Tryparedoxin Peroxidase of Leishmania donovani: Molecular Cloning, Heterologous Expression, Specificity, and Catalytic Mechanism

Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H₂O₂, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a k_cat of 11.2 s⁻¹ and limiting K_m values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 μM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173′ that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis.     

Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.     

Metabolic control analysis of the Trypanosoma cruzi peroxide detoxification pathway identifies tryparedoxin as a suitable drug target

Background

The principal oxidative-stress defense in the human parasite Trypanosoma cruzi is the tryparedoxin-dependent peroxide detoxification pathway, constituted by trypanothione reductase (TryR), tryparedoxin (TXN), tryparedoxin peroxidase (TXNPx) and tryparedoxin-dependent glutathione peroxidase A (GPxA). Here, Metabolic Control Analysis (MCA) was applied to quantitatively prioritize drug target(s) within the pathway by identifying its flux-controlling enzymes.

Methods

The recombinant enzymes were kinetically characterized at physiological pH/temperature. Further, the pathway was in vitro reconstituted using enzyme activity ratios and fluxes similar to those observed in the parasites; then, enzyme and substrate titrations were performed to determine their degree of control on flux. Also, kinetic characterization of the whole pathway was performed.

Results

Analyses of the kinetic properties indicated that TXN is the less efficient pathway enzyme derived from its high Km_app for trypanothione and low Vmax values within the cell. MCA established that the TXN–TXNPx and TXN–GPxA redox pairs controlled by 90–100% the pathway flux, whereas 10% control was attained by TryR. The Km_app values of the complete pathway for substrates suggested that the pathway flux was determined by the peroxide availability, whereas at high peroxide concentrations, flux may be limited by NADPH.

Conclusion

These quantitative kinetic and metabolic analyses pointed out to TXN as a convenient drug target due to its low catalytic efficiency, high control on the flux of peroxide detoxification and role as provider of reducing equivalents to the two main peroxidases in the parasite.

General Significance

MCA studies provide rational and quantitative criteria to select enzymes for drug-target development.     

High constitutive peroxidase activity and constitutive thermotolerance in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

 During the isolation of mutations in the heat-inducible hsp70-1 gene of Neurospora crassa by RIP (repeat-induced point mutations), several transformants were generated by electroporation of conidia with a plasmid harboring an incomplete copy of this gene. One isolate, designated E-45, containing ectopically integrated hsp70-1 DNA, exhibited a slow growth rate, low-temperature sensitivity, constitutive thermotolerance (without prior heat shock), and high constitutive peroxidase activity. The constitutive form of peroxidase (CP) was distinguishable from the heat-inducible form (HIP) by immunoinactivation employing polyclonal antiserum against the latter enzyme and by electrophoretic resolution in nondenaturing polyacrylamide gels. This enzyme was purified to near homogeneity and some of its properties examined. The relative molecular mass of native CP was in the range of 118–136 kDa, as estimated by gel filtration analysis on size exclusion matrices, whereas SDS-PAGE analysis yielded a size of ∼37 kDa for the polypeptide. Substrate saturation kinetics studies were conducted using ABTS [2,2′-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates: K _m, V _max, and K _cat values for H₂O₂ were ∼22 μM, ∼447 nmol mg⁻¹, and 0.33 s⁻¹, respectively, and those for ABTS were ∼55 μM, ∼453 nmol mg⁻¹, and 0.3 s⁻¹, respectively. Guaiacol was not used as a substrate by this enzyme. CP peroxidase was shown to be a heme-containing enzyme, stable at temperatures up to 58°C. Received: August 5, 2002 / Accepted: January 22, 2003 Acknowledgments This work was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to M.K.). The financial support provided to A. M. in the form of a graduate studentship award by the AHFMR (Alberta Heritage Foundation for Medical Research) and of a graduate teaching assistantship to A. S. by the Department of Biological Sciences, University of Calgary, is gratefully acknowledged. Correspondence to:M. Kapoor     

Nitroreductase activity of ferredoxin reductase BphA4 from <Emphasis Type="Italic">Dyella ginsengisoli</Emphasis> LA−4 by catalytic and structural properties analysis

