Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine onTrypanosoma cruzi

Summary The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes ofTrypanosoma cruzi, at concentrations lower than 100 M, and motility and infectivity of the bloodstream trypomastigote form at 200 M. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 M TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.     

Effect of trifluoperazine and calcium ions on gregarine gliding

Summary The drug trifluoperazine (TFP) inhibits the gliding motility of gregarine protozoans. This suggests that the Ca⁺⁺ binding protein, calmodulin, plays a role in the motility process. However the presence of extracellular Ca⁺⁺ was not required for gliding to occur.Acknowledgments. We should like to thank L. Cooper for technical assistance, Smith, Kline and French for the gift of trifluoperazine, and Terry Preston for valuable discussion.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production in Trypanosoma cruzi and Crithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis in Trypanosoma cruzi and stimulated O2 uptake and O2-. generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and O2-. generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained with Crithidia fasciculata despite the presence of catalase in this organism.     

Effect of tingenone, a quinonoid triterpene, on growth and macromolecule biosynthesis in Trypanosoma cruzi

Tingenone and horminone, two natural quinonoid substances, inhibited the in vitro growth of Trypanosoma cruzi, 30 microM drug concentration producing total inhibition of growth. Tingenone inhibited total uptake and incorporation of [3H]thymidine, [3H]uridine, L-[3H]leucine into parasite macromolecules. Other quinonoids assayed were either less effective (abruquinone A) or even quite inactive (visminone B and ferruginin B). Investigation of several mechanisms for the cytotoxic action of tingenone pointed to the interaction with DNA as the most likely factor involved. Tingenone also inhibited the growth of Crithidia fasciculata, but the drug was significantly less active on this organism than on T. cruzi.     

Hydrogen peroxide generation in Trypanosoma cruzi.

Homogenates from T. cruzi epimastigotes produced 3.4 pmoles H2O2/min 10(6) cells, as detected by the cytochrome c peroxidase assay. Addition of NADH or NADPH increased H2O2 production by a factor of 3 and 5, respectively. When supplemented with NADH and NADPH, the mitochondrial, microsomal and supernatant fractions produced H2O2, the soluble fraction and the mitochondrial membranes being apparently the main generators of H2O2. The epimastigote homogenates showed cyanide-sensitive superoxide dismutase activity, equivalent to 0.28 microgram bovine superoxide dismutase per mg homogenate protein.     

Geranylgeranylacetone protects human monocytes from mitochondrial membrane depolarization independently of Hsp70 expression

The anti-ulcer drug geranylgeranylacetone (GGA) has been shown to induce the expression of heat shock proteins (HSPs), in particular of Hsp70, in gastric and small intestine cells. In this study, we investigated whether GGA was able to induce Hsp70 in another cell type, human monocytes, which represent a well-established model of Hsp70 expression under oxidative stress. In these cells, GGA had no significant effect either on basal or tobacco smoke-induced Hsp70 expression. We further investigated the effects of GGA on mitochondria, a key organelle of oxidant-mediated cell injury and a putative target for GGA-mediated protection. GGA significantly increased basal mitochondrial membrane polarization and inhibited the decrease in mitochondrial membrane potential of human monocytes exposed to distinct sources of clinically relevant oxidants such as tobacco smoke and y-irradiation. Our results indicate that mitochondria are targets for GGA-mediated protection against oxidative stress in human monocytes, independently of Hsp70.     

Inducing effect of clofibrate on alkaline phosphatase and histidine-glyoxylate aminotransferase in rat liver

Summary The activity of plasma membrane alkaline phosphatase and both mitochondrial and peroxisomal histidineglyoxylate aminotransferase was significantly increased in the livers of male rats following treatment with the hypolipidemic drug clofibrate. Cycloheximide or puromycin administration to rats inhibited the effects of clofibrate.     

Effect of lipophilic cations on thiamine transport system in isolated rat hepatocytes

The effect of lipophilic cations such as triphenylmethylphosphonium, tetraphenylphosphonium and tetraphenylarsonium in addition to dibenzyldimethylammonium on thiamine transport in isolated rat hepatocytes was studied. Lipophilic cations at the concentration 10 microM almost completely inhibited thiamine uptake. Kinetic studies showed that these compounds were competitive inhibitors with a very high affinity. These results suggest that lipophilic cations in addition to quaternary ammonium compounds also share a common binding site for thiamine in isolated rat hepatocytes.     

