Succinate-ubiquinone Reductase Research Articles

Interaction of 6-Bromo- and 6-Chloro-Ubiquinone derivatives with mitochondrial electron transfer system

To understand the reaction mechanism of quinone-mediated electron transfer, a series of ubiquinone (Q)-derivatives with a bromine or chlorine atom at the 6-position and a different alkyl side chains at the 5-position of the benzoquinone ring were synthesized and characterized. The chemical properties and electron transfer activities were compared with the native ubiquinone, Q0C10. The redox midpoint potential of 6-Bromo- and 6-chloro-Q derivatives is 142 mV and 148 mV, respectively. These 6-halo-Q derivatives are reducible by ascorbate alone or by succinate in the presence of succinate-ubiquinone reductase. The reduced 6-halo-Q (6-halo-QH2) can be oxidized by cytochrome c alone or via ubiquinol-cytochrome c reductase. In the presence of cytochrome c oxidase and cytochrome c, 6-halo-QH2 is oxidizable by oxygen. 6-Halo-Q derivatives are reducible by succinate via succinate-ubiquinone reductase. They function as an electron-mediator connecting succinate-Q reductase and cytochrome c oxidase and bypass ubiquinol-cytochrome c reductase in the mitochondrial electron-transfer chain. This bypassing results in a decrease in energy coupling efficiency and a lower P/O ratio. These compounds might have therapeutic value in treating diseases that are caused by the oversupply of energy.

Baseline Sensitivity to and Succinate Dehydrogenase Activity and Molecular Docking of Fluxapyroxad and SYP-32497 in Rice Sheath Blight (Rhizoctonia solani AG1-IA) in China

Rice sheath blight caused by Rhizoctonia solani occurs worldwide and is mainly controlled by fungicides. SYP-32497 is a novel succinate dehydrogenase inhibitor, which interferes with the succinate ubiquinone reductase in the mitochondrial respiratory chain of fungi. This study aimed to evaluate the baseline sensitivity of R. solani from 13 major rice producing areas in China to SYP-32497 and fluxapyroxad. The study also explored the cause for the activity discrepancy between SYP-32497 and fluxapyroxad via an enzyme activity inhibition test and molecular docking. A total of 360 R. solani isolates were sensitive to SYP-32497 and fluxapyroxad. Baseline sensitivities were unimodally distributed with mean values of 0.00667 ± 0.00475 and 0.0657 ± 0.0250 μg mL−1, respectively, for SYP-32497 and fluxapyroxad. Enzyme activity assays and molecular docking results revealed that SYP-32497 exhibited a much higher SDH inhibition (IC50 = 0.300 μg mL−1) than to fluxapyroxad (IC50 = 1.266 μg mL−1) because of its excellent SDH binding ability via hydrogen bonding, π-cation, and hydrophobic interactions. These results suggest that SYP-32497 is a good suitable control agent for alternative rice sheath blight.

Succinate Ubiquinone Reductase Activity Predicts Multiple Myeloma Sensitivity to Venetoclax

