Molecular mechanisms of BK channel activation.

Large conductance, Ca(2+)-activated potassium (BK) channels are widely expressed throughout the animal kingdom and play important roles in many physiological processes, such as muscle contraction, neural transmission and hearing. These physiological roles derive from the ability of BK channels to be synergistically activated by membrane voltage, intracellular Ca(2+) and other ligands. Similar to voltage-gated K(+) channels, BK channels possess a pore-gate domain (S5-S6 transmembrane segments) and a voltage-sensor domain (S1-S4). In addition, BK channels contain a large cytoplasmic C-terminal domain that serves as the primary ligand sensor. The voltage sensor and the ligand sensor allosterically control K(+) flux through the pore-gate domain in response to various stimuli, thereby linking cellular metabolism and membrane excitability. This review summarizes the current understanding of these structural domains and their mutual interactions in voltage-, Ca(2+)- and Mg(2+)-dependent activation of the channel.

Endotoxin increases hepatic susceptibility to lipid peroxidation: a possible role of iron.

The purpose of this study was to investigate the possible mechanism by which endotoxin enhances peroxidative damage to membrane lipids. Male B6C3 mice were treated with endotoxin intraperitoneally 0 or 20 mg/kg body weight for 24 h. Freshly prepared liver homogenate was incubated with either 1-5 mM of reduced glutathione (GSH), glucose, H(2)O(2), ascorbic acid (AA), FeSO(4), FeCl(3), EDTA, FeCl(3) plus AA, AA plus EDTA or EDTA plus FeCl(3) in phosphate-buffered saline (PBS), pH 7.0, or PBS, at 37 degrees C for 60 min. The levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), were significantly higher in the liver of endotoxin-treated mice, and the values were markedly increased following incubation. Compared to PBS, incubation with H(2)O(2), FeCl(3), FeSO(4), and AA, but not glucose, significantly enhanced TBAR formation. The greatest increase of TBAR was found when AA and FeCl(3) were added together. On the other hand, EDTA and GSH inhibited the formation of TBAR during incubation. When added before AA, EDTA completely inhibited the peroxidative effect of AA or FeSO4, and when added subsequent to AA, EDTA partially prevented the adverse effect of AA. The results obtained suggest that ionic iron plays an important role in initiating endotoxin-induced peroxidative damage to membrane lipids, and that AA may be involved in releasing iron from its protein complex and/or maintaining ionic iron in a reduced or catalytic state.

Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice.

Manganese superoxide dismutase (Mn-SOD) plays an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine if increased expression of Mn-SOD gene alters intracellular redox status. Twelve week old male B6C3 mice, engineered to express human Mn-SOD in multiple organs, and their nontransgenic littermates were assessed for oxidative stress and antioxidant status in heart, brain, lung, skeletal muscle, liver, and kidney. Relative to their nontransgenic littermates, transgenic mice had significantly (p <.01) higher activity of Mn-SOD in heart, skeletal muscle, lung, and brain. Copper, zinc (Cu,Zn)-SOD activity was significantly higher in kidney, whereas catalase activity was lower in brain and liver. The activities of selenium (Se)-GSH peroxidase and non-Se-GSH peroxidase, and levels of vitamin E, ascorbic acid and GSH were not significantly different in any tissues measured between Mn-SOD transgenic mice and their nontransgenic controls. The levels of malondialdehyde were significantly lower in the muscle and heart of Mn-SOD mice, and conjugated dienes and protein carbonyls were not altered in any tissues measured. The results obtained showed that expression of human SOD gene did not systematical alter antioxidant systems or adversely affect the redox state of the transgenic mice. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids.

Oxidative stress and antioxidant status in mouse kidney: effects of dietary lipid and vitamin E plus iron.

The purpose of this study was to determine the effects of dietary fat, vitamin E, and iron on oxidative damage and antioxidant status in kidneys of mice. Sixty 1-month-old male Swiss-Webster mice were fed a basal vitamin E-deficient diet that contained either 8% fish oil + 2% corn oil or 10% lard with or without 1 g all-rac-alpha-tocopherol acetate or 0.74 g ferric citrate per kilogram of diet for 4 weeks. Significantly (P < 0.05) higher levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), and conjugated dienes were found in the kidneys of mice fed with fish oil compared with mice fed lard irrespective of vitamin E status. Mice maintained on a vitamin E-deficient diet had significantly higher renal levels of TBAR, but not conjugated dienes, than the supplemented group. Fish oil fed mice receiving vitamin E supplementation had lower levels of alpha-tocopherol than did mice in the lard fed group. Significantly higher levels of ascorbic acid were also found in the kidneys of mice fed with fish oil than were found in mice fed lard. The levels of protein carbonyls and glutathione (GSH), and activities of catalase, superoxide dismutase, selenium (Se)-GSH peroxidase, and non-Se-GSH peroxidase were not significantly altered by dietary fat or vitamin E. Dietary iron had no significant effect on any of the oxidative stress and antioxidant indices measured. The results obtained provide experimental evidence for the pro-oxidant effect of high fish oil intake in mouse kidney and suggest that dietary lipids play a key role in determining cellular susceptibility to oxidative stress.

