Substrate supplementation in permeabilized parasites partly decreases HDQ-mediated ΔΨm depolarization. RH strain tachyzoites were treated with 100 nM HDQ in the presence or absence of 250 μM uracil. HDQ treatment leads to a decreased ATP level.We further examined the influence of HDQ-mediated ΔΨm collapse on the parasitic ATP level. As expected, Complex I inhibitors had no effect on pfNDH2 enzyme activity (Table 2). The relative contribution of oxidative phosphorylation to total ATP synthesis is still a matter of debate for T. gondii, as it is for other apicomplexan parasites (24, 33). Previously described inhibitors of Complex I and NDH2 were evaluated for inhibition of recombinant pfNDH2 activity. The percentage of ΔΨm-positive parasites was determined by fluorescence microscopy after the fixation of at least 150 parasites. HDQ treatment leads to a collapse of the ΔΨm. The infected cultures were stained immediately before drug treatment with DiOC6(3), and the fraction of vacuoles containing ΔΨm-positive parasites was determined by fluorescence microscopy of at least 100 vacuoles at the indicated time periods. These results are in agreement with data from previous biochemical analyses in which the level of O2 consumption of digitonin-treated extracellular T. gondii was shown to be increased in the presence of ADP and decreased in the presence of the Fo subunit inhibitor oligomycin (39). When intracellular parasites were treated with 10 nM of the well-established complex III inhibitor atovaquone (1), the intensity of the DiOC6(3) staining decreased gradually over time, leading to a complete depolarization of the ΔΨm within 40 min in >60% of the parasites (Fig. A conventional function of the T. gondii FoF1-ATPase in coupling the proton gradient with ATP synthesis is consistent with our observation that (i) an HDQ-mediated depolarization of the inner mitochondrial membrane leads to a ∼30% reduction of the ATP level within 1 h and to a ∼70% reduction within 24 h, (ii) treatment with the Fo subunit inhibitor oligomycin leads to a ∼70% reduction of the ATP level, and (iii) oligomycin leads to a stabilization of the ΔΨm in the presence of HDQ. We supplemented the culture medium with 250 μM uracil and determined the T. gondii growth rate in the presence of 100 nM HDQ. (A) HDQ treatment decreases the ΔΨm of intracellular parasites. Crossing points were plotted against the log of cDNA dilutions, and amplification efficiencies (E) were calculated from the slopes of the obtained lines by the following formula: E = 10-1/slope. 8C). (B) Kinetics showing the influence of 10 nM complex III inhibitor atovaquone (ATO) and 10 nM, 100 nM, and 1 μM NDH2 inhibitor HDQ on the ΔΨm of individual parasites. The close link between energy metabolism and stage conversion is furthermore supported by our observation that long-term treatment with HDQ for 3 days leads to an upregulation of mRNA transcripts for bradyzoite markers. It was thus unclear whether the T. gondii F1 subunit is indeed associated with a putative membrane-bound Fo subunit or if this enzyme is localized in the mitochondrial matrix, as proposed previously for Plasmodium (29), which is lacking all three parts of the Fo subunit. Freshly released parasites were harvested and immediately stained in suspension with Mitotracker solution (see above), supplemented with 25 mM HEPES (pH 7.4), for 45 min at 37°C. HDQ growth inhibition is not mediated by pyrimidine starvation. Images were captured by using an AxioCam MRm camera and processed with Axiovision 4.6.3 software. DiOC6(3) specifically stained the mitochondria of the parasites and also the host cell mitochondria, which appear to be less intensely stained than the T. gondii mitochondria. It is the first enzyme (complex I) of the mitochondrial electron transport chain.. NADH + CoQ + 5H + → NAD + + CoQH 2 + 4H +. Drug-untreated controls were stained in parallel at the same time points. Results are represented as means ± SD of data from a representative experiment (n = 2). ut, untreated. Parasites in the supernatant were harvested by centrifugation and resuspended in 250 μl of 1% FCS-DMEM. The parasite appears to respond in a situation of energy starvation with a differentiation into the dormant stage. After incubation at 37°C for 