The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Delta and ubc7Delta) and ire1Delta, or expression of misfolded proteins show phenotypes similar to mutation of cell wall proteins, including hypersensitivity to cell wall-targeted molecules, alterations to cell wall protein layer, decreased cell wall thickness by electron microscopy, and increased cellular aggregation. Consistent with its important role in cell wall integrity, UPR is activated by signaling through the cell wall integrity mitogen-activated protein (MAP) kinase pathway during cell wall stress and unstressed vegetative growth. Both cell wall stress and basal UPR activity is mediated by Swi6p, a regulator of cell cycle and cell wall stress gene transcription, in a manner that is independent of its known coregulatory molecules. We propose that the cellular responses to ER and cell wall stress are coordinated to buffer the cell against these two related cellular stresses.

The identification of new antifungal molecules is an important goal of current anti-infective research. To achieve this goal, alternatives to traditional growth inhibition-based screening have been developed in recent years. In this study, we describe an assay to detect molecules that disrupt yeast cell integrity by using the release of adenylate kinase (AK) into culture medium as a reporter of yeast cell lysis. The protocol is applicable to 96- and 384-well microtiter plate formats; uses a commercially available luminescence assay kit to detect AK activity; is more sensitive than traditional growth-based assays; and is specific for fungicidal compounds. In the high-throughput setting, the procedure provides excellent Z' scores (0.75-0.9), making it a highly robust assay. The AK assay is performed in a single microtiter plate using an 'add and read' procedure that can be completed in a single work day.

Methylmercury (MeHg) exposure via fish in the diet remains a priority public health concern. Individual variation in response to a given MeHg exposure and the biotransformation of MeHg that follows complicate our understanding of this issue. MeHg elimination from the human body occurs slowly (elimination rate (kel) approximately 0.01 day(-1) or approximately 70 days half-life [t1/2]) and is a major determinant of the Hg body burden resulting from fish consumption. The underlying mechanisms that control MeHg elimination from the human body remain poorly understood. We describe here improved methods to obtain a MeHg elimination rate via longitudinal Hg analysis in hair using laser ablation-inductively coupled plasma-mass spectrometry. We measured MeHg elimination rates in eight individuals following the consumption of 3 fish meals in two 75-day trials separated by a 4-month washout period. In addition, since MeHg biotransformation to inorganic Hg (I-Hg) is associated with Hg excretion, we speciated Hg in feces samples to estimate individual MeHg de-methylation status. We observed a wide range of MeHg elimination rates between individuals and within individuals over time (kel = 0.0163-0.0054 day(-1); estimated t1/2 = 42.5-128.3 days). The ratio of MeHg and I-Hg in feces also varied widely among individuals. While the %I-Hg in feces was likely influenced by dental amalgams, findings with subjects who lacked amalgams suggest that faster MeHg elimination is associated with a higher %I-Hg in feces indicating more complete de-methylation. We anticipate these methods will contribute to future investigations of genetic and dietary factors that influence MeHg disposition in people.

Evaluating the potential for methylmercury (MeHg) toxicity relies on accurately predicting the mercury (Hg) body burden that results from eating fish. Hg body burden is directly determined by the slow elimination kinetics of MeHg in the human body (kel = 0.014 days-1 or t1/2 =50 days). Existing studies on MeHg half-life in humans demonstrate a wide range values (t1/2 = 30 to >150 days) and has lead to uncertainty in the derivation of a regulatory standard for acceptable daily oral intake. The causes of variation in MeHg toxicokinetics in humans remain little explored. Here we characterize variation in human MeHg metabolism and elimination rate (kel) in 37 adult volunteers who consumed 3 fish meals. We determined MeHg elimination rates via longitudinal Hg analysis in single hairs using laser ablation inductively coupled plasma mass spectrometry. We also measured MeHg metabolism (biotransformation) via speciation of fecal Hg. We find an average kel = 0.0157 days-1 (t1/2 = 44 days) amongst a more than 2-fold variation in kel across the cohort (0.0248-0.0112 days-1; t1/2 = 28-62 days). Although MeHg biotransformation varied widely between individuals, it showed a positive association with elimination rates across the cohort. A more than 2-fold change in kel over a period of 2 years was seen in some individuals. In 2 individuals, who received antibiotic for unrelated health issues, elimination rate was seen to slow significantly. Associations of kel with age, body mass index, gender, and fish eating habits were not observed. We establish that a measure of methylmercury metabolism and eliminaiton status (MerMES) can reduce uncertainty in determining an individual's MeHg toxicokinetics subsequent to eating fish.

A series of N-substituted and N′-substituted aminothiazole-derived morphinans (5) were synthesized for expanding the structure-activity relationships of aminothiazolo-morphinans. Although their affinities were somewhat lower than their prototype aminothiazolo-Ncyclopropylmorphinan (3), 3-aminothiazole derivatives of cyclorphan (1) containing a primary amino group displayed high affinity and selectivity at the κ and μ opioid receptors. [ 35 S]GTPγS binding assays showed that the aminothiazolomorphinans were κ agonists with mixed agonist and antagonist activity at the μ opioid receptor. These novel N′-monosubstituted aminothiazolederived morphinans may be valuable for the development of drug abuse medications.