Oxidative stress and antioxidant status in mouse liver: effects of dietary lipid, vitamin E and iron.

The purpose of this study was to determine the effects of dietary fat, vitamin E and iron on oxidative damage and antioxidant status. Male Swiss-Webster mice (1 mo old) were fed a basal vitamin E-deficient diet that contained either 8% fish oil + 2% corn oil or 10% lard with or without 1 g dl-alpha-tocopheryl acetate. The diets without vitamin E contained either 0.21 or 0.95 g ferric citrate/kg. Diets were fed for 4 wk/kg diet. Compared with the vitamin E-supplemented groups, mice fed diets without vitamin E (with or without supplemental iron) had significantly (P < 0.05) higher hepatic levels of thiobarbituric acid-reactive substances (TBARS), conjugated dienes and protein carbonyls when they were fed fish oil, but not lard. The levels of TBARS were further increased by iron supplementation in the mice fed fish oil. Significantly lower concentrations of alpha-tocopherol and higher glutathione (GSH) were found in the liver of mice fed fish oil and vitamin E than in those fed lard and vitamin E (P < 0.05). The activities of superoxide dismutase and glucose-6-phosphate dehydrogenase were lower in the fish oil-fed mice than in those fed lard (P < 0.05). The activities of Se-GSH peroxidase, non-Se-GSH peroxidase, catalase, and glutathione reductase were not altered by dietary fat or vitamin E/iron. The results obtained provide experimental evidence of the prooxidative effects of high dietary fish oil and iron, and suggest that vitamin E protects not only lipid-soluble compounds, but also water-soluble constituents, against oxidative damage. Further, dietary lipid plays a key role in determining cellular susceptibility to oxidative stress.

A limited survey of fumonisins in corn and corn-based products in Asian countries.

Natural occurrence of fumonisins B1 (FB1) and B2 (FB2), a promoter for hepato-carcinogenesis, was investigated in corn and corn - based products sampled in Japan, Nepal, and China by high - performance liquid chromatographic method. From the 9 imported corn kernel and 6 gluten feed samples, FB1 was detected in 8 corn (0.6 ∼ 4.1µg/g) and all gluten feed (0.3 ∼ 2.4µg/g) samples, while FB2 was found in the same corn (0.3 ∼ 10µg/g) and 3 gluten feed (0.8 ∼ 8.5µg/g) samples. ELISA analysis also revealed the contamination of aflatoxin B1 in 2 corn and all gluten feed samples along with fumonisins. Of 17 corn grit samples, 14 and 5 samples were contaminated with fumonisin B1 and B2, with maximum levels of 2.6 and 2.8µg/g, respectively. As for corn-based foodstuffs marketed in Japan, no significant contamination of fumonisins was observed. Among 24 corn kernel samples in Nepal, 12 and 7 samples were positive for FB1 and FB2, and averaged to 0.6 and 1.6µg/g, respectively. One sample showed the highest fumonisin contents as 4.6 and 5.5µg/g, respectively. In corn samples harvested at Shanghai and Beijing, China, FB1 and FB2 were detected in various concentrations. Mycological survey has also revealed the presence of a fumonisin - producing fungus in a crop field of Japan. These findings have for the first time demonstrated high levels of contamination of fumonisins in corn and corn - based products in Asian countries. Natural co - occurrence of fumonisins and aflatoxin B1 was also detected in raw materials for mixed feed.

Decontamination ofFusarium mycotoxins, nivalenol, deoxynivalenol, and zearalenone, in barley by the polishing process.