Ferredoxin reductase BphA4 was well known as a component of biphenyl dioxygenase. However, there was little information about whether it could utilize nonphysiological oxidants as electron acceptors. In the present study, we reported the novel nitroreductase activity of BphA4_LA−4. The homology model of ferredoxin reductase BphA4 from Dyella ginsengisoli LA−4 was constructed. According to the alignment of three-dimensional structures, it was supposed that BphA4_LA−4 could function as nitroreductase. Recombinant His-tagged BphA4_LA−4 was purified with a molecular mass of 49.6 ± 1 kDa. Biochemical characterization of purified BphA4_LA−4 possessed the nitroreductase activity with the optimal temperature 50°C and pH 8.0. The substrate spectrum and kinetics indicated BphA4_LA−4 could reduce several nitroaromatics with different apparent K _m values: m-dinitrobenzene (560 μM), o-dinitrobenzene (1,060 μM), o-nitroaniline (1,570 μM), m-nitrobenzoic acid (1,300 μM) and m-nitrophenol (67 μM). The nitroreductase activity was further explained by docking studies, which was indicated that Arg 288 should play an important role in binding nitroaromatics. Moreover, there existed a good linear correlation between lnK _m and calculated binding energy.     

Verification of the interaction of a tryparedoxin peroxidase with tryparedoxin by ESI-MS/MS

Tryparedoxin peroxidases (TXNPx) catalyze hydroperoxide reduction by tryparedoxin (TXN) by an enzyme substitution mechanism presumed to involve three catalytic intermediates: (i) a transient oxidation state having C52 oxidized to a sulfenic acid, (ii) the stable oxidized form with C52 disulfide-bound to C173', and (iii) a semi-reduced intermediate with C40 of TXN disulfide-linked to C173' from which the ground state enzyme is regenerated by thiol/disulfide reshuffling. This kinetically unstable form was mimmicked by a dead-end intermediate generated by cooxidation of TXNPx of Trypanosoma brucei brucei with an inhibitory mutein of TXN in which C43 was replaced by serine (TbTXNC43S). Cleavage of the isolated dead-end intermediate by trypsin plus chymotrypsin yielded a fragment that complied in size with the TbTXNC43S sequence 36 to 44 disulfide-linked to the TbTXNPx sequence 169 to 177. The presumed nature of the proteolytic fragment was confirmed by MS/MS sequencing. The results provide direct chemical evidence for the assumption that the reductive part of the catalysis is initiated by an attack of the substrate's solvent-exposed C40 on C173' of the oxidized peroxidase and, thus, confirm the hypothesis on the interaction of 2-Cys-peroxiredoxins with their proteinaceous substrates.     

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B₁ and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B₁. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B₁. Pyruvate was found to be the preferred co-substrate and amino group receptor (K _M = 490 μM at 10 μM hydrolyzed fumonisin B₁) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K _M = 1.1 μM and k _cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.     

Trichloroethylene degradation and mineralization by pseudomonads and Methylosinus trichosporium OB3b

 To examine the trichloroethylene (C₂HCl₃)-degrading capability of five microorganisms, the maximum rate, extent, and degree of C₂HCl₃ mineralization were evaluated for Pseudomonas cepacia G4, Pseudomonas cepacia G4 PR1, Pseudomonas mendocina KR1, Pseudomonas putida F1, and Methylosinus trichosporium OB3b using growth conditions commonly reported in the literature for expression of oxygenases responsible for C₂HCl₃ degradation. By varying the C₂HCl₃ concentration from 5 μM to 75 μM, V _max and K _m values for C₂HCl₃ degradation were calculated as 9 nmol/(min mg protein) and 4 μM for P. cepacia G4, 18 nmol/(min mg protein) and 29 μM for P. cepacia G4 PR1, 20 nmol/(min mg protein) and 10 μM for P. mendocina KR1, and 8 nmol/(min mg protein) and 5 μM for P. putida F1. This is the first report of these Michaelis-Menten parameters for P. mendocina KR1, P. putida F1, and P. cepacia G4 PR1. At 75 μM, the extent of C₂HCl₃ that was degraded after 6 h of incubation with resting cells was 61%–98%; the highest degradation being achieved by toluene-induced P. mendocina KR1. The extent of C₂HCl₃ mineralization in 6 h (as indicated by concentration of chloride ion) was also measured and varied from 36% for toluene-induced P. putida F1 to 102% for M. trichosporium OB3b. Since C₂HCl₃ degradation requires new bio-mass, the specific growth rate (μ_max) of each of the C₂HCl₃-degradation microorganisms was determined and varied from 0.080/h (M. trichosporium OB3b) to 0.864/h (P. cepacia G4 PR1). Received: 1 May 1995/Received revision: 11 July 1995/Accepted: 26 July 1995     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.     