Multiple myeloma (MM) is a plasma cell malignancy estimated to account for 12,590 deaths and 30,280 new diagnoses in the US in 2017. Despite the use of next generation immunomodulatory drugs (IMiDs), proteasome inhibitors and newer targeted therapies, the development of chemo-resistant MM is common, with approximately 20% of patients succumbing to aggressive treatment-refractory disease within a short period of diagnosis. Resistance is primarily associated with evasion of apoptosis connected to the inability to release sufficient pro-apoptotic BCL-2 proteins above a threshold required to elicit apoptosis. Thus, finding alternative strategies to shift a resistant cell closer to the apoptotic threshold could potentially circumvent resistance.Metabolites contribute to the development of resistance to apoptosis by regulating pro and anti-apoptotic BCL-2 family members. We previously reported that depriving cells of glutamine enhanced binding of BIM to BCL-2 thereby sensitizing myeloma cell lines and patient samples to the BH3 mimetic venetoclax (ABT-199), a highly selective, potent BCL-2 antagonist. ABT-199 is however effective only in 40% of MM patients exhibiting the (11;14) translocation, warranting investigation of strategies to increase the applicability of ABT-199. We were interested to explore if there were metabolic differences in ABT-199 sensitive t(11;14) and resistant cells that could inform us of a) predictors of sensitivity; and b) metabolic targets that could be inhibited to sensitize resistant cells to ABT-199.Given that, tumor cells differentially rely on glycolysis and mitochondrial oxidative phosphorylation we first evaluated oxygen consumption rates (OCR) in ABT-199 sensitive and resistant MM lines. All ABT-199 sensitive lines exhibited lower OCR in contrast to the resistant lines. Metabolite profiling and isotope tracing flux analyses of glutamine deprived myeloma cell lines revealed specific reduction of TCA cycle metabolites including succinate. TCA cycle intermediates are linked to mitochondrial respiration via electron transport chain (ETC) complexes (I-V). Among the five ETC complexes, only Complex II/succinate dehydrogenase (SDH) is directly connected to the TCA cycle and ETC. SDH facilitates the oxidation of succinate to fumarate in the TCA cycle through its subunit SDHA, and transfers the released electrons to ubiquinone via its SDHB, C and D subunits supporting the succinate ubiquinone reductase (SQR) activity of Complex II. Therefore, we sought to further interrogate succinate dehydrogenase in facilitating ABT-199 sensitization. We evaluated SDH ubiquinone reductase activity in ABT-199 sensitive and resistant myeloma lines and demonstrated that all ABT-199 sensitive cell lines had significantly lower SQR activity. Furthermore, inhibition of the SDH ubiquinone reductase with the Qp site inhibitor thenoyltrifluoroacetone (TTFA) sensitized resistant MM cells to ABT-199. Inhibition of Complex I with piericidin or the SDHA subunit with 3-NPA were not as effective in sensitizing MM to ABT-199. Protein expression and co-immunoprecipitation analyses demonstrated an induction of BIM and enhanced BIM-BCL-2 binding upon SDH inhibition with TTFA. TTFA treated cells exhibited a reduction of FOXO3a phosphorylation and concomitant nuclear localization upon SDH inhibition suggestive of FOXO3a playing a role in BIM induction. TTFA + ABT-199 co-treatment induced apoptosis in ABT-199 resistant myeloma patient cells without impacting viability of the associated normal populations.Our study thus identifies a tumor selective approach of metabolically-driven synthetic lethality that relies on inhibition of the mitochondrial enzyme SDH and use of the BCL-2 antagonist, ABT-199 for myeloma therapy. In addition, our results importantly suggest SDH activity predicts ABT-199 sensitivity and can be interrogated in MM patients as a predictive marker of ABT-199 sensitivity particularly within t(11;14) patients currently being enrolled for ABT-199 treatment. DisclosuresNooka:Amgen, Novartis, Spectrum, Adaptive: Consultancy. Boise:Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation

Heat-modified citrus pectin, a water-soluble indigestible polysaccharide fiber derived from citrus fruits and modified by temperature treatment, has been reported to exhibit anticancer effects. However, the bioactive fractions and their mechanisms remain unclear. In this current study, we isolated an active compound, trans-4,5-dihydroxy-2-cyclopentene-l-one (DHCP), from heat-treated citrus pectin, and found that is induces cell death in colon cancer cells via induction of mitochondrial ROS. On the molecular level, DHCP triggers ROS production by inhibiting the activity of succinate ubiquinone reductase (SQR) in mitochondrial complex II. Furthermore, cytotoxicity, apoptotic activity, and activation of caspase cascades were determined in HCT116 and HT-29 cell-based systems, the results indicated that DHCP enhances the sensitivity of cancer cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), with DHCP-induced ROS accounting for the synergistic effect between DHCP and TRAIL. Furthermore, the combination of DHCP and TRAIL inhibits the growth of HCT116 and HT-29 xenografts synergistically. ROS significantly increases the expression of TRAIL death receptor 5 (DR5) via the p53 and C/EBP homologous protein pathways. Collectively, our findings indicate that DHCP has a favorable toxicity profile and is a new TRAIL sensitizer that shows promise in the development of pectin-based pharmaceuticals, nutraceuticals, and dietary agents aimed at combating human colon cancer.

Electron transport chain activity is a predictor and target for venetoclax sensitivity in multiple myeloma

The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the ‘primed’ state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy.

