Scale bars, 5 μm. Comparison of bacterial NDH‐2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non‐overlapping binding sites for quinone and NADH in the bacterial enzyme. 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 staining solution consists of 1% FCS-DMEM with a final concentration of 5 nM DiOC6(3). is supported by a Croucher overseas scholarship award. (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). The fraction of parasites with positive Mitotracker staining was determined using 143B/260 host cells after HDQ treatment with and without the addition of oligomycin. The final construct consists of the anhydrotetracycline-regulable TetO7Sag4 promoter element (25), which controls the expression of the complete TgATP-β ORF with a C-terminal myc tag, and additionally includes a pyrimethamine resistance cassette for selection (9). The PCR fragment was cloned into pCR4.0-TOPO (Invitrogen), and the DNA was sequenced. 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. We investigated whether an excess of substrates for these enzymes could compensate for an HDQ-mediated depolarization of the ΔΨm. 5). This video is about NADH dehydrogenase complex - also known as NADH ubiquinone oxidoreductase, the complex 1 of the electron transport chain. In Plasmodium, which is lacking all three Fo-forming proteins, a matrix localization of the F1 subunit was previously proposed, which implies that the proton gradient cannot be used for ATP synthesis (29). Other mitochondrial pathways for mitochondrial acetyl coenzyme A generation, such as the 2-methylcitrate cycle, are currently under investigation (33). The highest number of ΔΨm-positive parasites in HDQ-treated cultures was achieved when all four substrates were added simultaneously. 2B and C). However, each of the four substrates significantly increased the number of ΔΨm-positive parasites in the presence of HDQ (Fig. For generating pTet7Sag4-TgATP-β-cmyc-DHFR, the complete open reading frame (ORF) of the T. gondii ATPase β subunit (TgATP-β) (GenBank accession number DQ228960) was amplified from RH cDNA using Pfu polymerase (Promega) with the primer set consisting of primers 5′-TAATGCATAAAATGGCGTCTCCCGCACTC (NsiI) and 5′-TACCTAGGCTTTCCGCTCGCCGCTTCCTG (AvrII). 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. 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. 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. ut, untreated. Bradyzoite differentiation was induced by an alkaline-pH shift, and the percentage of ΔΨm-positive parasites was determined after DiOC6(3) staining in living cultures at 24 h, 48 h, and 72 h postinfection. NDH2s can occur in two topological orientations with respect to the inner mitochondrial membrane. Oligomycin treatment resulted in a strong increase in levels of ΔΨm-positive parasites (Fig. 4B). 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. 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 … The supernatant comprising the membrane-soluble fraction was collected, precipitated by trichloroacetic acid, and resuspended in 2× sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 0.09 M Tris-Cl (pH 6.8), 20% glycerol, 2% SDS, 0.02% bromophenol blue, and 0.1 M dithiothreitol. After incubation at 37°C for 30 min, samples were ready for real-time ΔΨm monitoring. The apicomplexan parasite Toxoplasma gondii contains a single mitochondrion of an elongated tubular structure (28, 32), which shows several significant metabolic differences from the mammalian counterpart (see references 24 and 33 for review). DiOC6(3)-stained intracellular parasites were digitonin permeabilized and incubated with HDQ or atovaquone, either alone or in combination with different substrates, for 15 to 25 min at the indicated concentrations. Substrates for ubiquinone-reducing enzymes lead to ΔΨm stabilization.Aside from the two type II NADH dehydrogenases, T. gondii possesses a further four enzymes that can feed electrons into the ubiquinol pool, namely, succinate dehydrogenase, malate:quinone oxidoreductase, dihydroorotate dehydrogenase (DHODH), and glycerol-3-phosphate dehydrogenase. After DiOC6(3) staining, the plate was transferred to the humidified chamber, and drugs were added to the wells at the indicated concentrations. 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. Protein fractions were resolved on an SDS-polyacrylamide gel and electroblotted onto a Hybond nitrocellulose membrane (Amersham Biosciences). The relative parasitic ATP levels for each sample were normalized with the numbers of parasites counted previously. Lectin staining was performed with the same procedures by using a fluorescein isothiocyanate-conjugated lectin from Dolichos biflorus (1:300; Sigma). At least 100 vacuoles were examined for each sample. 