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Maresin 1 is a novel pro-resolving mediator derived from docosahexaenoic acid (DHA), with potent anti-inflammation effects against several animal models, including brain ischemia, sepsis, and lung fibrosis. However, its effect against motor neuron cell death is still not investigated. Therefore, we investigated the effects of maresin 1 on several stress-induced motor neuron cell death. Maresin 1 suppressed combinatorial stress which was evoked by superoxide dismutase 1 (SOD1) G93A and serum-free, -induced motor neuron cells death in a concentration-dependent manner, and had a stronger neuroprotective effective than DHA. Maresin 1 also had neuroprotective effects against transactivation response DNA-binding protein (TDP)-43 A315T and serum-free stress, H 2 O 2 , and tunicamycin-induced cell death. Maresin 1 reduced the reactive oxygen species (ROS) production caused by SOD1 G93A or TDP-43 A315T . Moreover, maresin 1 suppressed the NF-κB activation induced by SOD1 G93A and serum-free stress. These data indicate that maresin 1 has motor neuron protective effects against several stresses by reduction of ROS production or attenuation of the NF-κB activation. Maresin 1 also had neuroprotective effects against H 2 O 2 , and tunicamycin-induced cell death in a concentration-dependent manner. Finally, maresin 1 ameliorated the motor function deficits of spinal muscular atrophy model in which endoplasmic reticulum stress was upregulated. Thus, maresin 1 may be beneficial to protect against motor neuron diseases.

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20-25 micromol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.

Acid phosphatase (APase) activity of the yeast Yarrowia lipolytica increased with increasing Cu2+ concentrations in the medium. Furthermore, the enzyme in soluble form was stimulated in vitro by Cu2+, Co2+, Ni2+, Mn2+ and Mg2+ and inhibited by Ag+ and Cd2+. The most effective ion was Cu2+, especially for the enzyme from cultures in medium containing Cu2+, whereas APase activity in wall-bound fragments was only slightly activated by Cu2+. The content of cellular phosphate involving polyphosphate was decreased by adding Cu2+, regardless of whether or not the medium was rich in inorganic phosphate. Overproduction of the enzyme stimulated by Cu2+ might depend on derepression of the gene encoding the APase isozyme.

The Cu2+-tolerant yeast Yarrowia lipolytica accumulated Cu2+ until the late logarithmic phase. Thereafter, Cu2+ was temperature-dependently extruded into phosphate-limited culture medium containing high concentrations of heavy metal ions but not into 10 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer (pH 6.0). Peptone in the culture medium played an important role in the extrusion, which proceeded even when peptone was substituted with cysteine or histidine, but not with any other amino acid tested.

Phytochelatin-related peptides were analyzed in chickpea plants exposed to six different heavy-metal ions. Cadmium and arsenic stimulated phytochelatin and homophytochelatin synthesis in roots but other metals did not. These metals, however, caused an overall increase in the precursors, glutathione, homoglutathione and cysteine. These changes may be different biochemical indexes for heavy-metal contamination.

The possible roles of phytochelatin (PC) and glutathione (GSH) in the heavy metal detoxification in plants were examined using two varieties (CSG-8962 and C-235) of chickpea ( Cicer arietinumL.). The seedlings were grown for 5 days and the roots were treated with 0-20 micro M CdSO(4) for 3 days. The CSG-8962 seedlings exhibited more Cd-tolerant characteristics than did the C-235, where the roots, rather than shoots, suffered from more toxic effects by Cd. Both the seedlings synthesized the large amounts of PCs and homo-phytochelatins (hPCs) in roots, but only a little in shoots in response to Cd. The Cd treatments also caused a marked increase in the levels of GSH and cysteine in both the root and shoot tissues, suggesting that Cd may activate the GSH biosynthesis and, hence, enhance PC synthesis in the plants. Such a Cd-sensitive PC synthesis in chickpea plants does not explain the difference in Cd sensitivity in the varieties, but can be used as a biochemical indicator for Cd contamination in various environments. In the chickpea plants, possible PC-dependent and independent mechanisms for Cd tolerance are discussed.