Abstract 2715: Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma

Abstract Multiple myeloma (MM) is the second most common hematologic malignancy. In 2017, MM accounted for approximately 30,770 new diagnoses and 12770 deaths in the US. Although, advancements in treatment options have increased survival rates and life expectancy, MM remains incurable due to development of resistance. Venetoclax is a highly selective, potent BCL-2 antagonist, currently in phase I/II trials for MM and FDA approved for the treatment of CLL patients exhibiting 17p deletion. Venetoclax is effective in eliciting cell death as a single agent in a subset of MM with the (11;14) translocation (which we henceforth designate “sensitive”) in contrast to the majority of MM that is resistant (which we henceforth designate “resistant”). We previously reported that glutamine deprivation increases BIM binding to BCL-2 thereby sensitizing MM to venetoclax, while alpha-ketoglutarate supplementation reversed this sensitivity. We were therefore interested to explore if there was a metabolic basis for t(11;14)-myeloma sensitivity to single agent venetoclax to aid in 1) identifying venetoclax sensitive MM and; 2) inform us of metabolic targets that could be inhibited to sensitize resistant MM to venetoclax. We first queried the CoMMpass MM patient RNAseq trial data and cell lines for electron transport chain (ETC) and TCA cycle gene expression differences in t(11;14) vs non-t(11;14) patients and cell lines. All sensitive cells exhibited varied suppression of TCA/ETC genes, and reduced TCA cycle metabolites and oxygen consumption rates (OCR) compared to resistant cells. Examination of TCA cycle activities connected to the ETC and OCR showed significant decrease in Complex II Succinate ubiquinone reductase (SQR) activity in sensitive cell lines and purified CD38+ve MM patient cells and elevated SQR activity in resistant cells. Furthermore, inhibition of SQR with thenoyltrifluoroacetone (TTFA) sensitized resistant cells to venetoclax. Consistent with SQR inhibition leading to ABT-199 sensitivity, overexpression of an SQR mutant (SDHCR72C) in SDHC-knockout resistant MM, increased venetoclax sensitivity, identifying a unique role for SQR in regulating BCL-2 dependence. In interrogating the mechanistic underpinnings of SQR inhibition-induced sensitivity to venetoclax, we identified increased expression of ATF4 and BIM upon SQR inhibition. ATF4KD or BIMKO restored viability in TTFA and venetoclax co-treated MM further confirming the role of SQR inhibition-induced ATF4 and BIM in venetoclax sensitivity. In testing translationally relevant compounds, we determined that the histone deacetylase inhibitor, panobinostat reduced SQR activity in a dose dependent manner and sensitized MM cells to venetoclax. Our study thus identifies SQR as a novel target and predictive marker to aid in identifying ABT-199-responsive MM patients in a functional biomarker informed manner. Citation Format: Richa Bajpai, Abhinav Achreja, Changyong Wei, Arusha Siddiqa, Shannon M. Matulis, Vikas Gupta, Samuel K. McBrayer, Anjali Mittal, Manali Rupji, Hsiao-Rong Chen, Jeanne Kowalski, Sagar Lonial, Ajay K. Nooka, Lawrence H. Boise, Deepak Nagrath, Mala Shanmugam. Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2715.

Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine

The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.

Sensitivity of Rhizoctonia solani causing rice sheath blight to fluxapyroxad in China

Fluxapyroxad,3-(difluoromethyl)-1-methyl-N-(3’,4’,5’-trifluorobiphenyl-2-yl) pyrazole-4-carboxamide (C18H12F5N3O, Fig. 1), is a SDHI fungicide, which is a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. Between 2008 and 2010, a total of 128 isolates of Rhizoctonia solani from Anhui Province of China were characterized for the baseline sensitivity to fluxapyroxad. The isolates collected between 2008 and 2010 all showed similar sensitivity to fluxapyroxad. Baseline sensitivity was distributed as unimodal curves with an average EC50 value of 0.054 ± 0.014 μg ml−1. However, EC50 values of boscalid for inhibition of mycelial growth of R. solani isolates ranged from 1.89 to 2.68 μg/ml and the average (±SE) EC50 value was 2.212 ± 0.228 μg/ml, indicating that the R. solani isolates were less sensitive to boscalid than that of fluxapyroxad. Fluxapyroxad exhibited excellent protective and curative activity against rice sheath blight and provided 82.6–94.2 % protective or curative control efficacy. In field trials, control efficacy of fluxapyroxad at 100 g a.i/ha 15 days and 30 day after second application was 83.4–88.0 %, suggesting excellent activity against sheath blight. Control efficacy of boscalid at a dosage of 600 g a.i/ha 15 days and 30 day after second application was about 51.7–57.0 %. There was a significant difference in the efficacy between fluxapyroxad and boscalid or jinggangmycin. These results suggested that fluxapyroxad is a good alternative fungicide to jinggangmycin for the control of rice sheath blight.