Expansion of a direct shoot organogenesis system in peanut (Arachis hypogaea L.) to include US cultivars

Agrobacterium-mediated transformation, employing direct shoot organogenesis, allows for mature transgenic plants to be obtained quickly (3–4 mo). In this study, peanut (Arachis hypogaea L.) cultivars Florida-07, Georgia Green, Georgia Brown, New Mexico Valencia A, and VC-2 were selected to test their shoot induction response for use in future transformation experiments. Two types of cotyledon explants were examined, those that previously had an attached embryo axis upon cotyledon separation (explant A) and those that were embryo axis-free upon separation (explant B). Explants were placed onto a shoot induction medium with N ⁶-benzyladenine concentrations ranging from 10–80 μM for Florida-07, Georgia Green, and VC-2; 10–20 μM for Georgia Brown; and 10–640 μM for New Mexico Valencia A. Following a 4-wk culture period, explants were visually rated based on a scale of 1–4, where 1 indicated slight greening, but no growth, and 4 indicated greening, adventitious bud formation, as well as small leaf expansion. A difference in shoot induction was observed for the cotyledon explants examined (P > t = <0.0001). Explant A had greater shoot induction with a visual rating of 1.8 ± 0.1; explant B had a rating of 1.6 ± 0.1 (P > t = <0.0001). Additionally, cultivars responded to the culture conditions differently (cultivar × N ⁶-benzyladenine interaction). Georgia Green on 10 μM N ⁶-benzyladenine produced the most shoot buds (24.6%) and the highest visual rating (2.1), followed by VC-2 on 10 μM N ⁶-benzyladenine (22.1%, 1.8), New Mexico Valencia A on 640 μM N ⁶-benzyladenine (21.4%, 1.8), Georgia Brown on 80 μM N ⁶-benzyladenine (9.0%, 1.7), and Florida-07 on 40 μM N ⁶-benzyladenine (7.1%, 1.8). Of the tested varieties, Georgia Green, New Mexico Valencia A, and VC-2 were best suited for future transformation experiments based on their shoot bud production.     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Leishmania infantum: provision of reducing equivalents to the mitochondrial tryparedoxin/tryparedoxin peroxidase system

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)₂] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)₂ can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)₂ is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.     

Domoic acid neurotoxicity in hippocampal slice cultures

Summary.  The neurotoxicity of domoic acid was studied in 2–3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1–100 μM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC₅₀ values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC₅₀ value of 6 μM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 μM NBQX against 3 and 10 μM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 μM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 μM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Kinetics and redox-sensitive oligomerisation reveal negative subunit cooperativity in tryparedoxin peroxidase of Trypanosoma brucei brucei

Tryparedoxin peroxidases (TXNPx) are peroxiredoxin-type enzymes that detoxify hydroperoxides in trypanosomatids. Reduction equivalents are provided by trypanothione [T(SH)2] via tryparedoxin (TXN). The T(SH)2-dependent peroxidase system was reconstituted from TXNPx and TXN of T. brucei brucei (TbTXN-Px and TbTXN). TbTXNPx efficiently reduces organic hydroperoxides and is specifically reduced by TbTXN, less efficiently by thioredoxin, but not by glutathione (GSH) or T(SH)2. The kinetic pattern does not comply with a simple rate equation but suggests negative co-operativity of reaction centers. Gel permeation of oxidized TbTXNPx yields peaks corresponding to a decamer and higher aggregates. Electron microscopy shows regular ring structures in the decamer peak. Upon reduction, the rings tend to depolymerise forming open-chain oligomers. Co-oxidation of TbTXNPx with TbTXNC43S yields a dead-end intermediate mimicking the catalytic intermediate. Its size complies with a stoichiometry of one TXN per subunit of TXNPx. Electron microscopy of the intermediate displays pentangular structures that are compatible with a model of a decameric TbTXNPx ring with ten bound TbTXN molecules. The redox-dependent changes in shape and aggregation state, the kinetic pattern and molecular models support the view that, upon oxidation of a reaction center, other subunits adopt a conformation that has lower reactivity with the hydroperoxide.     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000     