30 min, samples were ready for real-time ΔΨm monitoring. Real-time monitoring of the T. gondii ΔΨm.Mitotracker comprises a chloromethyl moiety that forms covalent bonds with the SH groups of mitochondrial membrane proteins, which allows the fixation of the stained samples before microscopic analysis. The bacterial NDH‐2 structure establishes a framework for the structure‐based design of small‐molecule inhibitors. At least 100 vacuoles were examined for each sample. These results are consistent with previously reported observations of the mitochondrial membrane depolarization effect of atovaquone in T. gondii (39) and thus demonstrate the suitability of DiOC6(3) staining for real-time ΔΨm imaging. This is in agreement with previously reported observations that inhibitors of the respiratory chain and of oxidative phosphorylation lead to an induction of bradyzoite differentiation (4, 37). Parasites were fixed with 4% paraformaldehyde-PBS for 10 min after washing, and a 10-μl aliquot of the parasite suspension was air dried on a glass slide and mounted with Moviol. 4B). The mode of action of HDQ in T. gondii is thus an inhibition of oxidative phosphorylation. The depolarization kinetics of 10 nM HDQ were similar to those of 10 nM atovaquone. 1A and B) for the last 6 h. In the remaining parasites, which were classified as being ΔΨm positive, the intensity of the staining appeared to be less than that for the untreated controls. Immunofluorescence assay.Samples were first fixed with 4% paraformaldehyde-PBS for 10 min and then permeabilized with 0.25% Triton X-100-PBS for another 15 min. One hundred microliters of the parasite suspension, containing 4 × 106 parasites, was mixed thoroughly with the same volume of BacTiter-Glo reagent and incubated at room temperature for 5 min. Inhibition of membrane-associated T. gondii FoF1-ATPase attenuates HDQ-mediated ΔΨm depolarization. At 24 h, 48 h, and 72 h postinfection, living samples were stained with DiOC6(3) and analyzed by immunofluorescence microscopy, followed by fixation and BAG1 and Dolichos biflorus lectin staining. (BO 1557/3-1). Type II NADH dehydrogenase inhibitor 1-hydroxy-2-dodecyl-4(1H)quinolone leads to collapse of mitochondrial inner-membrane potential and ATP depletion in Toxoplasma gondii. Lectin staining was performed with the same procedures by using a fluorescein isothiocyanate-conjugated lectin from Dolichos biflorus (1:300; Sigma). More than 100 extracellular parasites were analyzed for each sample. The ATP level of the harvested parasites was determined using a luminescence assay, and the obtained values were normalized for parasite numbers. HFFs were infected with tachyzoites and cultivated in the presence of 100 nM and 1 μM HDQ for 72 h. Enolase 1 and bag1 mRNA transcripts were determined by real-time PCR. One consequence of inhibiting Ndh is an increase in the NADH/NAD + ratio toward a higher reducing potential. Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 1535-9778; Online ISSN: 1535-9786, Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany, Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1. The kinetics revealed that HDQ leads to a gradual decrease of the parasite's total ATP level, resulting in a ∼30% reduction after 1 h and a ∼70% reduction after 24 h (Fig. Subsequently, the lysates were separated after centrifugation at 13,000 × g for 45 min. Antimycins. For ΔΨm detection of intracellular parasites, infected HFFs in a 24-well plate were incubated with staining solution (1 ml per well) at 37°C for 45 min. In order to determine the kinetics of an HDQ-mediated ΔΨm collapse in intracellular T. gondii, we first searched for a ΔΨm-sensitive dye that allows real-time imaging of the T. gondii ΔΨm. Mitochondrial localization of myc-tagged ATPase-β was confirmed by colocalization with Mitotracker fluorescence. The pH shift medium (pH 8.3) was exchanged daily to maintain a constant pH. Results are expressed as means ± SD of data from duplicate wells from a representative experiment (n = 3). The percentage of ΔΨm-positive parasites was determined from pictures taken by fluorescence microscopy after a 15- to 25-min incubation period at 37°C. 143B/206 cells, which lack mitochondrial DNA (20), and the parental 143B cell line were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 μg/ml), 1% glutamine, sodium pyruvate (110 μg/ml), and uridine (50 μg/ml). This observation clearly indicates that HDQ acts as an ETC inhibitor upstream of the atovaquone target, which is complex III, at the level of ubiquinone reduction. It is conceivable that a reserve energy system of limited capacity contributes to a stable ATP amount within the first minutes after the inhibition of oxidative phosphorylation. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. We do not retain these email addresses. DiOC6(3)-stained intracellular parasites were digitonin permeabilized and treated with 1 μM HDQ (A) or 1 μM atovaquone (ATO) (B) either alone or in combination with 10 mM malate (MAL); 10 mM succinate (SUC); 10 mM dihydroorotate (DHO); 1 mM glycerol-3-phosphate (G-3-P); 10 mM oxaloacetate (OAA); a mixture of malate, succinate, dihydroorotate, and glycerol-3-phosphate (SUB); and 0.2 mM TMPD-1.5 mM ascorbate (TMPD/ASC). Relative ATP levels were normalized for parasite numbers. However, the parasite appears to possess an unusual Fo subunit, since from the three proteins (Fo-a, Fo-b, and Fo-c) that typically form the Fo subunit, obvious homologues for Fo-a and Fo-b are lacking (23). ^, P < 0.002; *, P < 0.005; **, P < 0.03; ***, P < 0.02; #, P < 0.01; ##, P < 0.001 (determined by a Student's t test) (A). The only high-affinity NDH2 inhibitors described so far are 1-hydroxy-2-alkyl-4(1H)quinolones with long alkyl-site chains, for example, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) (C 12), which possesses structural similarity to ubiquinone. This article provides an updated overview of the plethora of complex I inhibitors. 8A). Determination of the intracellular ATP level.The parasite ATP level was determined by using the BacTiter-Glo microbial cell viability assay (Promega). We use cookies to help provide and enhance our service and tailor content and ads. β-Tubulin mRNA levels were used for the normalization of enolase 1 and bag1 transcript levels in HDQ-treated and -untreated samples. A likely candidate for such an energy buffer system is the adenylate kinase reaction, which converts two ADP molecules into ATP and AMP in a reversible reaction. Complex I functions in the transfer of electrons from NADH to the respiratory chain. Determination of the intracellular ATP level. (B) Comparison of the Mitotracker staining patterns from a sample in which the 6-h HDQ treatment period was started 16 h postinfection (top) to those from an untreated control (bottom). NDH2s can occur in two topological orientations with respect to the inner mitochondrial membrane. The PCR fragment was cloned into pCR4.0-TOPO (Invitrogen), and the DNA was sequenced. In contrast to Plasmodium, T. gondii possesses a pyrimidine salvage pathway, and parasites deficient in de novo pyrimidine synthesis can be rescued with high uracil concentrations (19). HDQ was kindly provided by W. Oettmeier (University of Bochum). A. Adessi, R. De Philippis, in Encyclopedia of Biological Chemistry (Second Edition), 2013. Substrates for ubiquinone-reducing enzymes lead to ΔΨm stabilization. Myc-tagged TgATP-β of stably transfected parasites was targeted to the mitochondrion, as shown by the colocalization with the Mitotracker signal (Fig. Instead, P. falciparum expresses one isoform (2) and T. gondii expresses two isoforms (22) of so-called “alternative” or type II NADH dehydrogenases (NDH2s). (C) Quantification of mitochondrial membrane depolarization kinetics after treatment with 10 nM, 100 nM, and 1 μM HDQ; 10 nM atovaquone (ATV); and a combination of 10 nM HDQ and 10 nM atovaquone. After fractionation of the T. gondii lysate, ATPase-β was found exclusively in the membrane fraction and was absent in the soluble fraction, suggesting that T. gondii possesses a typical, membrane-associated mitochondrial F1-ATPase (Fig. Internal enzymes are facing with their active site toward the mitochondrial matrix and use mitochondrial NAD(P)H as the electron donor, while external enzymes use cytosolic NAD(P)H. Up to now, the orientation of the apicomplexan isoforms is unknown. The addition of succinate, dihydroorotate, glycerol-3-phosphate, or malate to digitonin-permeabilized cells stabilized the ΔΨm in the presence of HDQ, whereas these substrates did not influence atovaquone-mediated depolarization. S.S.L. The HDQ-mediated depolarization of the ΔΨm occurs within minutes, while the onset of the ATP decrease started with a delay of ∼30 min. In brief, tachyzoites were freshly released by syringe passage, and 3 × 104 parasites were inoculated onto a confluent HFF monolayer grown on a 24-well imaging plate. Required for proper complex I assembly (PubMed: 28671271). By continuing you agree to the use of cookies. Enter multiple addresses on separate lines or separate them with commas. T. gondii cultivation, in vitro stage conversion, and cell lines.Tachyzoites were propagated in human foreskin fibroblasts (HFFs) as previously described (30). In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) in T. gondii. Significant inhibition of NADH-DH was seen following incubation of brain slices with very low concentration of L-BOAA (0.1 pM). The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. This study has been supported by a grant from the Deutsche Forschungsgemeinschaft to W.B. Scale bars, 5 μm. ATP levels from each sample were measured as duplicates in a flat-bottomed 96-well plate. HDQ growth inhibition is not mediated by pyrimidine starvation.HDQ possesses structural similarities to ubiquinol and is believed to interact with the ubiquinol binding site of type II NADH dehydrogenases (13). Results are expressed as means ± SD of data from duplicate samples from a representative experiment. *, P < 0.003 (determined by a Student's t test) (B). The frequency of Mitotracker-positive parasites in the BAG1-positive population was less than 10%, compared to ∼80% Mitotracker-positive parasites in freshly released extracellular tachyzoites (Fig. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. Mitotracker staining was applied for the released parasites, followed by fixation, permeabilization, and BAG1 staining. The pellet fraction was also resuspended in the same buffer. *, P < 0.002; **, P < 0.05 (determined by a Student's t test). NAD’ is an effective competitive inhibitor of the reaction (K, = 20 PM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the- DCIP reductase assay. HDQ induces bradyzoite differentiation.HDQ was shown to effectively inhibit parasite replication (31). Thank you for sharing this Eukaryotic Cell article. In drug-untreated controls, more than 80% of the parasites displayed a strong ΔΨm, confirming that the digitonin treatment itself did not affect the ΔΨm (Fig. This respiratory entry point affects the amount of ATP produced per NADH/O 2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. We demonstrate in this study that the treatment of intracellular tachyzoites with HDQ, a quinolone-like compound that was previously shown to inhibit TgNDH2-I (22), leads to a fast, dose-dependent collapse of the ΔΨm and subsequently to a decrease in the intracellular ATP level. A recent study reported that HDQ inhibits DHODH of P. falciparum (10). The Fo subunit is typically composed of three proteins (Fo-a, Fo-b, and Fo-c); however, the T. gondii genome has no obvious homologues for the Fo-a and Fo-b proteins (23). Plasmids.Plasmid tub-S9-RFP/sag-CAT was constructed by replacing the FNR fragment from BglII/AvrII-digested ptub-FNR-RFP/sag CAT (36) with a BglII/AvrII S9 fragment from ptub-S9-GFP/sag-CAT (8). Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction. Results are expressed as means ± SD of data from duplicate samples from a representative experiment (n = 2). Stable transgenic parasite lines expressing TgATP-β and S9-red fluorescent protein (RFP) were selected with 1 μM pyrimethamine and 20 μM chloramphenicol, respectively. The NADH dehydrogenase Ndh has no homolog in humans, so Mtb Ndh inhibitors could be developed with limited toxicity risk. We demonstrate in this study that HDQ treatment in nanomolar concentrations leads to a depolarization of the T. gondii ΔΨm within minutes. In contrast to yeast or plants, where the presence of exogenous NADH dehydrogenases is not a matter of discussion, the existence of an enzyme of that kind in animals, namely in heart, is a source of debate and few studies have been made so … Since physiological substrate concentrations in non-digitonin-treated cells are not sufficient to compensate