Type II Fp of human mitochondrial respiratory complex II and its role in adaptation to hypoxia and nutrition-deprived conditions

The flavoprotein (Fp) subunit of human mitochondrial succinate-ubiquinone reductase (SQR, complex II) has isoforms (type I, type II). Type II Fp is predominantly expressed in some cancer and fetal tissues and those tissues are often exposed to ischemia. The present study shows that complex II with type II Fp has lower optimal pH than complex II with type I Fp, and type II Fp mRNA expression was induced by ischemia. The result suggests complex II with type II Fp may function in cells with low mitochondrial matrix pH caused by ischemia and its function is related to cellular adaptation to ischemia.

Baseline sensitivity and efficacy of thifluzamide in Rhizoctonia solani

Thifluzamide is a SDHI (succinate dehydrogenase inhibitor) fungicide, which interferes with succinate ubiquinone reductase in the mitochondrial electron transport chain of fungi. Presently, jinggangmycin is the major fungicide extensively used for the control of rice sheath blight caused by Rhizoctonia solani and resistance to jinggangmycin was first reported to occur in China. A total of 128 isolates of R. solani from Anhui Province of China were characterised for the baseline sensitivity to thifluzamide. The isolates were very sensitive to thifluzamide and the baseline sensitivity curve was unimodal with an average EC50 value of 0.058 ± 0.012 µg mL−1. However, EC50 values of boscalid (another SDHI fungicide) for inhibition of mycelial growth of 22 arbitrarily selected R. solani isolates ranged from 1.89 to 2.68 µg mL−1. Thifluzamide applied at 110 µg mL−1 exhibited excellent protective and curative activity against rice sheath blight and provided 81.1–91.0% protective or curative control efficacy. In field trials in 2010 and 2011, control efficacies of thifluzamide at 82 g.a.i ha−1 15 and 30 days after second application were 84.2% and 86.7%, respectively, suggesting excellent activity against sheath blight. There was a statistically significant difference in the efficacy between thifluzamide and boscalid or jinggangmycin. These results suggested that thifluzamide should be a good alternative fungicide to jinggangmycin for the control of rice sheath blight.

Toward understanding the role of mitochondrial complex II in the intraerythrocytic stages of Plasmodium falciparum: Gene targeting of the Fp subunit

Malaria parasites in human hosts depend on glycolysis for most of their energy production, and the mitochondrion of the intraerythrocytic form is acristate. Although the genes for all tricarboxylic acid (TCA) cycle members are found in the parasite genome, the presence of a functional TCA cycle in the intraerythrocytic stage is still controversial. To elucidate the physiological role of Plasmodium falciparum mitochondrial complex II (succinate-ubiquinone reductase (SQR) or succinate dehydrogenase (SDH)) in the TCA cycle, the gene for the flavoprotein subunit (Fp) of the enzyme, pfsdha (P.falciparum gene for SDH subunit A, PlasmoDB ID: PF3D7_1034400) was disrupted. SDH is a well-known marker enzyme for mitochondria. In the pfsdha disruptants, Fp mRNA and polypeptides were decreased, and neither SQR nor SDH activity of complex II was detected. The suppression of complex II caused growth retardation of the intraerythrocytic forms, suggesting that complex II contributes to intraerythrocytic parasite growth, although it is not essential for survival. The growth retardation in the pfsdha disruptant was rescued by the addition of succinate, but not by fumarate. This indicates that complex II functions as a quinol-fumarate reductase (QFR) to form succinate from fumarate in the intraerythrocytic parasite.

