| 1. |
- Struglics, André, et al.
(författare)
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Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria
- 2000
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Ingår i: FEBS Letters. - 0014-5793. ; 475:3, s. 213-217
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Tidskriftsartikel (refereegranskat)abstract
- Inside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with [γ-32P]ATP. Proteins were separated by SDS-PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of [γ- 32P]ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins. (C) 2000 Federation of European Biochemical Societies.
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| 2. |
- Fredlund, Kenneth M., et al.
(författare)
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Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes
- 1994
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Ingår i: Plant Physiology. - 0032-0889 .- 1532-2548. ; 106:3, s. 1103-1106
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Tidskriftsartikel (refereegranskat)abstract
- The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.
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| 3. |
- Fredlund, Kenneth M., et al.
(författare)
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Oxidation of external NAD(P)H by purified mitochondria from fresh and aged red beetroots (Beta vulgaris L.)
- 1991
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 97:1, s. 99-103
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria were isolated from fresh beetroots (Beta vulgaris L. cvs Rubria and Nina) by differential centrifugation followed by Percoll gradient centrifugation. These purified mitochondria oxidized external NADH, although relatively slowly (20-40 versus 100-120 nanomoles oxygen per minute times milligram protein for NADH and succinate oxidation, respectively), with respiratory control ratios of two to three and ADP/O ratios of 1.2 to 1.6. NADPH was also oxidized, but even more slowly and with little or no coupling. The optimum for both NADH and NADPH oxidation by fresh beetroot mitochondria was pH 6. The rate of external NADH oxidation by isolated mitochondria was enhanced threefold during storage of the intact tubers at 10°C for 12 weeks. The optimum of the induced NADH oxidation was approximately pH 6.8. Succinate and malate oxidation only increased by 30% during the same period and NADPH oxidation was constant. This is strong evidence that NADH and NADPH oxidation are catalyzed by different enzymes at least in beetroots. Activity staining of nondenaturing polyacrylamide gels with NADH and Nitro Blue Tetrazolium did not show differences in banding pattern between mitochondria isolated from fresh and stored beetroots. The induction is discussed in relation to physiological aging processes.
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| 4. |
- Møller, Ian M., et al.
(författare)
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NAD(P)H-ubiquinone oxidoreductases in plant mitochondria
- 1993
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Ingår i: Journal of Bioenergetics and Biomembranes. - 0145-479X. ; 25:4, s. 377-384
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Tidskriftsartikel (refereegranskat)abstract
- Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca2+ with a K0.5 of about 1 μM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca2+ with a K0.5 of 3 μM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.
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| 5. |
- Rasmusson, Allan G., et al.
(författare)
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Purification of a rotenone-insensitive NAD(P)H dehydrogenase from the inner surface of the inner membrane of red beetroot mitochondria
- 1993
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - 0005-2728. ; 1141:1, s. 107-110
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Tidskriftsartikel (refereegranskat)abstract
- The soluble fraction of disrupted red beetroot mitochondria was resolved by anion-exchange chromatography. Three NADH-oxidising activities were found, including one duroquinone reductase oxidising both NADH and NADPH. This NAD(P)H-duroquinone reductase, which we assign as the internal rotenone-insensitive NAD(P)H dehydrogenase, was further purified by affinity chromatography into a 26 kDa polypeptide.
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| 6. |
- Struglics, André, et al.
(författare)
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Phosphoproteins and protein kinase activities intrinsic to inner membranes of potato tuber mitochondria
- 1999
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Ingår i: Plant and Cell Physiology. - 0032-0781. ; 40:12, s. 1271-1279
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Tidskriftsartikel (refereegranskat)abstract
- Inside-out submitochondrial particles (IO-SMP) were isolated and purified from potato (Solanum tuberosum L. cv.) tubers. When these IO-SMP were incubated with [γ32P]ATP more then 20 proteins became labelled as a result of phosphorylation. The 32P incorporation was stimulated by the oxidizing reagent ferricyanide. Except for a 17 kDa protein which was phosphorylated only in the absence of divalent cations, the protein phosphorylation required Mg2+. The time for half-maximum 32P incorporation was 4 min for the 22 kDa phospho-F1 δ-subunit and 2 min for the 28 kDa phospho-F0 b-subunit of the proton-ATPase. The K(m) for ATP for the detected phosphoproteins was between 65 μM and 110 μM. The pH optimum for protein phosphorylation in inner membranes was between pH 6 and 8, and for the F1 δ-subunit and the F0 b-subunit the pH optima were 6.5-8 and pH 8, respectively. A 37 kDa phosphoprotein was phosphorylated on a histidine residue while the remainder of the inner membrane proteins were phosphorylated on serine or threonine residues. Two autophosphorylated putative kinases were identified: one at 16.5 kDa required divalent cations for autophosphorylation, while another at 30 kDa did not. A 110 kDa protein was labelled only with [α-32P]ATP, suggesting adenylylation.
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| 7. |
- Struglics, André, et al.