for the HDQ-mediated ΔΨm depolarization, a major role of NDH2 activity in providing the ETC with reduction equivalents can be assumed under the assumption that HDQ is not affecting any other targets of the ETC. Incubation of sagittal slices of mouse brain with L-BOAA resulted in dose and time-dependent inhibition of mitochondrial NADH-dehydrogenase (NADH-DH). DiOC6(3) was found to be the most suitable dye for this purpose, since it resulted in an intense and specific mitochondrial staining pattern, which perfectly colocalized with a mitochondrially targeted RFP (S9-RFP) (Fig. This dormant stage is likely to be less dependent on the ΔΨm, since ΔΨm-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Since a reduction in the level of parasite replication in T. gondii is often associated with stage differentiation (4), we investigated whether HDQ treatment leads to bradyzoite differentiation. The subsequent lack of oxidative phosphorylation leads to a ∼70% reduction of the intracellular ATP level within 24 h. This suggests an indispensable role of NDH2 activity in the maintenance of the ΔΨm and in energy metabolism in the tachyzoite stage. A common response to the inhibition of oxidative phosphorylation, which might also occur in T. gondii, is an increased metabolic flux through other energy-generating pathways, like glycolysis. When mock controls were stained with Mitotracker at different time points from 7 to 32 h postinfection, 75 to 80% of all intracellular parasites showed the typical intense staining of the single T. gondii mitochondrion (Fig. (B) Kinetics showing the decrease of DiOC6(3)-positive vacuoles and the increase of BAG1-positive and lectin-positive vacuoles during bradyzoite differentiation. 2A). A combination of 10 nM HDQ with 10 nM atovaquone resulted in a significantly faster collapse of the ΔΨm than treatment with 10 nM of the individual drugs, which is in agreement with a synergistic mode of action between HDQ and atovaquone (31). The ΔΨm of living, intracellular parasites was monitored by DiOC6(3) staining. The … (A) Fluorescence images from a 72-h sample showing a DiOC6(3)-negative/BAG1-positive/lectin-positive vacuole (top) and a vacuole that is weakly DiOC6(3) positive (bottom). inhibitor(13)] and(iii) mitochondrialNADHdehydrogenase [NADH:(acceptor)oxidoreductase, EC 1.6.99.3] energy-con- servingsite 1 of theelectron transportsystem, the targetofro- Members of the NADH dehydrogenase family and analogues are commonly systematically named using the format NADH:acceptor oxidoreductase. This indicates either a high degree of divergence in the lacking parts or an unusual composition of the Fo subunit. (B) Parasites expressing myc-tagged ATPase-β were fractionated into a soluble fraction (S) and a membranous fraction (P). HFFs were infected with RH strain parasites and, after 24 h, treated with 1 μM HDQ for the indicated time periods or with 1 μM oligomycin for 24 h. Intracellular parasites were released by syringe passages, and the ATP level was quantified as photons per second (CPS) in a luminescence assay. 4C), suggesting that the HDQ-mediated ΔΨm depolarization can be attenuated by preventing protons from reentering the mitochondrial matrix. The dye could be used for up to 1.5 h in real-time imaging, with a maximum of 10 to 15 exposures taken within this time. Their suitability as a drug target in Plasmodium is controversial and has been the subject of discussion (16, 17, 38). The highest number of ΔΨm-positive parasites in HDQ-treated cultures was achieved when all four substrates were added simultaneously. Cells were treated for this purpose with 2 μM digitonin, a concentration which was shown previously to selectively permeabilize the parasite's plasma membrane for metabolites without disturbing the function of the respiratory chain (39). We compared the cationic fluorophores TMRE (tetramethylrhodamine ethyl ester), JC-1 (3,3′-tetraethylbenzimidazolcarbocyanine iodine), and DiOC6(3) for their abilities to specifically stain the mitochondrion of intracellular tachyzoites with no or only weak staining of the plasma membrane. The observed synergism of 10 nM HDQ in combination with 10 nM atovaquone on ΔΨm depolarization is in agreement with the synergism of these drugs to inhibit parasite