In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperaturesensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substratebinding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2. INTRODUCTIONAccumulation of unfolded proteins in the endoplasmic reticulum (ER) results in the transcriptional induction of various genes including those encoding ER-resident chaperones and folding enzymes. This cellular mechanism is called the unfolded protein response (UPR), and several important features of the UPR signaling pathway have been revealed initially through studies in the budding yeast Saccharomyces cerevisiae. Yeast Ire1 is a 1115-amino acid transmembrane protein that transmits the unfolded protein signal across the ER membrane (Cox et al., 1993;Mori et al., 1993). The cytoplasmic domain (C-terminal half) of Ire1 possesses both serine/threonine kinase and site-specific endoribonuclease activities Sidrauski and Walter, 1997). Ire1 dimerizes in response to the accumulation of unfolded proteins, resulting in its trans-autophosphorylation and activation (Shamu and Walter, 1996;Welihinda and Kaufman, 1996). Activated Ire1 in turn promotes splicing of HAC1 precursor mRNA to produce the mature form (Cox and Walter, 1996;Sidrauski and Walter, 1997), which is effectively translated into a functional transcription factor (Mori et al., 2000;Ruegsegger et al., 2001). Mature form of Hac1 efficiently induces transcription of UPR target genes containing the UPR element (UPRE) in their promoter region (Mori et al., 1992;Kohno et al., 1993).In mammalian cells, accumulation of unfolded proteins initiates signaling from the ER via more complicated pathways. Two homologues of Ire1, Ire1␣ and Ire1␤, have been identified (Tirasophon et al., 1998;Wang et al., 1998;Iwawaki et al., 2001). According to recent reports (Yoshida et al., 2001;Calfon et al., 2002), IRE1 functions to promote splicing of an mRNA encoding ...

The Ca2+‐dependent response to oxidative stress caused by H2O2 or tert‐butylhydroperoxide (tBOOH) was investigated in Saccharomyces cerevisiae cells expressing transgenic cytosolic aequorin, a Ca2+‐dependent photoprotein. Both H2O2 and tBOOH induced an immediate and short‐duration cytosolic Ca2+ increase that depended on the concentration of the stressors. Sublethal doses of H2O2 induced Ca2+ entry into the cytosol from both extracellular and vacuolar sources, whereas lethal H2O2 shock mobilized predominantly the vacuolar Ca2+. Sublethal and lethal tBOOH shocks induced mainly the influx of external Ca2+, accompanied by a more modest vacuolar contribution. Ca2+ transport across the plasma membrane did not necessarily involve the activity of the Cch1p/Mid1p channel, whereas the release of vacuolar Ca2+ into the cytosol required the vacuolar channel Yvc1p. In mutants lacking the Ca2+ transporters, H2O2 or tBOOH sensitivity correlated with cytosolic Ca2+ overload. Thus, it appears that under H2O2‐induced or tBOOH‐induced oxidative stress, Ca2+ mediates the cytotoxic effect of the stressors and not the adaptation process.

One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated waters. In this study, the heavy metal hypersensitive yeast strain pmr1Delta was investigated for the ability to remove Mn2+, Cu2+, Co2+, or Cd2+ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in removing Mn2+, Cu2+, or Co2+ from synthetic effluents containing 1-2 mM cations, with a selectivity and also in removing Mn2+ and Cd2+ from synthetic effluents containing 20-50 microM cations, with a selectivity Mn2+ > Cd2+.

Homocysteine (Hcy), a sulfur amino acid, is the only direct precursor for L-methionine synthesis through a reaction that requires vitamin B₁₂, representing a connection with "one-carbon" units metabolism. Hcy catabolism requires vitamin B₆ and as a consequence, alteration in folic acid and B vitamins status impairs Hcy biotransformation. Numerous studies have indicated that Hcy is an independent risk factor for cardio- and cerebrovascular diseases. In the last decade, several clinical trials have investigated the possible correlation between the use of folic acid and vitamins B₆ and B₁₂ for lowering Hcy plasma concentration and the reduced risk of stroke or its recurrence. This review is aimed to present some aspects of Hcy biochemistry, as well as the mechanisms through which it exerts the toxic effects on the vascular endothelium. We also discuss the results of some of the clinical trials developed to investigate the beneficial effects of vitamin therapy in the prevention and management of stroke.

The role of protein phosphatase 2B (PP2B/calcineurin) of Saccharomyces cerevisiae in the tolerance to divalent cations was investigated. PP2B-deficient mutants were found to be sensitive to MnCl2, but not to ZnCl2, CuCl2, NiCl2 and CoCl2. By measuring both manganese uptake and its efflux, it was found that the sensitivity of the mutant cells was due to an increase in manganese uptake and that the wild-type cells were able to prevent manganese entry into the cells, rather than export it in a more efficient manner. In the presence of the immunosuppressant FK506, the behavior of wild-type cells became similar to that of PP2B mutants. Out of various divalent cations tested, externally added magnesium ions were able to block manganese uptake in both wild-type and PP2B mutant strains.

Complexes with mixed ligands [Cu(N-N)2(pmtp)](ClO4)2 ((1) N-N: 2,2′-bipyridine; (2) L: 1,10-phenanthroline and pmpt: 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine) were synthesized and structurally and biologically characterized. Compound (1) crystallizes into space group Pa and (2) in P-1. Both complexes display an intermediate stereochemistry between the two five-coordinated ones. The biological tests indicated that the two compounds exhibited superoxide scavenging capacity, intercalative DNA properties, and metallonuclease activity. Tests on various cell systems indicated that the two complexes neither interfere with the proliferation of Saccharomyces cerevisiae or BJ healthy skin cells, nor cause hemolysis in the active concentration range. Nevertheless, the compounds showed antibacterial potential, with complex (2) being significantly more active than complex (1) against all tested bacterial strains, both in planktonic and biofilm growth state. Both complexes exhibited a very good activity against B16 melanoma cells, with a higher specificity being displayed by compound (1). Taken together, the results indicate that complexes (1) and (2) have specific biological relevance, with potential for the development of antitumor or antimicrobial drugs.

Cerebrovascular accidents are currently the second major cause of death and the third leading cause of disability in the world, according to the World Health Organization (WHO), which has provided protocols for stroke prevention. Although there is a multitude of studies on the health benefits associated with anthocyanin (ACN) consumption, there is no a rigorous systematization of the data linking dietary ACN with stroke prevention. This review is intended to present data from epidemiological, in vitro, in vivo, and clinical studies dealing with the stroke related to ACN-rich diets or ACN supplements, along with possible mechanisms of action revealed by pharmacokinetic studies, including ACN passage through the blood-brain barrier (BBB).

To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca in mediating the cell response to high Cu was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu was accompanied by elevations in cytosolic Ca with patterns that were influenced not only by Cu concentration but also by the oxidative state of the cell. When Ca channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca pool under Cu stress was the vacuolar Ca channel, Yvc1, also activated by the Cch1-mediated Ca influx. Using yeast mutants defective in the Cu transport across the plasma membrane, it was found that the Cu-dependent Ca elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu and HO acted in synergy to induce Ca-mediated responses to external stress.