Induction for the expression of the metallothionein gene, CUP1, in the yeast Saccharomyces cerevisiae by cobalt was examined using a reporter gene with the promoter of this gene fused to the coding region of lacZ. The expression of the gene was induced by cobalt as well as by copper and silver ions. The activity of beta-galactosidase showed high levels after treatment with 1.0 mM cobalt chloride. It has been reported that the induction for the transcription of CUP1 by copper and silver is mediated by the Ace1 transcription factor. However, the expression of the gene by cobalt occurred in yeast cells lacking the Ace1 factor. These results suggest the presence of a novel cobalt-specific transcription factor for the CUP1 gene.

Yeast cells carrying the CAD2 gene exhibit a resistance to cadmium. We cloned this gene and demonstrated that it was a mutated form derived from the gene of a putative copper-transporting ATPase (PCA1). By site-directed mutagenesis, it appeared that the mutation conferring cadmium resistance was a R970G-substitution in the C-terminal region of Pca1 protein. The intracellular cadmium level of cells carrying CAD2 was lower than that of cells carrying either PAC1 or delta cad2. Furthermore, cells with overexpression of CAD2 showed a much lower intracellular cadmium level than that of cells with a single-copy CAD2. From these results, we conclude that the Cad2 protein controls the intracellular cadmium level through an enhanced cadmium efflux system.

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu2+ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd2+ and Mn2+. By adding Cd2+ and Mn2+ at 10 microM concentration, the beta-galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO4, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu2+ and 1 microM-Cd2+. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd2+ environment. In contrast, the presence of Mn2+ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu2+. The recovery from growth inhibition by Mn2+ was due to decreased Cu2+ accumulation. Inhibitory concentrations of Co2+, Ni2+ and Zn2+ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd2+ and Mn2+. These results are discussed in relation to Cu2+ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.

Certain mutants of the yeast Saccharomyces cerevisiae show copper or cadmium resistance. Both copper- and cadmium-resistant strains produce the same metallothionein with 53 amino acid residues which causes metal detoxification by chelating copper or cadmium. The metal detoxification role is the only known function of the metallothionein in yeast. The MT is encoded by the CUP1 gene on chromosome VIII which is expressed by induction with metals. The CUP1 is amplified to 3-14 copies with 2 kb-tandem-repeat units in the metal-resistant strains, whereas the wild-type strain contains only a single copy of the CUP1. Although transcription of CUP1 is inducible by metals, the ACE1 protein serves a dual function as a sensor for copper and an inducer for CUP1 transcription in the copper-resistant strain. In the cadmium-resistant strain, the heat-shock factor having a point mutation may be the regulator for CUP1 transcription. Therefore, it has been clarified that production of MT in yeast is controlled by two systems, the amplification of CUP1 and its transcriptional regulation.

A cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein encoded by the CUP1 gene as does a copper-resistant strain. The mechanism of expression of the gene is inducible by copper ions in the copper-resistant strain. However, assays of CUP1-specific mRNA revealed that the transcription of the CUP1 gene in the cadmium-resistant strain is constitutive and the rate of transcription is further increased by exposure to cadmium or copper ions. This result was confirmed by the appearance of constitutive-expression segregants from diploid crosses between the cadmium-resistant strain and a strain with a reporter gene having the promoter of CUP1.

A cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein with the same characteristics as the copper metallothionein that is encoded by CUP1 in a copper-resistant strain. The structural gene for metallothionein from the cadmium-resistant strain resembles CUP1 in terms of the fragmentation patterns generated by restriction enzymes. Furthermore, the gene may be amplified as 2.0 kb repeating units in both the cadmium-resistant and the copper-resistant strains. However, transformants with a plasmid that carried the metallothionein gene from the cadmium-resistant strain were resistant to copper but not to cadmium. It appears that the same metallothionein gene, CUP1, is amplified in both cadmium- and copper-resistant yeasts. However, the mechanism for the cadmium-specific inducibility of the gene may be restricted to the cadmium-resistant strain.