An anticancer agent, pyrvinium pamoate inhibits the NADH–fumarate reductase system—a unique mitochondrial energy metabolism in tumour microenvironments

Increased glycolysis is the principal explanation for how cancer cells generate energy in the absence of oxygen. However, in actual human tumour microenvironments, hypoxia is often associated with hypoglycemia because of the poor blood supply. Therefore, glycolysis cannot be the sole mechanism for the maintenance of the energy status in cancers. To understand energy metabolism in cancer cells under hypoxia-hypoglycemic conditions mimicking the tumour microenvironments, we examined the NADH-fumarate reductase (NADH-FR) system, which functions in parasites under hypoxic condition, as a candidate mechanism. In human cancer cells (DLD-1, Panc-1 and HepG2) cultured under hypoxic-hypoglycemic conditions, NADH-FR activity, which is composed of the activities of complex I (NADH-ubiquinone reductase) and the reverse reaction of complex II (quinol-FR), increased, whereas NADH-oxidase activity decreased. Pyrvinium pamoate (PP), which is an anthelmintic and has an anti-cancer effect within tumour-mimicking microenvironments, inhibited NADH-FR activities in both parasites and mammalian mitochondria. Moreover, PP increased the activity of complex II (succinate-ubiquinone reductase) in mitochondria from human cancer cells cultured under normoxia-normoglycemic conditions but not under hypoxia-hypoglycemic conditions. These results indicate that the NADH-FR system may be important for maintaining mitochondrial energy production in tumour microenvironments and suggest its potential use as a novel therapeutic target.

Hereditäre Paragangliome

Zusammenfassung Hereditäre Paragangliome/Phäochromozytome werden autosomal-dominant vererbt. Es lassen sich 3 Formen, PGL1, PGL3 und PGL4 unterscheiden. Sie werden verursacht durch Mutationen in den Genen SDHD, SDHC und SDHB, welche für Komponenten des Komplexes II der mitochondrialen Atmungskette (Succinat-Ubiquinon-Reduktase, SDH) kodieren. Bei allen 3 Formen findet sich „loss of heterozygosity“ (LOH) der Region des mutierten Gens in Tumor-DNA. Dies führt zu Funktionsverlust der SDH, Anhäufung von Succinat sowie Sauerstoffradikalen. Dadurch werden hypoxieabhängige Stoffwechselwege aktiviert, welche zur Tumorbildung führen könnten. Während PGL3 und PGL4 sowohl durch maternal als auch durch paternal vererbte Keimbahnmutationen der Gene SDHC bzw. SDHB verursacht werden, findet sich PGL1 fast ausschließlich bei paternaler Transmission des mutierten SDHD-Gens. Diese Beobachtung lässt sich erklären durch partielle Inaktivierung (Imprinting) des maternalen SDHD-Gens und Induktion hypoxieabhängiger Gene in Paragangliengewebe, wodurch der Verlust des gesamten maternalen Chromosoms 11 durch Non-Disjunction begünstigt werden könnte.

The NADH‐fumarate reductase system, a novel mitochondrial energy metabolism, is a new target for anticancer therapy in tumor microenvironments

Since deficiencies of critical nutrients and hypoxia are observed in hypovascular tumors, glycolysis alone cannot explain how cancer cells maintain their required energy levels. To study energy metabolism in cancer cells within such tumor microenvironments, we examined the NADH-fumarate reductase system, which is found in anaerobic organisms, such as parasitic helminthes. In human cancer cells cultured under tumor microenvironment-mimicking conditions, mitochondrial NADH-fumarate reductase activity increased in parallel with an increase in fumarate reductase activity, which is the reverse reaction of succinate-ubiquinone reductase and is regulated by the phosphorylation of its subunit. Pyrvinium pamoate, an anthelmintic drug, has an anticancer effect within tumor-mimicking microenvironments. We found that one of the biological mechanisms of pyrvinium is the inhibition of the NADH-fumarate reductase system. Therefore, the NADH-fumarate reductase system might be important for maintaining mitochondrial energy metabolism within the tumor microenvironments and might represent a novel target for anticancer therapies.

Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil.

In adult Ascaris suum (roundworm) mitochondrial membrane-bound complex II acts as a rhodoquinol-fumarate reductase, which is the reverse reaction to that of mammalian complex II (succinate-ubiquinone reductase). The adult A. suum rhodoquinol-fumarate reductase was crystallized in the presence of octaethyleneglycol monododecyl ether and n-dodecyl-beta-D-maltopyranoside in a 3:2 weight ratio. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 123.75, b = 129.08, c = 221.12 A, and diffracted to 2.8 A resolution using synchrotron radiation. The presence of two molecules in the asymmetric unit (120 kDa x 2) gives a crystal volume per protein mass (V(M)) of 3.6 A(3) Da(-1).

