Plant metallothioneins – metal chelators with ROS scavenging activity?

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K(m) for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.     

Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.     

Age, sex ratio and timing of the catch of kelts and ascending Atlantic salmon in the subarctic River Teno

By 15 June, 82% of the catch of Atlantic salmon Salmo salar kelts had been taken from the middle part of River Teno, northern Scandinavia. The median date of capture was 4 June for males and 8 June for females. Salmon of 1–4 sea–winters (SW) of both sexes survived spawning to return to sea as kelts. Among males, 1 SW kelts were caught earliest in the spring and 3 SW latest, but among females 4 SW were earliest, then 3 SW and finally 1 and 2 SW. There were 17 river and sea–age combinations among the kelts compared with 23 among the ascending salmon. The smolt age distribution and the mean smolt age differed significantly only between female 2 SW ascending salmon (3·97 years) and kelts (4·14 years). The proportion of 1 SW females was higher and that of 3 SW males lower among kelts than among ascending salmon. The proportion of males among 1 SW ascending salmon was 80% but among kelts only 57%. Similarly, the proportion of males among 3 SW fish was 21% for ascending salmon but only 7% for kelts. Hence overwinter mortality was higher among males. Male and female kelts of 1 and female kelts of 2 SWhad a greater mean length than ascending salmon in corresponding groups indicating a better survival of larger fish within an age group. Grilse ascend rivers after most kelts have left, but the main catch of ascending 2–3 SW salmon takes place concurrently with kelts leaving the river, inadvertently targeting kelts in the fishery.     

Metabolic compartmentation of pyruvate in the isolated perfused rat heart.

1. Prompted by the finding of markedly differing specific radioactivities of tissue alanine and lactate in isolated rat hearts perfused with [1-14C]pyruvate, a more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken. Isolated rat hearts were perfused by the once-through Langendorff technique under metabolic and isotopic steady-state conditions but with various routes of radioactive label influx, and the specific radioactivities of pyruvate, lactate and alanine were determined. An enzymic method was devised to determine the specific radioactivity of C-1 of pyruvate. 2. Label introduction as [1-14C]pyruvate resulted in a higher specific radioactivity of tissue alanine and mitochondrial pyruvate than of lactate, and a higher specific radioactivity of perfusate lactate than of tissue lactate. Label introduction as [1-14C]lactate resulted in a roughly similar isotope dilution into the tissue and perfusate pyruvate and the tissue alanine. Label introduction as [3,4-14C]glucose resulted in the same specific radioactivity of tissue lactate and alanine and a roughly similar specific radioactivity of mitochondrial pyruvate. 3. The results can be reconciled with a metabolic model containing two cytosolic functional pyruvate pools. One pool (I) communicates more closely with the glycolytic system, whereas the other (II) communicates with extracellular pyruvate and intracellular alanine. Pool II is in close connection with intramitochondrial pyruvate. The physical identity of the cytosolic subcompartments of pyruvate is discussed.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.     

Cytoskeletal structure of myoblasts with the mitochondrial DNA 3243A-->G mutation and of osteosarcoma cells with respiratory chain deficiency

The cytoskeleton, mainly composed of actin filaments, microtubules, and intermediate filaments, is involved in cell proliferation, the maintenance of cell shape, and the formation of cellular junctions. The organization of the intermediate filaments is regulated by phosphorylation and dephosphorylation. We examined cell population growth, apoptotic cell death, and the morphology of cytoskeletal components in myoblast cultures derived from patients with the 3243A-->G mutation in mitochondrial DNA (mtDNA) and from control subjects by means of assays detecting cellular nucleic acids, histone-associated DNA fragments and by immunolabeling of cytoskeletal components. Population growth was slower in the 3243A-->G myoblast cultures, with no difference in the amount of apoptotic cell death. The organization of vimentin filaments in myoblasts with 3243A-->G was disturbed by randomization of filament direction and length, whereas no disturbances were observed in the other cytoskeletal proteins. Vimentin filaments formed large bundles surrounding the nucleus in mtDNA-less (rho(0)) osteosarcoma cells and in osteosarcoma cells after incubation with sodium azide and nocodazole. We conclude that defects in oxidative phosphorylation lead to selective disruption of the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases.