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The branched respiratory electron transport chain of plants contains a non-phosphorylating alternative pathway consisting of type II NAD(P)H dehydrogenases on both sides of the inner membrane linked through the ubiquinone pool to ...
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The branched respiratory electron transport chain of plants contains a non-phosphorylating alternative pathway consisting of type II NAD(P)H dehydrogenases on both sides of the inner membrane linked through the ubiquinone pool to an alternative oxidase (AOX). T-DNA and RNA interference (RNAi) were used to reduce gene expression to characterize the external NAD(P)H dehydrogenase NDB4 in Arabidopsis. The ndb4 lines showed different levels of suppression of NDB4 protein, leading to increases in NBD2 and AOX1a mRNA and protein levels in all lines. These changes were associated with lower reactive oxygen species formation and an altered phenotype, including changes in growth rate, root : shoot ratios and leaf area. The general growth pattern for the ndb4 mutants was decreased leaf area early in development (6-15 d) followed by a prompt subsequent increase in leaf area that exceeded the leaf area of the wild type by maturity (the 10-12 rosette stage). This pattern was most evident for the RNAi lines that had increased mitochondrial electron transport capacity. The RNAi lines also exhibited better tolerance to salinity stress, with better growth rates and lower shoot Na+ content compared with controls when grown under saline conditions. We hypothesize that these differences reflect the enhanced expression of NDB2 and AOX in the ndb4 mutant plants.
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SUMMARY The study of plant mitochondria started in earnest around 1950 with the first isolations of mitochondria from animal and plant tissues. The first 35?years were spent establishing the basic properties of plant mitochondria ...
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SUMMARY The study of plant mitochondria started in earnest around 1950 with the first isolations of mitochondria from animal and plant tissues. The first 35?years were spent establishing the basic properties of plant mitochondria and plant respiration using biochemical and physiological approaches. A number of unique properties (compared to mammalian mitochondria) were observed: (i) the ability to oxidize malate, glycine and cytosolic NAD(P)H at high rates; (ii) the partial insensitivity to rotenone, which turned out to be due to the presence of a second NADH dehydrogenase on the inner surface of the inner mitochondrial membrane in addition to the classical Complex I NADH dehydrogenase; and (iii) the partial insensitivity to cyanide, which turned out to be due to an alternative oxidase, which is also located on the inner surface of the inner mitochondrial membrane, in addition to the classical Complex IV, cytochrome oxidase. With the appearance of molecular biology methods around 1985, followed by genomics, further unique properties were discovered: (iv) plant mitochondrial DNA (mtDNA) is 10–600 times larger than the mammalian mtDNA, yet it only contains approximately 50% more genes; (v) plant mtDNA has kept the standard genetic code, and it has a low divergence rate with respect to point mutations, but a high recombinatorial activity; (vi) mitochondrial mRNA maturation includes a uniquely complex set of activities for processing, splicing and editing (at hundreds of sites); (vii) recombination in mtDNA creates novel reading frames that can produce male sterility; and (viii) plant mitochondria have a large proteome with 2000–3000 different proteins containing many unique proteins such as 200–300 pentatricopeptide repeat proteins. We describe the present and fairly detailed picture of the structure and function of plant mitochondria and how the unique properties make their metabolism more flexible allowing them to be involved in many diverse processes in the plant cell, such as photosynthesis, photorespiration, CAM and C4 metabolism, heat production, temperature control, stress resistance mechanisms, programmed cell death and genomic evolution. However, it is still a challenge to understand how the regulation of metabolism and mtDNA expression works at the cellular level and how retrograde signaling from the mitochondria coordinates all those processes.
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Abnormalities of mitochondrial energy metabolism may play a role in normal aging and certain neurodegenerative disorders. In this regard, complex I of the electron transport chain has received substantial attention, especially in ...
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Abnormalities of mitochondrial energy metabolism may play a role in normal aging and certain neurodegenerative disorders. In this regard, complex I of the electron transport chain has received substantial attention, especially in Parkinson's disease. The conventional method for studying complex I has been quantitation of enzyme activity in homogenized tissue samples. To enhance the anatomic precision with which complex I can be examined, we developed an autoradiographic assay for the rotenone site of this enzyme. [3H]dihydrorotenone ([3H]DHR) binding is saturable (KD = 15-55 nM) and specific, and Hill slopes of 1 suggest a single population of binding sites. Nicotinamide adenine dinucleotide (NADH) enhances binding 4- to 80-fold in different brain regions (EC50 = 20-40 microM) by increasing the density of recognition sites (Bmax). Nicotinamide adenine dinucleotide phosphate also increases binding, but NAD+ does not. In skeletal muscle, heart, and kidney, binding was less affected by NADH. [3H]DHR binding is inhibited by rotenone (IC50 = 8-20 nM), meperidine (IC50 = 34-57 microM), amobarbitol (IC50 = 375-425 microM), and MPP+ (IC50 = 4-5 mM), consistent with the potencies of these compounds in inhibiting complex I activity. Binding is heterogeneously distributed in brain with the density in gray matter structures varying more than 10-fold. Lesion studies suggest that a substantial portion of binding is associated with nerve terminals. [3H]DHR autoradiography is the first quantitative method to examine complex I with a high degree of anatomic precision. This technique may help to clarify the potential role of complex I dysfunction in normal aging and disease.
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BACKGROUND: Genotype announcement may be one of the effective methods to induce smoking cessation, but the studies are limited throughout the world. METHODS: Subjects were smokers who attended a health checkup examination provided...