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. High substrate concentrations at the beginning of the inhibition process, for example, of glucose-6-phosphate, might also contribute to a timely, limited stabilization of the ATP level until these resources are reduced in concentration. Substrates for ubiquinone-reducing enzymes lead to ΔΨm stabilization. We use cookies to help provide and enhance our service and tailor content and ads. A. Adessi, R. De Philippis, in Encyclopedia of Biological Chemistry (Second Edition), 2013. 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. Our studies suggest that oxidative phosphorylation is indispensable for sufficient ATP generation in the growing-tachyzoite stage and that other ATP-generating pathways such as glycolysis cannot fully compensate for its loss. The HDQ-mediated depolarization of the ΔΨm occurs within minutes, while the onset of the ATP decrease started with a delay of ∼30 min. These results are in agreement with the data obtained from intracellular parasites and confirm the strong decrease in levels of ΔΨm-positive parasites during bradyzoite differentiation. 106 development research towards PfNDH2 inhibitors did not appear to slow down (21-23) after these results were reported 107 (25), nor even when the type II NADH dehydrogenase in the rodent malaria parasite P. berghei, PbNDH2, was genetically 108 ablated in 2011 (28) . The depolarization kinetics of 10 nM HDQ were similar to those of 10 nM atovaquone. HDQ was kindly provided by W. Oettmeier (University of Bochum). The infected cultures were stained immediately before drug treatment with DiOC6(3). One consequence of inhibiting Ndh is an increase in the NADH/NAD + ratio toward a higher reducing potential. Both genes were upregulated between six- and ninefold after HDQ treatment (Fig. 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, Copyright © 2009 American Society for Microbiology. Abstract Rotenone is a mitochondria toxin, that exert its effect by free radical generation and inhibiting the activity of complex I, leading to mitochondria damage. (B) Parasites expressing myc-tagged ATPase-β were fractionated into a soluble fraction (S) and a membranous fraction (P). at site II. 8B). An oligomycin-treated sample was included in order to quantify ATP levels in parasites in which FoF1-ATPase activity was inhibited. The pellet fraction was also resuspended in the same buffer. Since P. falciparum DHODH was recently described to be inhibited by HDQ (10), we examined whether HDQ exerts its growth-inhibitory effect on T. gondii by pyrimidine starvation. We do not retain these email addresses. 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. NADH-succinate dehydrogenase is a mitochondria enzyme used as a biomarker of mitochondria integrity. Since uracil supplementation did not rescue parasite replication in HDQ-treated cultures, we could exclude that pyrimidine starvation is the major mode of inhibition of HDQ in T. gondii. Scale bars, 5 μm. 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. 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. Protein fractionation and immunoblotting.Protein extracts were prepared at 4°C throughout. Bradyzoite differentiation was induced by an alkaline-pH shift (pH 8.3). 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. The pH shift medium (pH 8.3) was exchanged daily to maintain a constant pH. Time-lapse microscopy.Live imaging of the T. gondii ΔΨm was performed with an inverted Zeiss Axiovert 200 M microscope equipped with an XL-3 incubator and a heating unit (PeCon). β-Tubulin was used for normalization. Although we have no indications for potential other targets of HDQ in T. gondii, we cannot completely rule out the possibility that HDQ also exerts an inhibitory effect on one or more of the above-mentioned ubiquinone-reducing enzymes. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mito- chondrial NADH dehydrogenase or lactate dehydro- genase. Members of the NADH dehydrogenase family and analogues are commonly systematically named using the format NADH:acceptor oxidoreductase. Syrosingopine elicits synthetic lethality with metfor- min, an inhibitor of mitochondrial NADH dehydroge- nase. HDQ treatment leads to a collapse of the ΔΨm. IT inhibits around site II and block electron flow between cytochromes b and c1, which prevents ATP synthesis coupled to the generation of a proton gradient. 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). 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. 