Sensitivity of Sclerotinia sclerotiorum from oilseed crops to boscalid in Jiangsu Province of China

Between 2006 and 2008, 120 isolates of Sclerotinia sclerotiorum from the Jiangsu Province of China were characterized for the baseline sensitivity to boscalid, a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. The isolates collected between 2006 and 2008 all showed similar sensitivity to boscalid. Baseline sensitivities were distributed as unimodal curves with mean EC 50 values of 0.17 ± 0.09 μg ml −1 for inhibition of mycelial growth. Inhibition of mycelial growth of isolates whether they had resistance to carbendazim or, dimethachlon or dual resistance to both carbendazim and dimethachlon was similar, suggesting that there was no cross-resistance between boscalid and MBC/dimethachlon already in commercial use. In field trials, the treatments boscalid (50% WG), at 125, 187.5 and 250 g ai ha −1 provided higher control efficacies of 65.2, 69.0 and 75.1%, respectively, than the treatments carbendazim (50% WP), at 750 g ai ha −1 and dimethachlon (46% WP), at 690 g ai ha −1 with control efficacies of 18.9 and 44.6%, respectively.

Regulation of succinate-ubiquinone reductase and fumarate reductase activities in human complex II by phosphorylation of its flavoprotein subunit

Complex II (succinate-ubiquinone reductase; SQR) is a mitochondrial respiratory chain enzyme that is directly involved in the TCA cycle. Complex II exerts a reverse reaction, fumarate reductase (FRD) activity, in various species such as bacteria, parasitic helminths and shellfish, but the existence of FRD activity in humans has not been previously reported. Here, we describe the detection of FRD activity in human cancer cells. The activity level was low, but distinct, and it increased significantly when the cells were cultured under hypoxic and glucose-deprived conditions. Treatment with phosphatase caused the dephosphorylation of flavoprotein subunit (Fp) with a concomitant increase in SQR activity, whereas FRD activity decreased. On the other hand, treatment with protein kinase caused an increase in FRD activity and a decrease in SQR activity. These data suggest that modification of the Fp subunit regulates both the SQR and FRD activities of complex II and that the phosphorylation of Fp might be important for maintaining mitochondrial energy metabolism within the tumor microenvironment.

Protein Tyrosine Nitration of the Flavin Subunit Is Associated with Oxidative Modification of Mitochondrial Complex II in the Post-ischemic Myocardium

Increased O(2)* and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart ( Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640-32654 ). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide ((77)AAFGLSEAGFNTACVTK(93)) of the 70-kDa protein as a chimeric epitope incorporating a "promiscuous" T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.

S13.26 Divergence in structure of mitochondrial respiratory complex II (succinate–ubiquinone reductase) revealed by protozoan enzymes

S13.26 Divergence in structure of mitochondrial respiratory complex II (succinate–ubiquinone reductase) revealed by protozoan enzymes

A rapid method for gene expression analysis of Borna disease virus in neurons and astrocytes using laser microdissection and real-time RT-PCR

Laser microdissection combined with real-time RT-PCR represents a powerful method to analyse the transcription efficiency of defined cell types. Therefore, a RNA-preserving immunolabelling method was established to identify neurons and astrocytes in persistently BDV-infected rat brain sections for subsequent laser microdissection and quantitation of viral gene products by real-time RT-PCR. Firstly, to ensure an accurate measurement of viral RNA after immunolabelling, different reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], succinate-ubiquinone reductase [SDHA], hypoxanthine phosphoribosyl-transferase-1 [HPRT]) were tested. Only normalisation with GAPDH yielded a stable relative expression of viral RNA encoding the nucleoprotein (BDV-N), the matrixprotein and the glycoprotein (intron I and intron II). The two remaining reference genes biased the ratios of BDV-transcripts in the immunolabelled brain sections significantly. Secondly, 100 immunolabelled neurons and astrocytes were harvested using laser microdissection and amplification of all viral transcripts revealed 681 and 168 (BDV-N), 573 and 254 (intron I), 324 and 133 (intron II) and 161 and 36 (GAPDH) absolute copy numbers in neurons and astrocytes, respectively. Thus, laser microdissection combined with real-time RT-PCR provides an effective tool for the analysis of cell-specific viral transcription efficiency and allows elucidating virus–host-interactions and virus persistence mechanisms in the CNS.