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BACKGROUND: Genotype announcement may be one of the effective methods to induce smoking cessation, but the studies are limited throughout the world. METHODS: Subjects were smokers who attended a health checkup examination provided by a local government in Hokkaido, Japan, 2003. Those who agreed to know their genotypes were informed of the genotypes of glutathione S-transferease (GST) M1 present/null, GSTT1 present/null, and NAD(P)H:quinone oxidoreductase 1 (NQO1) C609T (Pro187Ser). RESULTS: Out of 143 smokers (92 males and 51 females), 101 individuals participated in the present study. A postal questionnaire one year after the genotype announcement found that 8 persons (6 males and 2 females) of 41 respondents had quitted smoking. Two of 8 quitters stated that they had quitted smoking due to the announcement. There were none who regretted the genotype tests. CONCLUSION: Although the cessation rate, 7.9% (8/101) at least, was not marked, no harmful effects were observed among the respondents.
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A new butenylflavanone, (2S)-5-hydroxy-7-methoxy-8-[(E)-3-oxo-1-butenyl]flavanone (1), and a new rotenoid, 4',5'-dihydro-11,5'-dihydroxy-4'-methoxytephrosin (2), as well as three active flavonoids of previously known structure, is...
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A new butenylflavanone, (2S)-5-hydroxy-7-methoxy-8-[(E)-3-oxo-1-butenyl]flavanone (1), and a new rotenoid, 4',5'-dihydro-11,5'-dihydroxy-4'-methoxytephrosin (2), as well as three active flavonoids of previously known structure, isoliquiritigenin (3), genistein (4), and chrysoeriol (5), along with nine known inactive compounds, alpha-toxicarol (6), sumatrol, 6a,12a-dehydro-alpha-toxicarol, 11-hydroxytephrosin, obovatin, marmesin, lupenone, benzyl benzoate, and benzyl trans-cinnamate, were isolated from an ethyl acetate-soluble extract of the stems of Tephrosia toxicaria, using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa 1c1c7 mouse hepatoma cells to monitor chromatographic fractionation. The structures of compounds 1 and 2 were elucidated by spectroscopic data interpretation. All isolates were evaluated for their potential cancer chemopreventive properties utilizing an in vitro assay to determine quinone reductase induction. Selected compounds were tested in a mouse mammary organ culture assay to evaluate the inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesions.
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Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a p...
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Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a ping-pong mechanism in which the NAD(P)H and the substrate occupy alternatively overlapping regions of the same binding site and participate in a double hydride transfer: one from NAD(P)H to the FAD of the enzyme, and one from the FADH2 of the enzyme to the quinone substrate. The main function of QR1 is probably the detoxification of dietary quinones but it may also contribute to the reduction of vitamin K for its involvement in blood coagulation. In addition, the same reaction that QRi uses in the detoxification of quinones, activates some compounds making them cytotoxic. Since QRi is elevated in many tumors, this property has encouraged the development of chemotherapeutic compounds that become cytotoxic after reduction by QR1.
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The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX)...
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The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)(+) ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.
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In this study, the kinetic parameters, V(max) and K(m), of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no corr...
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In this study, the kinetic parameters, V(max) and K(m), of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no correlation between the experimentally determined rates of p-benzoquinone reduction by DT-diaphorase and the calculated chemical reactivity of the examined substrates as expressed by the energy of the lowest unoccupied molecular orbital, E(LUMO). However, a reasonable correlation was found between the natural logarithm of V(max)/K(m) and the partition coefficient of the p-benzoquinones (r=0.81). Furthermore, tetrachloro-p-benzoquinone, one of the tested quinones is shown to be an inhibitor of rat DT-diaphorase. The presence of bovine serum albumin (BSA) in the incubation mixture protects DT-diaphorase against the inactivation by tetrachloro-p-benzoquinone, probably by interacting with the quinone. Maldi-Tof analysis of the incubation mixture of the purified DT-diaphorase and tetrachloro-p-benzoquinone showed that every subunit of the enzyme shifted about +414 amu, whereas the dimer shifted about +849 amu relative to control values. This indicates a covalent modification of the rat liver DT-diaphorase by tetrachloro-p-benzoquinone.
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Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca2+ influx and mobilization of Ca2+ from internal stores. Here, we show t...
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Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca2+ influx and mobilization of Ca2+ from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51 kDa and 14 kDa subunits) or the Ca2+-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca2+ response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca2+ signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca2+ oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca2+ further supported the involvement of these organelles in staurosporine-induced Ca2+ signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca2+ dynamics and bioenergetics. (C) 2015 Elsevier B.V. All rights reserved.
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Activity-guided fractionation of the petroleum ether and ethyl acetate extracts of the stem bark of Pongamia pinnata, using cultured Hepa 1c1c7 mouse hepatoma cells to evaluate quinone reductase (QR) inducing activity, led to the ...
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Activity-guided fractionation of the petroleum ether and ethyl acetate extracts of the stem bark of Pongamia pinnata, using cultured Hepa 1c1c7 mouse hepatoma cells to evaluate quinone reductase (QR) inducing activity, led to the isolation of four new flavanone derivatives (1-4), one new flavone (5), one new chalcone (6), and 13 known compounds of the flavonoid, terpenoid, and fatty acid types. The structures of 1-6 were characterized on the basis of the interpretation of their spectroscopic data. The absolute stereochemistry of compounds 1-4 was determined from their CD data and by Mosher ester determination. All isolates obtained were evaluated in the quinone reductase induction assay.
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