and NAFLD is thought to be the hepatic representation. Those with NAFLD have an increased risk of death (3) and it is an independent predictor of atherosclerosis and cardiovascular disease (1). Liver biopsy is considered the gold standard for diagnosis, (4), and grading and staging, of non-alcoholic fatty liver disease. Fatty-liver is diagnosed when there is macrovesicular steatosis with displacement of the nucleus to the edge of the cell and at least 5% of the hepatocytes are seen to contain fat (4).Steatosis represents fat accumulation in liver tissue without inflammation. However, it is only called non-alcoholic fatty liver disease when alcohol - >20gms-30gms per day (5), has been excluded from the diet. Both non-alcoholic and alcoholic fatty liver are identical on histology. (4).LFT’s are indicative, not diagnostic. They indicate that a condition may be present but they are unable to diagnosis what the condition is. When a patient presents with raised fasting blood glucose, low HDL (high density lipoprotein), and elevated fasting triacylglycerols they are likely to have NAFLD. (6) Of the imaging techniques MRI is the least variable and the most reproducible. With CT scanning liver fat content can be semi quantitatively estimated. With increasing hepatic steatosis, liver attenuation values decrease by 1.6 Hounsfield units for every milligram of triglyceride deposited per gram of liver tissue (7). Ultrasound permits early detection of fatty liver, often in the preclinical stages before symptoms are present and serum alterations occur. Earlier, accurate reporting of this condition will allow appropriate intervention resulting in better patient health outcomes. References 1. Chalasami N. Does fat alone cause significant liver disease: It remains unclear whether simple steatosis is truly benign. American Gastroenterological Association Perspectives, February/March 2008 www.gastro.org/wmspage.cfm?parm1=5097 Viewed 20th October, 2008 2. Booth, M. George, J.Denney-Wilson, E: The population prevalence of adverse concentrations with adiposity of liver tests among Australian adolescents. Journal of Paediatrics and Child Health.2008 November 3. Catalano, D, Trovato, GM, Martines, GF, Randazzo, M, Tonzuso, A. Bright liver, body composition and insulin resistance changes with nutritional intervention: a follow-up study .Liver Int.2008; February 1280-9 4. Choudhury, J, Sanysl, A. Clinical aspects of Fatty Liver Disease. Semin in Liver Dis. 2004:24 (4):349-62 5. Dionysus Study Group. Drinking factors as cofactors of risk for alcohol induced liver change. Gut. 1997; 41 845-50 6. Preiss, D, Sattar, N. Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogenesis and treatment considerations. Clin Sci.2008; 115 141-50 7. American Gastroenterological Association. Technical review on nonalcoholic fatty liver disease. Gastroenterology.2002; 123: 1705-25

A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed

Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6 gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.

Whole-body substrate metabolism is associated with disease severity in patients with non-alcoholic fatty liver disease

Objectives In non-alcoholic fatty liver disease (NAFLD), hepatic steatosis is intricately linked with a number of metabolic alterations. We studied substrate utilisation in NAFLD during basal, insulin-stimulated and exercise conditions, and correlated these outcomes with disease severity. Methods 20 patients with NAFLD (mean±SD body mass index (BMI) 34.1±6.7 kg/m2) and 15 healthy controls (BMI 23.4±2.7 kg/m2) were assessed. Respiratory quotient (RQ), whole-body fat (Fatox) and carbohydrate (CHOox) oxidation rates were determined by indirect calorimetry in three conditions: basal (resting and fasted), insulin-stimulated (hyperinsulinaemic–euglycaemic clamp) and exercise (cycling at an intensity to elicit maximal Fatox). Severity of disease and steatosis were determined by liver histology, hepatic Fatox from plasma β-hydroxybutyrate concentrations, aerobic fitness expressed as , and visceral adipose tissue (VAT) measured by computed tomography. Results Within the overweight/obese NAFLD cohort, basal RQ correlated positively with steatosis (r=0.57, p=0.01) and was higher (indicating smaller contribution of Fatox to energy expenditure) in patients with NAFLD activity score (NAS) ≥5 vs <5 (p=0.008). Both results were independent of VAT, % body fat and BMI. Compared with the lean control group, patients with NAFLD had lower basal whole-body Fatox (1.2±0.3 vs 1.5±0.4 mg/kgFFM/min, p=0.024) and lower basal hepatic Fatox (ie, β-hydroxybutyrate, p=0.004). During exercise, they achieved lower maximal Fatox (2.5±1.4 vs. 5.8±3.7 mg/kgFFM/min, p=0.002) and lower (p<0.001) than controls. Fatox during exercise was not associated with disease severity (p=0.79). Conclusions Overweight/obese patients with NAFLD had reduced hepatic Fatox and reduced whole-body Fatox under basal and exercise conditions. There was an inverse relationship between ability to oxidise fat in basal conditions and histological features of NAFLD including severity of steatosis and NAS

Monitoring food and non-alcoholic beverage promotions to children

Food and non-alcoholic beverage marketing is recognized as an important factor influencing food choices related to non-communicable diseases. The monitoring of populations' exposure to food and non-alcoholic beverage promotions, and the content of these promotions, is necessary to generate evidence to understand the extent of the problem, and to determine appropriate and effective policy responses. A review of studies measuring the nature and extent of exposure to food promotions was conducted to identify approaches to monitoring food promotions via dominant media platforms. A step-wise approach, comprising ‘minimal’, ‘expanded’ and ‘optimal’ monitoring activities, was designed. This approach can be used to assess the frequency and level of exposure of population groups (especially children) to food promotions, the persuasive power of techniques used in promotional communications (power of promotions) and the nutritional composition of promoted food products. Detailed procedures for data sampling, data collection and data analysis for a range of media types are presented, as well as quantifiable measurement indicators for assessing exposure to and power of food and non-alcoholic beverage promotions. The proposed framework supports the development of a consistent system for monitoring food and non-alcoholic beverage promotions for comparison between countries and over time.

Monitoring the health-related labelling of foods and non-alcoholic beverages in retail settings

Food labelling on food packaging has the potential to have both positive and negative effects on diets. Monitoring different aspects of food labelling would help to identify priority policy options to help people make healthier food choices. A taxonomy of the elements of health-related food labelling is proposed. A systematic review of studies that assessed the nature and extent of health-related food labelling has been conducted to identify approaches to monitoring food labelling. A step-wise approach has been developed for independently assessing the nature and extent of health-related food labelling in different countries and over time. Procedures for sampling the food supply, and collecting and analysing data are proposed, as well as quantifiable measurement indicators and benchmarks for health-related food labelling.

Molecular basis for lysine specificity in the yeast ubiquitin-conjugating enzyme Cdc34

Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation

Cleavage and polyadenylation factor (CPF) is a multi‐protein complex that functions in pre‐mRNA 3′‐end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′‐end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′‐end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C‐terminal domain (CTD) of RNAP II plays a major role in coupling 3′‐end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre‐mRNA 3′ end processing, binds to the C‐terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3′ end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3′ end processing activity of Pcf11p and a deficiency of Pcf11p in 3′ end processing did not prevent CTD binding. Transcriptional run‐on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.

The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities

Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite's cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90 alpha or Hsp90 beta, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90's. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window.