Examination of the subcellular distribution of nickel in a Ni-resistant strain N08 of Saccharomyces cerevisiae showed that 70% of the nickel is distributed in the vascular fraction, which contains large amounts of histidine. The nickel taken up by cells grown in medium containing a high concentration of histidine was preferentially distributed to the vacuole. Arginine and lysine did not affect the intracellular distribution of Ni. In a Ni-sensitive strain 0605-S6, the distribution of nickel into the vacuole was lower than that observed in strain N08. Strain 0605-S6 exhibited no increase in the histidine content of the vacuolar fraction when grown in a Ni-supplemented medium. The Ni-resistant mechanism appears to involve the sequestration of nickel to the vacuole, and histidine could play an important role in the reduction of free nickel in the vacuole by the formation of histidine-nickel complexes.

A mutant strain of Saccharomyces cerevisiae (NR 6), which can be associated with a reduction in the accumulation of Mg2+ has been isolated. This mutant strain displays resistance to both Ni2+ and Co2+, but not, however, towards Cu2+, Cd2+, Mn2+, Zn2+ and Cr2O7(2-). Both Co2+ and Ni2+ uptake by the mutant strain is less than by the wild type (CMR-50). The inhibitory effects of Ni2+ and Co2+ on the growth of both strains NR-6 and CMR-50 can be cancelled by increasing the concentrations of Mg2+ in the medium and to a lower extent by the addition of Ca2+ which results in a decreased uptake of these metals. It therefore appears that the resistant mechanisms of this mutant strain NR-6 is due to a reduction in the uptake of Co2+ and Ni2+ via a Mg2+ transport system.

A cadmium-resistant strain, X3382-3A, which is able to grow in a medium containing 0.2 mM cadmium sulfate, was picked out from our laboratory stock strains of Saccharomyces cerevisiae. The cadmium resistance of this strain is controlled by a single dominant nuclear gene, denoted as CAD2. The locus of CAD2 was mapped by gene linkage to a site 15.5 centimorgans to the right of the his7 locus on the right arm of chromosome II. The cadmium resistance of the strain carrying CAD2 was evaluated for its properties of cadmium uptake, cadmium distribution and cadmium-metallothionein formation, in comparison with those of some other strains. The results suggest that the novel type of cadmium resistance controlled by CAD2 does ot involve production of a cadmium-metallothionein.

When a nickel resistant strain N08 of S. cerevisiae was grown in a Ni-supplemented medium, approximately 70% of the nickel is distributed in the soluble fraction. The soluble fraction was chromatographed on Sephadex G-10 and the fraction contained both nickel and large amounts of histidine. When cells were grown in medium containing various combinations of nickel and magnesium and which exhibited approximately 50% growth inhibition, a molar ratio of intracellular histidine and nickel contents remained constant at 1.2-1.4, indicating that the increase in histidine content is correlated with nickel accumulation. The wild type strain 0605-S6, however, exhibits no increase in histidine content when grown in a Ni-supplemented medium, and, therefore, a nickel-resistant mechanism of yeast appears to be the formation of histidine-nickel complexes.

The Cd2+ accommodation mechanism of S. cerevisiae has been examined. In a synthetic medium containing 1 microM CdSO4, cell proliferation was observed following a time lag period of approximately 18 h. During the time lag period, the cell number and viable cell count did not change and the cells did not excrete organic acid as a Cd-chelator, neither did they produce inorganic sulphide in the form of CdS. Whereas approximately 30% of Cd2+ taken up by Cd-accommodated cells was distributed in the cytosol, only a small percentage of Cd2+ was found in the cytosol of unaccommodated cells following 1 h incubation in 1 microM Cd-medium. Furthermore, some Cd2+ in the soluble fraction from Cd-accommodated cells was bound to low molecular weight (less than 30,000) protein(s), while unaccommodated cells only contained small amounts of these proteins. Therefore, it is proposed that Cd2+ binds to the low molecular weight protein(s) present in the cytosol, resulting in a decrease of Cd-toxicity, although the exact mechanism of accommodation is not clear.