(författare)
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The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane
- 1993
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 88:1, s. 19-28
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Tidskriftsartikel (refereegranskat)abstract
- Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−-dependent; EC 1.1.1.37) latency.The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)−1 min−1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane.When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated.
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| 8. |
- Struglics, André, et al.
(författare)
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Two subunits of the F0F1-ATPase are phosphorylated in the inner mitochondrial membrane
- 1998
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 243:3, s. 664-668
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Tidskriftsartikel (refereegranskat)abstract
- Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [γ-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the δ'-subunit of the F1-ATPase and the b-subunit of the F0-ATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1 and F0 and regulate energy coupling in oxidative phosphorylation.
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| 9. |
- Unemo, M, et al.
(författare)
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Experiences with the new genetic variant of Chlamydia trachomatis in Orebro county, Sweden - proportion, characteristics and effective diagnostic solution in an emergent situation
- 2007
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Ingår i: Eurosurveillance. - 1560-7917 .- 1025-496X. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- A Chlamydia trachomatis variant that contains a 377 bp deletion in the cryptic plasmid was recently reported in Sweden. This deletion includes the targets for Cobas Amplicor, Cobas TaqMan48, and Abbott m2000. We examined the proportion and characteristics of this variant in Örebro county, Sweden and developed an effective diagnostic solution. In total, 2,401 consecutive C. trachomatis culture samples and 536 PCR samples from symptomatic and asymptomatic patients and screened females were included. Culture, Cobas Amplicor, and LightMix 480HT were used for diagnosis. A mutant-specific PCR, plasmid sequencing, omp1 sequencing and multilocus sequence typing (MLST) were used to identify and characterise mutants. In total, 162 (6.7%) of the cultured samples were positive for C. trachomatis. However, 61 (38%) of those were negative when using Cobas Amplicor, and 60 of these were subsequently confirmed as the new variant. 13 of these mutant isolates were further characterised genetically, and all were of identical genotype E and the unique MLST sequence type: 21, 19, 1, 2, 1. Of all culture-positive samples, 161 of 162 were positive in the LightMix 480HT assay. The single negative sample was only weakly positive in culture, and negative in all PCRs. Of the 536 PCR samples, 37 were positive in both Cobas Amplicor and LightMix 480HT, 13 were only positive in LightMix 480HT (mutants), and two were only positive in Cobas Amplicor. Mutated C. trachomatis were prevalent in Örebro county in the period from October 2006 to February 2007, and it appeared to be a single clone. LightMix 480HT seemed sensitive, specific, and enabled high throughput diagnostics. However, rare low positive samples may be false-negative. Frequent surveillance and evaluations of diagnostic methods worldwide are crucial.
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| 10. |
- Liu, Yunjun, et al.
(författare)
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A Redox-Mediated Modulation of Stem Bolting in Transgenic Nicotiana sylvestris Differentially Expressing the External Mitochondrial NADPH Dehydrogenase.
- 2009
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548. ; 150, s. 1248-1259
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Tidskriftsartikel (refereegranskat)abstract
- Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato NDB1 displayed early bolting whereas sense-suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance, but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+)-ratio was unaffected, the stem NADPH/NADP(+)-ratio was altered following the genetic modification and strongly correlated to the bolting phenotype. Metabolic profiling of the stem displayed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars and hexose phosphates. Consistent with the phenotype, the modified NDB1 level also affected expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+)-ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are present in green stems. The results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.
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| 11. |
- Schmidt, John, et al.
(författare)
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Substrate and plant genotype strongly influence the growth and gene expression response to trichoderma afroharzianum T22 in sugar beet
- 2020
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Ingår i: Plants. - : MDPI AG. - 2223-7747. ; 9:8, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- Many strains of Trichoderma fungi have beneficial effects on plant growth and pathogen control, but little is known about the importance of plant genotype, nor the underlying mechanisms. We aimed to determine the effect of sugar beet genotypic variation on Trichoderma biostimulation. The effect of Trichoderma afroharzianum T22 on sugar beet inbred genotypes were investigated in soil and on sterile agar medium regarding plant growth, and by quantitative reverse transcriptase-linked polymerase chain reaction (qRT-PCR) analysis for gene expression. In soil, T22 application induced up to 30% increase or decrease in biomass, depending on plant genotype. In contrast, T22 treatment of sterile-grown seedlings resulted in a general decrease in fresh weight and root length across all sugar beet genotypes. Root colonization of T22 did not vary between the sugar beet genotypes. Sand-and sterile-grown roots were investigated by qRT-PCR for expression of marker genes for pathogen response pathways. Genotype-dependent effects of T22 on, especially, the jasmonic acid/ethylene expression marker PR3 were observed, and the effects were further dependent on the growth system used. Thus, both growth substrate and sugar beet genotype strongly affect the outcome of inoculation with T. afroharzianum T22.
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| 12. |
- Unemo, Magnus, et al.
(författare)
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The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization
- 2010
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Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 156, s. 1394-1404
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms - the genes for central metabolism, development cycle and virulence are conserved - or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population.
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