Korean dehusked and unhusked barley naturally contaminated withFusarium mycotoxins were polished using a Satake Grain Testing Mill. The pearled barley and bran fractions with different degrees of polishing were analyzed for nivalenol (NIV) and deoxynivalenol (DON) by gas chromatography with an electron capture detector, and for zearalenone (ZEN) by high-performance liquid chromatography with a fluorescence detector. NIV was detected in all the pearled barley fractions, but DON and ZEN were not detected in ≥27 % pearled barley fractions from dehusked barley and ≥36% pearled barley fractions from unhusked barley. However, for all degrees of polishing, NIV, DON, and ZEN were detected in bran fractions. The levels of NIV, DON, and ZEN in the bran fractions increased several fold over the original barley. Polishing was effective in removing DON and ZEN from the naturally contaminated barley, but not NIV.

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.

A limited survey of Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in 1984 UK harvested wheat and barley.

A limited survey for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in 1984 UK-grown cereals (31 samples) have been carried out using a new procedure, which is a rapid and sensitive method for Fusarium mycotoxins. NIV, DON and ZEN were detected in 17 (55%), 20 (65%) and 4 (13%) out of 31 samples, and average levels in positive samples were 101 micrograms/kg, 31 micrograms/kg and 1 microgram/kg, respectively. Additional surveys on two wheat and eight barley samples harvested in Scotland have shown that 30%, 60% and 100% of the samples were contaminated with NIV, DON and ZEN, respectively. The contents averaged 391 micrograms/kg of NIV, 39 micrograms/kg of DON and 9 micrograms/kg of ZEN. The results of this survey show that UK-grown cereals were significantly contaminated with NIV, DON and ZEN in a similar way to that observed in Japan, Korea and China. This is the first evidence of the natural occurrence of NIV in UK cereals.

Further survey on the Fusarium mycotoxins in Korean cereals.

Fifty-one samples of cereals from the 1984 harvest from Korea were analyzed for nivalenol (NIV), fusarenon-X (FX), deoxynivalenol (DON) and 3-acetyl-DON by gas chromatography (GC) utilizing a 63Ni electron capture detector (ECD), and were quantitated for zearalenone (ZEN) by high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). Trichothecenes and ZEN in the positive samples were confirmed by GC-mass spectrometry (MS). Out of 51 samples, 51, 46 and 42 were positive for NIV, DON and ZEN, respectively, and one malt sample was heavily contaminated with NIV (2675 ng/g) and DON (246 ng/g), and one wheat sample was heavily contaminated with NIV (3169 ng/g). Neither FX nor 3-acetyl-DON was detected in any of the samples. The data reported here indicates that Korean cereals harvested in 1984 are simultaneously contaminated with NIV, DON and ZEN, and the incidences and levels are similar to those observed in the cereals harvested in 1983.

Examination of Chinese and U.S.S.R. cereals for the Fusarium mycotoxins, nivalenol, deoxynivalenol and zearalenone.

Cereals, foods and feeds sampled in Taiwan, China and the U.S.S.R. were contaminated with nivalenol, deoxynivalenol and zearalenone. The frequencies and levels of contamination are similar to those observed in the cereals of Japan and Korea. This is the first report on the natural occurrence of nivalenol, deoxynivalenol and zearalenone in Chinese and U.S.S.R. cereals, foods and feeds.

The coexistence of the Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in Korean cereals harvested in 1983.

A survey for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in Korean cereals (totalling 53 samples) harvested in 1983, showed that 96%, 72% and 57% of the samples were contaminated with NIV, DON and ZEN, respectively. Average concentrations (micrograms/kg) in unpolished barley were 546 (NIV), 117 (DON) and 110 (ZEN), and those in polished barley were 130 (NIV) and 21 (DON). The ZEN levels were below the detection limit (1 microgram/kg). Malt, wheat and rye were also heavily contaminated with these Fusarium mycotoxins. The results of this survey show that Korean cereals harvested in 1983 were significantly contaminated with NIV, DON and ZEN, and the incidence and levels, where observed, are similar to those reported in Japan.

Rapid and sensitive determination of zearalenone in cereals by high-performance liquid chromatography with fluorescence detection.

Zearalenone, an estrogenic mycotoxin of Fusarium species, in cereals can be extracted with acetonitrile-water (3:1), purified on a Florisil column, resolved by high-performance liquid chromatography (HPLC) with a Nucleosil 50-10 column using 90% water-saturated chloroform-cyclohexane-acetonitrile-ethanol (50:15:2:1) and quantitated by fluorescence measurement. This method is rapid, simple and reproducible, and detects zearalenone in wheat, barley, corn and other cereals with picogram sensitivity. A combination of this HPLC method with a gas-liquid chromatographic method for trichothecenes may be applied to the simultaneous detection of Fusarium mycotoxins (zearalenone, nivalenol and deoxynivalenol) in cereals.