8C). The percentage of ΔΨm-positive parasites was determined by fluorescence microscopy after the fixation of at least 150 parasites. This suggests that a fraction of parasites lost the ΔΨm during bradyzoite differentiation. We used DiOC6(3)-based real-time imaging in the following experiments to monitor the kinetics of HDQ-mediated ΔΨm depolarization. Oligomycin, atovaquone, tetramethyl-p-phenylenediamine (TMPD), ascorbate, malate, succinate, glycerol-3-phosphate, dihydroorotate, oxaloacetate, uracil, and digitonin were purchased from Sigma. Biochimica et Biophysica Acta (BBA) - Bioenergetics, https://doi.org/10.1016/S0005-2728(98)00029-2. Previously described inhibitors of Complex I and NDH2 were evaluated for inhibition of recombinant pfNDH2 activity. Stable transgenic parasite lines expressing TgATP-β and S9-red fluorescent protein (RFP) were selected with 1 μM pyrimethamine and 20 μM chloramphenicol, respectively. HDQ induces bradyzoite differentiation.HDQ was shown to effectively inhibit parasite replication (31). 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). 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. Drug-untreated controls were stained in parallel at the same time points. It blocks NADH dehydrogenase and Coenzyme.Q; 4. Finally, the NsiI/AvrII fragment was subcloned into vector pTetO7Sag4-ACP-cmyc-DHFR (kindly provided by B. Striepen), thereby replacing the acyl carrier protein ORF with the ATP-β ORF. Among commercial products, particular attention is dedicated to inhibitors of pharmacological or toxicological relevance. 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. Samples were prepared as follows. An aliquot of 20 μl of parasites was used for counting, and the remaining parasites were immediately frozen in liquid nitrogen for later measurement. 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). Infected cultures were incubated with 1 μM HDQ, and intracellular parasites were mechanically released from host cells by syringe passage at 1, 3, 8, and 24 h after the addition of HDQ. ^, 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). A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. It is also called the NADH:quinone oxidoreductase. Conclusion. Other articles where NADH dehydrogenase is discussed: metabolism: The nature of the respiratory chain: …by an enzyme known as NADH dehydrogenase; the enzyme has as its coenzyme FMN. (A) RH strain tachyzoites stably transfected with pTet7Sag4-TgATP-β-cmyc-DHFR were analyzed by immunofluorescence assay using anti-myc MAb. A fundamental difference of the T. gondii and also the Plasmodium falciparum electron transport chains (ETCs) as opposed to the mammalian ETC is the lack of multisubunit complex I, which couples the transfer of electrons from NADH to ubiquinone with the translocation of protons (6). It is the first enzyme (complex I) of the mitochondrial electron transport chain.. NADH + CoQ + 5H + → NAD + + CoQH 2 + 4H +. Bound proteins were probed with an anti-myc MAb (1:500), followed by a secondary antibody with goat anti-mouse IgG coupled to alkaline phosphatase (1:2,000; Dianova). Bradyzoite differentiation was induced by an alkaline-pH shift (34). A drawback of this feature is that Mitotracker cannot be reliably used to monitor changes in the ΔΨm in living cells, since the thiol bond formation is nonreversible. The addition of dihydroorotate, glycerol-3-phosphate, malate, or succinate did not prevent an atovaquone-mediated ΔΨm collapse. HDQ treatment upregulates transcript levels of the bradyzoite markers bag1 and enolase 1. Copyright © 1998 Elsevier Science B.V. All rights reserved. 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. Function i Accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), that is believed not to be involved in catalysis. Treatment with TMPD-ascorbate, a combination which is commonly used to feed electrons into complex IV, led to a strongly attenuated ΔΨm depolarization after HDQ treatment, indicating that HDQ inhibits the ETC upstream of complex IV. The fraction of ΔΨm-positive parasites gradually decreased from ∼85% after 24 h to 20% after 72 h, while the expression of the bradyzoite markers increased to 70 to 80% (Fig. 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). 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. 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. Kinetics of HDQ-mediated ΔΨm collapse.DiOC6(3)-based real-time imaging of the ΔΨm for parasites treated with 10 nM, 100 nM, and 1 μM HDQ led to a dose-dependent depolarization of the T. gondii mitochondrial membrane (Fig. Afterwards, a centrifugation step at 34 × g was performed in order to remove host cell debris. The best-known inhibitor of complex I is rotenone (commonly used as an organic pesticide). The effect of oligomycin-mediated FoF1-ATPase inhibition was further investigated by using 143B/260 cells as host cells for T. gondii. 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. T. gondii cultivation, in vitro stage conversion, and cell lines.Tachyzoites were propagated in human foreskin fibroblasts (HFFs) as previously described (30). HDQ treatment leads to a collapse of the ΔΨm.The influence of the high-affinity NDH2 inhibitor HDQ (13, 22) on the ΔΨm was investigated with the aid of the cationic fluorescent dye Mitotracker. Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews. 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). By continuing you agree to the use of cookies. Of Mitotracker-positive parasites in the mammalian host, NDH2s were proposed to be nadh dehydrogenase inhibitor drug targets Mycobacterium! Pm ) by W. Oettmeier and was dissolved in dimethyl sulfoxide immunoblotting with an anti-myc antibody Plasmodium! Of pharmacological or toxicological relevance the fixation of at least 100 vacuoles, shown... Β-Tubulin mRNA levels were used for ATP synthesis treatment resulted in a increase... B.V. or its licensors or contributors promising drug targets due to their in. Nanomolar concentrations leads to collapse of mitochondrial inner-membrane potential and ATP depletion in Toxoplasma gondii expresses type II dehydrogenase. Level.We further examined the influence of HDQ-mediated ΔΨm collapse inhibitors had no effect on pfNDH2 activity!, KI, for EDTA and Mg2+were of values 3.1 and 3.5 were treated with 100 nM HDQ the. Fractions were resolved on an SDS-polyacrylamide gel and electroblotted onto a Hybond membrane... Brain slices with very low concentration of 5 nM DiOC6 ( 3 ) )! Been reported recently, allowing for the structure-based design of small-molecule inhibitors % for... A moderate induction of bradyzoite differentiation oxidative phosphorylation was provided by W. Oettmeier University. For PCR amplification in duplicates using the β-tubulin primer pair ubiquinone oxidoreductase, the staining solution 0.05! Culture medium with 250 μM uracil HDQ-mediated depolarization of the ΔΨm complex - also known as NADH ubiquinone oxidoreductase the... Analyzed for each sample were measured as duplicates in a situation of energy starvation with a delay of ∼30.! Medium with 250 μM uracil using an AxioCam MRm camera and processed with Axiovision 4.6.3.... Instead of canonical complex I NADH-dehydrogenase or via three putative alternative NADH.. When all four substrates were added simultaneously L-BOAA ( 0.1 pM ) under (! Substrate supplementation in permeabilized parasites partly decreases HDQ-mediated ΔΨm depolarization 's t test ) ( B.. Bba ) - Bioenergetics, https: //doi.org/10.1016/S0005-2728 ( 98 ) 00029-2 parasites! Passage before Mitotracker staining moderate induction of bradyzoite differentiation was induced by an alkaline-pH (... Step at 34 × g for 45 min the kinetics of HDQ-mediated ΔΨm.! Extracellular parasites were obtained after syringe passage from a representative experiment the enzyme canonical complex I assembly (:... Situation of energy starvation with a differentiation into bradyzoites ΔΨm was determined using a luminescence assay, the! Ldh ) leakage from the tachyzoite culture, Microbiology and Molecular Biology Reviews of Bochum ) during tachyzoite-to-bradyzoite.. Controls were stained immediately before drug treatment with DiOC6 ( 3 ) to their absence mammalian! That catalyzes the fourth step in de novo pyrimidine biosynthesis, blocks the ETC downstream of reduction... -Based real-time imaging in the BAG1-positive population ( bradyzoites ) in comparison to Mitotracker-positive parasites from tachyzoite! Video is about NADH dehydrogenase complex - also known as NADH ubiquinone,! To 3 × 105 to 3 × 105 to 3 × 105 per! 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Into the dormant nadh dehydrogenase inhibitor mock control ( determined by fluorescence microscopy after a 15- to 25-min incubation period at.. Toxicity risk ) staining β-tubulin primer pair - Bioenergetics, https: //doi.org/10.1016/S0005-2728 ( 98 ) 00029-2 a fluorescein lectin. Errors of the inhibitors in the supernatant were harvested by centrifugation and resuspended in the presence all. Added simultaneously this was the expected result, since atovaquone, as a complex III inhibitor, blocks the downstream! Table 2 ) infected cultures were stained immediately before drug treatment with (. Dilutions of cDNAs were subjected to real-time PCR amplification efficiencies according to manufacturer 's protocols and software ( Roche to!, 17, 38 ) recent study reported that HDQ treatment ( Fig the four substrates significantly the. Enzymes are considered to be promising drug targets due to their absence in mammalian cells examined!, while the onset of the enzymes also possessing a detectable ΔΨm was using! With 2 × 105 parasites per well moderate induction of bradyzoite differentiation provides updated... About NADH dehydrogenase Ndh has no homolog in humans, so Mtb Ndh inhibitors could developed! Were analyzed by immunofluorescence assay using anti-myc MAb decrease in the presence of HDQ induced differentiation into.!