Previous studies showed that, in wild-type (MATa) cells, a-factor causes an essential rise in cytosolic Ca2 . We show that calcineurin, the Ca2 /calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal. Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca2+-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca2+-deficient medium. Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin. Although calcineurin inhibits intracellular Ca sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2 transport bypasses the requirement for calcineurin during the pheromone response. These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels.Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, farl) show decreased dependence on calcineurin during treatment with pheromone. Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest. Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response. INTRODUCTIONThe signaling role of Ca21 is highly conserved among eukaryotic cells. Several distinct Ca2+-dependent signaling pathways exist in higher eukaryotes, and some components of these pathways have also been identified in the lower eukaryote Saccharomyces cerevisiae. Protein kinase C, for example, is required for maintenance of yeast cell integrity (for review, Levin and Errede, 1995), and phospholipase C, encoded by the PLC1 gene, is required for a diverse set of metabolic processes (Flick and Thorner, 1993;Payne and Fitzgerald-Hayes, 1993;Yoko-o et al., 1993). Yeast cells also possess calmodulin, and this protein is required for vegetative growth (Davis et al., 1986 (Cyert et al., 1991;Kuno et al., 1991;Liu et al., 1991;Cyert and Thorner, 1992). Calcineurin mutants fail to ,grow in the presence of high concentrations of Na+, Li+, Mn2+, or OH-, and the intracellular concentrations of several ions are altered in these cells (Nakamura et al., 1993;Breuder et al., 1994;Mendoza et al., 1994;Farcasanu et al., 1995;Pozos et al., 1996). Calcineurin may regulate the activity and/or the level of ion transporters in yeast cells. For example, the transcriptional induction of several P-type ATPases (ENA1, PMC1, PMR1) is reduced in calcineurin mutants (Mendoza et al., 1994;Cunningham and Fink, 1996). In addition, the expression of FKS2, a gene whose product is required for synthesis of 13-1,3-glucan in the cell wall, is regulated by calcineurin (Mazur et al., 1995). Several studies have indicated that Ca2+-dependent signal...

The prevalence and concentration of many foodborne pathogens exhibit seasonal patterns at different stages of the farm-to-table continuum. Escherichia coli O157:H7 is one such pathogen. While numerous studies have described the seasonal trend of E. coli O157:H7 in live cattle, ground beef, and human cases, it is difficult to relate the results from these different studies and determine the interrelationships that drive the seasonal pattern of beef-related human illnesses. This study uses a common modeling approach, which facilitates the comparisons across data sets, to relate prevalence in live cattle to raw ground beef and human illness. The results support an intuitive model where a seasonal rise of E. coli O157:H7 in cattle drives increased ground beef prevalence and a corresponding rise in the human case rate. We also demonstrate the use of these models to assess the public health impact of consumer behaviors. We present an example that suggests that the probability of illness, associated with summertime cooking and handling practices, is not substantially higher than the baseline probability associated with more conventional cooking and handling practices during the remainder of the year.

The U.S. Department of Agriculture Food Safety Inspection Service is responsible for ensuring the safety of meat, poultry, and egg products consumed in the United States. Here we describe a risk assessment method that provides quantitative criteria for decision makers tasked with developing food safety policies. To demonstrate the utility of this method, we apply it to a hypothetical case study on the use of an Escherichia coli O157:H7 cattle vaccine to prevent human illness caused by consuming beef. A combination of quantitative risk assessment methods and marginal economic analysis are used to describe the maximum cost per unit that would still allow the vaccine to be a cost-effective intervention as well as the minimum effectiveness it could have at a fixed cost. We create two economic production functions where the input is number of vaccinated cattle and the output is human illnesses prevented. The production functions are then used for marginal economic analysis to assess the cost/benefit ratio of using the vaccine to prevent foodborne illness. In our case study, it was determined that vaccinating the entire U.S. herd at a cost of between $2.29 and $9.14 per unit (depending on overall effectiveness of the vaccine) would be a cost-effective intervention for preventing E. coli O157:H7 illness in humans. In addition, we determined that vaccinating only a given fraction of the herd would be cost effective for vaccines that are less effective or more costly. For example, a vaccine costing $9.00 per unit that had a 100% efficacy but required 100% herd coverage for immunity would be cost effective for use in about 500,000 cattle each year-equating to an estimated 750 human illnesses prevented per annum. We believe this approach could be useful for public health policy development in a wide range of applications.

Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types. In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH−, and for maintaining viability during prolonged treatment with mating pheromone. In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+. We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Δ or cna1Δ cna2Δ). Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone. ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance. Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants. pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+. We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type. Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular [Na+] and [Ca2+]. Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity. These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis.

The use of genomic technologies at the U.S. Department of Agriculture could enhance inspection, monitoring, and risk assessment capabilities within its Food Safety and Inspection Service (FSIS). Molecular assays capable of detecting hundreds of microbial DNA sequences within a single food sample that identify food-borne pathogens of concern and characterize their traits most relevant to human health risk are of great interest for FSIS. For example, a high-density assay, or combination of assays, could screen FSIS inspected food for pathogens relevant to public health (e.g., Salmonella, Listeria, and toxic E. coli) as well as their associated virulence factors and antibiotic resistance genes. Because most genotype assays can be completed in one working day with a minimum of reagents, use of such assays could potentially save FSIS a significant amount of cost/time for analyses. Further, a genotype assay can detect specific microbial traits relevant to human health risk based on the DNA sequence of toxin producing genes, antibiotic resistance alleles, and more. By combining rapid analysis with specific data on human health risks, information from such high-density genotype assays could provide expanded support for test and hold situations, recalls, outbreak management, and microbial risk assessments (e.g., provide data needed for food-borne illness source attribution). Environ. Mol. Mutagen.