Saccharomyces Cerevisiae as a biotechnological tool for ageing research:studies on translation and metabolism

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*. encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a nonethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Eastern Orthodox Christian fasting in northeastern Pennsylvania

The purpose of this research was to examine the fasting practices of Eastern Orthodox Christians (EOCs) in northeastern Pennsylvania. Fasting, according to Eastern Orthodox Church doctrine, is primarily abstinence from meat, dairy products, fish and certain other foods during Easter Lent and other periods, for approximately 180 days annually. Goals were to discern what EOCs consider their fasting rules to be, their actual fasting practices, what factors influence this practice and the relationship of fasting to nutrition. Methodology included 29 months of ethnographic fieldwork at local parishes, content analysis of local written materials and semi-structured interviews of 58 core church members. A pile sort was conducted whereby subjects classified various foods according to fasting or non-fasting status and then sorted the fasting foods into a hierarchy of avoidance. Data were analyzed using ANTHROPAC and NVivo software. Results included identification of a cognitive hierarchy of avoidance, with meat the most important to avoid, followed by dairy and alcoholic beverages. An important finding was the differences in subjects' knowledge of Church doctrine and a wide variation in their actual fasting practices. Contrary to Church doctrine, fish was not usually perceived as a food to abstain from. A historic Byzantine Catholic presence in the area (with a different fasting doctrine), family members who did not fast, and health concerns were some factors that affected fasting practices. A conclusion is that while meat, dairy and alcoholic beverages were usually categorized as foods to avoid during fasts, it is not possible to generalize with regard to actual practices or the impact of fasting on nutrition, due to individual variation. It was demonstrated that qualitative data could provide information that can be crucial to know prior to conducting quantitative nutrition research or counseling. Findings of this study suggest that one cannot assume subjects who belong to a given religion that has prescribed food avoidance practices are following them homogeneously and/or according to official doctrine. ^

Managers' Perception of Alcohol Service Training Programs

In order to serve alcoholic beverages in a responsible manner, most managers turn to alcohol server training programs to provide effective standardized training for the alcohol server. Whether or not the programs are perceived by hospitality managers as effective has not been established. The author presents the results of a study of Oklahoma hospitality managers who perceive alcohol server training as becoming less effective.

Current Status of Collectibility of Gaming-Related Credit Dollars

In her piece entitled - Current Status Of Collectability Of Gaming-Related Credit Dollars - Ruth Lisa Wenof, Graduate Student at Florida International University initially states: “Credit is an important part of incentives used to lure gamblers to gaming establishments. However, a collection problem exists in casinos retrieving gaming-related credit losses of individuals living in states where gambling is illegal. The author discusses the history of this question, citing recent cases related to Atlantic City.” This author’s article is substantially laden with legal cases associated with casinos in New Jersey; Atlantic City to be exact. The piece is specific to the segment of the gaming industry that the title suggests, and as such is written in a decidedly technical style. “Legalized casino gaming, which was approved by the citizens of New Jersey on November 8, 1976, has been used as a unique tool of urban redevelopment for Atlantic City,” Wenof says in providing some background on this ‘Jersey shore municipality. “Since Resorts International opened its casino…revenues from gambling have increased rapidly. Resorts' gross win in 1978 was $134 million,” Wenof says. “Since then, the combined gross win of the city's 11 casinos has been just shy of $7.5 billion.” The author points out that the competition for casino business is fierce and that credit dollars play an integral role in soliciting such business. “Credit plays a most important part in every casino hotel. This type of gambler is given every incentive to come to a particular hotel,” says the author. “Airplanes, limousines, suites, free meals, and beverages all become a package for the person who can sign a marker. The credit department of a casino is similar to that of a bank. A banker who loans money knows that it must be paid back or his bank will fail. This is indeed true of a casino,” Wenof warns in outlining the potential problem that this article is fundamentally designed around. In providing further background on credit essentials and possible pitfalls, Wenof affords: “…on the Casino Control Act the State Commission of Investigation recommended to the legislature that casinos should not be allowed to extend credit at all, by reason of a concern for illicit diversion of revenues, which is popularly called skimming within the industry…” Although skimming is an after-the-fact problem, and is parenthetic to loan returns, it is an important element of the collective [sic] credit scheme. “A collection problem of prime importance is if a casino can get back gaming-related credit dollars advanced by the casino to a gambler who lives in a state where gambling is illegal,” is a central factor to consider, Wenof reveals. This is a primary focus of this article. Wenof touches on the social/societal implications of gambling, and then continues the discussion by citing a host of legal cases pertaining to debt collection.

Manager's Perceptions of Alcohol Server Staffing and Training Methods

Over-consumption of alcoholic beverages is a concern of managers of hotels and motels with a club/lounge, restaurant, and tavern. The authors surveyed members of two industry associations in Oklahoma to ascertain alcohol server training methods and managers' perception of the value of such programs.