replication in vitro (31). The frequency of ΔΨm-positive parasites decreases during bradyzoite differentiation. Oligomycin treatment resulted in a strong increase in levels of ΔΨm-positive parasites (Fig. Other mitochondrial pathways for mitochondrial acetyl coenzyme A generation, such as the 2-methylcitrate cycle, are currently under investigation (33). Afterwards, a centrifugation step at 34 × g was performed in order to remove host cell debris. NADH dehydrogenase; inhibitor; carvedilol. Detection of ΔΨm in T. gondii.The T. gondii ΔΨm was monitored after staining with the fluorophore Mitotracker or DiOC6(3) (3,3′-dihexyloxacarbocyanine iodine; Invitrogen). Due to their absence in the mammalian host, NDH2s were proposed to be promising drug targets against Mycobacterium tuberculosis (40). Digitonin permeabilization.Substrate supplementation was performed on digitonin-permeabilized intracellular parasites using a modification of a previously described protocol that was established for extracellular parasites (39). It blocks NADH dehydrogenase and Coenzyme.Q; 4. The inner mitochondrial membrane is impermeable to protons, and the only possibility for protons to reenter the mitochondrial matrix is through the Fo proton channel, which can be inhibited by oligomycin. To exclude this possibility, we verified the results using freshly harvested extracellular bradyzoites, which were released from their parasitophorous vacuoles and the emerging cyst wall by extensive syringe passage. (C) Extracellular parasites were obtained after syringe passage from a 72-h bradyzoite culture and a 24-h tachyzoite culture. 7), indicating that HDQ leads to a moderate induction of bradyzoite differentiation. The inhibitory effect of rhein against NADH dehydrogenase-2 activity was non-competitive with ferricyanide [K 3 Fe(CN) 6] with a K i value of 3.5-4.5 µm. Scale bars, 5 μm. Scale bars, 5 μm. Together, these results suggest that HDQ possesses a different mode of action compared to that of atovaquone and inhibits the ETC upstream of complex III at the level of ubiquinone reduction. Oxalacetate was used as a control and did not result in an increased frequency of ΔΨm-positive parasites. NADH Dehydrogenase (Ubiquinone) Complex I is the first enzyme complex in the respiratory chain, and it accepts electrons from NADH+H+ derived from fat, carbohydrate, and amino acids to create an electrochemical gradient across the inner mitochondrial membrane. The effect of oligomycin-mediated FoF1-ATPase inhibition was further investigated by using 143B/260 cells as host cells for T. gondii. To detect bradyzoites, a bradyzoite-specific anti-BAG1 MAb (7E5) (1:500) (3) and a Cy3-conjugated anti-mouse IgG antibody (1:300; Dianova) were used instead. Infected HFFs were washed once with PBS supplemented with protease inhibitors (protease inhibitor cocktail; Roche) to remove extracellular parasites. Reactive proteins were detected by alkaline phosphatase staining solution containing 0.05% bromo-4-chloro-3-indolyl phosphate and 0.5% nitroblue tetrazolium (Sigma) as substrates. However, the overall contribution of oxidative phosphorylation to energy production in relation to other ATP-generating pathways has not been satisfactorily clarified for intracellular T. gondii so far. A flavoprotein and iron sulfur-containing oxidoreductase that catalyzes the oxidation of NADH to NAD. PCR amplification was carried out using the following parameters: 10 min at 95°C followed by 40 cycles of denaturation (95°C for 10 s), annealing (60°C for 5 s), and extension (72°C for 15 s). From: Mitochondrial Case Studies, 2016. While both of the inhibitors will decrease the activity of NADH dehydrogenase significantly (both suppresses enzyme activity to about 40% of the control, uninhibited enzyme), at limiting substrate concentrations, Mg2+will inhibit the enzyme more efficiently than EDTA. The rate of bradyzoite differentiation was determined using the same samples after fixation and staining with a bradyzoite-specific anti-BAG1 antibody, which detects a cytosolic small heat shock protein, and with a fluorescein isothiocyanate-conjugated Dolichos biflorus lectin, which detects a carbohydrate structure on the emerging cyst wall (Fig.
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