Hydrogen peroxide and hydroxyl radical involvement in the activation of caspase-3 in chemically induced apoptosis of HL-60 cells

Apoptosis of HL-60 cells induced by actinomycin D, H7, or daunorubicin was shown to involve the activation of caspase-3-like protease, 2 h after the addition of these drugs, based on microassay of enzyme activity by high-performance liquid chromatography. Catalase and a spin trap, N-t-butyl--phenylnitrone, which effectively inhibited the apoptosis induced by these drugs, also inhibited the activation of caspase-3-like protease. These results suggest that hydrogen peroxide and the hydroxyl radical are common mediators of caspase-3 activation caused by these chemicals, with apparently different functional mechanisms. Based on mitochondrial activity determined by oxygen consumption, complexes I, II, and IV were inhibited by actinomycin D. H7 inhibited complexes I and IV, 1 and 1.5 h respectively, after the addition of the drug to HL-60 cells. Daunorubicin inhibited complex IV, 1.5 h after the addition of the drug to HL-60 cells. Inhibition of complex IV by actinomycin D, H7, and daunorubicin were almost fully restored by the addition of cytochrome c. The release to the cytosol of cytochrome c by these drugs was also demonstrated by Western blot analysis. Addition of catalase inhibited the depression of complex IV activity induced by actinomycin D and H7. These observations indicate a direct relationship between hydrogen peroxide and the release of cytochrome c during apoptosis caused by actinomycin D, H7, and daunorubicin. Received 24 November 2000; received after revision 2 January 2001; accepted 30 January 2001     

The effects of sodium and amiloride on the motility of the caudal epididymal spermatozoa of the rat

Summary The forward motility of the rat caudal epididymal spermatozoa has been studied in different Na⁺ concentrations. When spermatozoa were suspended in a completely Na⁺-free solution, the forward motility suffered a progressive fall and after 3 h was completely suppressed. This effect was fully reversible on resuspending the spermatozoa in a solution containing Na⁺. Amiloride caused a fall in motility and the effect was similar to that of Na⁺ removal. The inhibition by amiloride of the motility was concentration dependent and the dose response curve showed an IC₅₀-value of about 5×10^–5 M. The role of Na⁺ influx in the maintenance of sperm motility was discussed.This work was supported by the World Health Organization.The technical assistance of Mr C.M. Li and the gift of amiloride from Merck, Sharp and Dohme are gratefully acknowledged.     

New evidence on the robust identification of news shocks: Role of revisions in utilization‐adjusted TFP series and term structure data

Data revisions and selections of appropriate forwarding‐looking variables have a major impact on true identification of news shocks and quality of research findings derived from structural vector autoregression (SVAR) estimation. This paper revisits news shocks to identify the role of different vintages of total factor productivity (TFP) series and term structure of interest rates as major prognosticators of future economic growth. There is a growing strand of literature regarding the use of utilization‐adjusted TFP series, provided by Fernald (Federal Reserve Bank of San Francisco, Working Paper Series, 2014) for identification of news shocks. We reestimate Barsky and Sims' (Journal of Monetary Economics, 2011, 58, 273–289) empirical analysis by employing 2007 and 2015 vintages of TFP data. We find substantial quantitative as well as qualitative differences among impulse response functions when using 2007 and 2015 vintages of TFP data. Output and hours initially decline, followed by quick reversal of both variables. In sharp contrast to results achieved by the 2007 vintage of TFP data, results achieved by the 2015 vintage of TFP data depict that output and hours will increase in response to positive TFP shock. By including term structure data in our VAR specification, total surprise technology shock and news shock account for 97% and 92% of the forecast error variance in total TFP and total output respectively. We find that revisions in TFP series over time ultimately impact the conclusion regarding news shocks on business cycles. Our results support the notion that term structure data help in better identification of news shock as compared to other forward‐looking variables.     

Prolongation of the duration of motility and fertilizing ability of rainbow trout spermatozoa by the addition of theophylline to the dilution medium

After rainbow Trout sperm were diluted in 0.01 and 0.001 M theophylline media, motility and fertilizing ability were prolonged as compared to the control media without theophylline. However, the motility observed was lower and of a different type than normal motility. This suggests that other factors besides cyclic nucleotides intervene in the motility of Trout spermatozoa.     

Inhibition of xanthine oxidase by liquiritigenin and isoliquiritigenin isolated from Sinofranchetia chinensis

The methanol extract of the stem of Sinofranchetia inhibited the activity of xanthine oxidase in vitro. Bioassay-guided purification led to the isolation ofliquiritigenin and isoliquiritigenin as the main xanthine oxidase inhibitors. This inhibition of enzyme activity was found to be dose dependent, with an IC50 value of approximately 49.3 microM for liquiritigenin and 55.8 microM for isoliquiritigenin. Lineweaver-Burk transformation of the inhibition data indicated that the inhibition was of a mixed type for both liquiritigenin and isoliquiritigenin. For liquiritigenin, the Ki and K(I) were determined to be 14.0 microM and 151.6 microM, respectively. For isoliquiritigenin, the Ki and K(I) were determined to be 17.4 microM and 81.9 microM, respectively. These results suggest that these natural products could be used to treat conditions where the inhibition of xanthine oxidase is warranted.     

Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth

Axons of primary olfactory neurons are intimately associated with olfactory ensheathing cells (OECs) from the olfactory epithelium until the final targeting of axons within the olfactory bulb. However, little is understood about the nature and role of interactions between OECs and axons during development of the olfactory nerve pathway. We have used high resolution time-lapse microscopy to examine the growth and interactions of olfactory axons and OECs in vitro. Transgenic mice expressing fluorescent reporters in primary olfactory axons (OMP-ZsGreen) and ensheathing cells (S100ß-DsRed) enabled us to selectively analyse these cell types in explants of olfactory epithelium. We reveal here that rather than providing only a permissive substrate for axon growth, OECs play an active role in modulating the growth of pioneer olfactory axons. We show that the interactions between OECs and axons were dependent on lamellipodial waves on the shaft of OEC processes. The motility of OECs was mediated by GDNF, which stimulated cell migration and increased the apparent motility of the axons, whereas loss of OECs via laser ablation of the cells inhibited olfactory axon outgrowth. These results demonstrate that the migration of OECs strongly regulates the motility of axons and that stimulation of OEC motility enhances axon extension and growth cone activity.     

Cyclic CMP (cytidine 3′,5′-monophosphate) suppresses changes in human sperm amplitude of lateral head displacement and hyperactivation

Summary The dibutyryl analog of cCMP suppressed sperm amplitude of lateral head displacement and hyperactivation. Sperm motility was inhibited by dibutyryl cCMP with a shift toward less linear trajectory sperm movements. The results suggest a role of cCMP as an inhibitory signal on sperm motility patterns related to sperm capacitation.     

Cyclic CMP (cytidine 3',5'-monophosphate) suppresses changes in human sperm amplitude of lateral head displacement and hyperactivation

The dibutyryl analog of cCMP suppressed sperm amplitude of lateral head displacement and hyperactivation. Sperm motility was inhibited by dibutyryl cCMP with a shift toward less linear trajectory sperm movements. The results suggest a role of cCMP as an inhibitory signal on sperm motility patterns related to sperm capacitation.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production inTrypanosoma cruzi andCrithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis inTrypanosoma cruzi and stimulated O₂ uptake and generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H₂O₂ production were obtained withCrithidia fasciculata despite the presence of catalase in this organism.Acknowledgments. This work was aided by grants from the University of Buenos Aires, the Scientific Office of the American States Organization and CEDIQUIFA (Buenos Aires). S.G.G. and M.P.M.P. are Research Fellows and A.O.M.S. is Career Investigator of CONICET (Argentina). L. T. Viñas and M. G. Gutierrez lent able technical assistance.     

Fenretinide induces mitochondrial ROS and inhibits the mitochondrial respiratory chain in neuroblastoma

Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.     

Hydrogen peroxide generation inTrypanosoma cruzi

Summary Homogenates from T. cruzi epimastigotes produced 3.4 pmoles H₂O₂/min 10⁶ cells, as detected by the cytochrome c peroxidase assay. Addition of NADH or NADPH increased H₂O₂ production by a factor of 3 and 5, respectively. When supplemented with NADH and NADPH, the mitochondrial, microsomal, and supernatant fractions produced H₂O₂, the soluble fraction and the mitochondrial membranes being apparently the main generators of H₂O₂. The epimastigote homogenates showed cyanide-sensitive superoxide dismutase activity, equivalent to 0.28 g bovine superoxide dismutase per mg homogenate protein.This investigation was supported by grants from Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET) Argentina and the Scientific Office, American States Organization.Career Investigator of CONICET.     

<Emphasis Type="Italic">Dictyostelium</Emphasis> dynamin B modulates cytoskeletal structures and membranous organelles

Dictyostelium discoideum cells produce five dynamin family proteins. Here, we show that dynamin B is the only member of this group of proteins that is initially produced as a preprotein and requires processing by mitochondrial proteases for formation of the mature protein. Our results show that dynamin B-depletion affects many aspects of cell motility, cell-cell and cell-surface adhesion, resistance to osmotic shock, and fatty acid metabolism. The mature form of dynamin B mediates a wide range and unique combination of functions. Dynamin B affects events at the plasma membrane, peroxisomes, the contractile vacuole system, components of the actin-based cytoskeleton, and cell adhesion sites. The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system. Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.