Studies on succinate-tetrazolium reductase systems

Slater, T. F.; Sawyer, Barbara; Straeuli, U

doi:10.1016/0006-3002(63)90513-4

Cited by 679 publications

(170 citation statements)

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“…This would explain why mutants selected for the inability to reduce tetrazolium red have the variety of changes observed in the respiratory chain. Work with Escherichia coli [37] or animal mitochondria [38] indicates that the site of tetrazolium red reduction occurs after ubiquinone but thc exact site or whether there is more than one site has not been unambiguously determined [37]. Since whole cells of othcr mutants lacking ascorbate -Ph(NMe), oxidase including those that lack c-type cytochromes reduce tetrazolium red at rates comparable to the wild-type strain, this suggests that the site of tetrazolium reduction in whole cells of A .…”

Section: Discussionmentioning

confidence: 99%

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

et al. 1984

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A method is described for the isolation of respiratory mutants of Azotobacter cinelundii with incrcascd amounts of cl-type cytochrome by selecting for the inability to reduce tctrazolium red. Five stable mutants were obtained that had six-fold higher levels of cytochrome d, increased amounts of b-type and lower amounts of o-type and c-type cytochromes than the wild-type strain. Spectral alterations in cytochrome al were also observed in the mutants. N A D H and succinate oxidase activities of membrane particles were about two fold higher in the mutants compared to the wild-type strain. Ascorbate-N,N,N',N'-tetramethyl-p-phenylene diaminc oxidase activity was barely detectable in membranc particles of the mutants. These results are consistent with an increasc in the amount of the cytochrome d oxidase branch and a decrease in the amount and activity of the cytochrome 0, a1 oxidase branch in the mutants. Growth rates under oxygen-excess conditions and respiratory-linked proton translocation ratios of the mutant and wild-type strains were similar as were the photochemical spectral and kinetic properties of cytochrome d.The respiratory chain of Azotobacter vinelundii has been extensively studied [l, 21. The components of the respiratory chain include highly active flavin-dependent NADH and malate dehydrogenases, hydrogenase, ubiquinone-8, and at least seven spectroscopically detectable cytochromes, b-560. [3,9: 141. Sites of cncrgy conservation occur at the level of NADH dchydrogenase, hydrogenase, the ubiquinone region [4,9,7,16,17,33]. Recent studies diminish the possibility that the cytochrome c-al, o branch is involved in the energy conservation cfficicncy under non-oxygen-limiting growth conditions [11,12.13:15].In addition to its role in energy conservation, it has been proposed that the high activity of the respiratory chain protects the oxygen-sensitive nitrogenase complex in Azotohacter from inactivation [18 -201. Respiratory protection of nitrogenase at higher dissolved oxygen tensions is provided by an increase in whole cell respiration rate which is accompanied by increases in N A D H oxidase activity and levels of cytochrome d [18 -201. The study of respiratory mutants of A . uinelandii has proven to be useful in determining the contribution of each branch of the respiratory chain in the oxidation of physiological substrates and in the efficiency of encrgy conservation. Biochemical studies of mutants unable to reoxidize Ph(NMe,), could not demonstrate a significant role for the MATbRIALS A N D METHODS Bacterial strainsThe wild-type strain AVOP of Azobacter rinelandii was used. Strains AV90 and AVZ 07 are Ph(NMe,),-oxidasenegative mutants of AVOP [I1 -131 (Hoffman and Dcr Vartanian, unpublished results). Mt.dia and conditions of cultiuatiorzEach strain was routinely grown in 1.5 1 of modified Burk's medium [21] with 2 '4 sucrose (wiv) without an added nitrogen source. Other ctrbon sourccs were added at a final concentration of 0.2 ,d (wlv) and nitrogen sources were added to givc a final combined niiro...

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Section: Discussionmentioning

confidence: 99%

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

et al. 1984

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“…Under anaerobic conditions, the electrons of the respiratory chain can be transferred by cytochrome c oxidase to TTC, which is then reduced to red formazan (32). Contrary to the deleted recipient strain dum11, which remains green in the dark when overlaid with TTC, strains that possess an intact cytochrome pathway of respiration (functional complex III and complex IV) become brown.…”

Section: Methodsmentioning

confidence: 99%

High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

Remacle

Cardol

Coosemans

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

157

131

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Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100 -250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This ⌬nd4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.green alga ͉ mitochondrial DNA mutagenesis ͉ telomere ͉ complex I assembly ͉ respiratory-deficient mutant

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“…3B). Of note, conversion of MTT is widely used for cell proliferation assay, although it was originally adopted as a functional analysis of mitochondrial reductase activity (26). Furthermore, the decreased mitochondrial function in U87-SC cells was also reflected by a lower mitochondrial membrane potential as measured by rodmine-123 staining in comparison with U87 cells (Fig.…”

Section: U87-sc Cells Exhibited Stem-like Properties and Drugmentioning

confidence: 96%

Metabolic Alterations in Highly Tumorigenic Glioblastoma Cells

Zhou¹,

Zhou²,

Shingu³

et al. 2011

Journal of Biological Chemistry

223

127

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Recent studies suggest that a small subpopulation of malignant cells with stem-like properties is resistant to chemotherapy and may be responsible for the existence of residual cancer after treatment. We have isolated highly tumorigenic cancer cells with 100-fold increase in tumor initiating capacity from the tumor xenografts of human glioblastoma U87 cells in mice. These cells exhibit stem-like properties and show unique energy metabolic characteristics including low mitochondrial respiration, increased glycolysis for ATP generation, and preference for hypoxia to maintain their stemness and tumor forming capacity. Mechanistically, mitochondrial depression in the highly tumorigenic cells occurs mainly at complex II of the electron transport chain with a down-regulation of the succinate dehydrogenase subunit B, leading to deregulation of hypoxia-inducible factors. Under hypoxia, the stem-like cancer cells are resistant to conventional anticancer agents but are sensitive to glycolytic inhibition. Furthermore, combination of glycolytic inhibition with standard therapeutic agents is effective in killing the tumor-initiating cells in vitro and inhibits tumor formation in vivo. Our study suggests that stem-like cancer cells prefer a low oxygen microenvironment and actively utilize the glycolytic pathway for ATP generation. Inhibition of glycolysis may be an effective strategy to eradicate residual cancer stem cells that are otherwise resistant to chemotherapeutic agents in their hypoxic niches.

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Studies on succinate-tetrazolium reductase systems

Cited by 679 publications

References 15 publications

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

Metabolic Alterations in Highly Tumorigenic Glioblastoma Cells

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