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H₂O₂, either alone or together. Cells were pretreated with medium containing 1, 10, or 100 μM equol for 1 h prior to the addition of 1 mM H₂O₂ for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H₂O₂ caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 μM) but not high concentrations of equol (10 μM) inhibited cell damage, while 100 μM equol caused more serious damage than H₂O₂ alone. Pretreatment with 1 μM equol had no effect on cell viability, while pretreatment with 10 and 100 μM equol significantly decreased cell viability in a dose-dependent manner. Compared with H₂O₂ alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 μM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H₂O_2, except for an obvious increase in GSH-Px activity caused by 10 μM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H₂O₂, while pretreatment with high-dosage equol shows a synergistic effect with H₂O₂ in inducing cell damage.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

Inhibitors of animal phospholipase A2 enzymes are selective inhibitors of auxin-dependent growth. Implications for auxin-induced signal transduction

Auxin and elicitors reportedly activate phospolipase A. A number of inhibitors known to inhibit animal phospholipase A₂ were tested for their ability to inhibit hormone and fusicoccin-induced growth. To this end, growth induced by indolyl-3-acetic acid and 2,4-dichlorophenoxyacetic acid in hypocotyl segments of etiolated zucchini (Cucurbita pepo L.) seedlings was determined in the presence of the inhibitors nordihydroguajaretic acid (NDGA), aristolochic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), PB_x (a prostaglandin derivative), and oleylethyl phosphocholine. Each chemical proved inhibitory to auxin-induced growth, oleylethyl phosphocholine being the least effective. The effects of the first three inhibitors were investigated in more detail. Growth induced by 10 μM 2,4-dichlorophenoxyacetic acid or 1 μM indolyl-3-acetic acid was inhibited 50% by about 30–50 μM NDGA, by about 25 μM aristolochic acid, and by about 10–20 μM EYTA. Growth inhibition was reversible and became apparent 0.5–1 h after inhibitor addition. Growth induced by 0.5 or 1 μM fusicoccin was much less inhibited by NDGA and by ETYA, whereas aristolochic acid was only slightly less effective on fusicoccin-induced than on auxin-induced growth. These three inhibitors were also tested for their effects on gibberellin-induced growth in light-grown peas (Pisum sativum L.) and on cytokinin-induced expansion growth in excised cotyledons from radish (Raphanus sativum L.) seedlings. In both tests, aristolochic acid had toxic side-effects although gibberellin-induced growth was still apparent. In the gibberellin test, neither NDGA at up to 100 μM nor ETYA at 80 μM was inhibitory to hormone-induced growth. Moreover, 40 μM ETYA was not inhibitory to kinetin-induced growth. We hypothesize that the selectivity of phospholipase A₂ inhibitors for auxin-induced growth implies a different signal transduction pathway for each of the different signal substances tested, and that auxins might use fatty acid(s) and/or lysophospholipid(s) or their derivatives as the preferred second messengers. Received: 24 September 1996 / Accepted: 18 January 1997     

Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.

Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl (DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg²⁺. Kinetic data for chorismate in the forward reaction (K _m = 807 μM, V _max = 6.2 pkat · mg⁻¹) and for isochorismate in the reverse reaction (K _m = 675 μM, V _max = 5.9 pkat · mg⁻¹) were determined. Received: 18 November 1996 / Accepted: 28 December 1996     

The crystal structures of the tryparedoxin-tryparedoxin peroxidase couple unveil the structural determinants of leishmania detoxification pathway

Leishmaniasis is a neglected disease caused by Leishmania, an intracellular protozoan parasite which possesses a unique thiol metabolism based on trypanothione. Trypanothione is used as a source of electrons by the tryparedoxin/tryparedoxin peroxidase system (TXN/TXNPx) to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is not only unique to the parasite but is also essential for its survival; therefore, it constitutes a most attractive drug target. Several forms of TXNPx, with very high sequence identity to one another, have been found in Leishmania strains, one of which has been used as a component of a potential anti-leishmanial polyprotein vaccine. The structures of cytosolic TXN and TXNPx from L. major (LmTXN and LmTXNPx) offer a unique opportunity to study peroxide reduction in Leishmania parasites at a molecular level, and may provide new tools for multienzyme inhibition-based drug discovery. Structural analyses bring out key structural features to elucidate LmTXN and LmTXNPx function. LmTXN displays an unusual N-terminal α-helix which allows the formation of a stable domain-swapped dimer. In LmTXNPx, crystallized in reducing condition, both the locally unfolded (LU) and fully folded (FF) conformations, typical of the oxidized and reduced protein respectively, are populated. The structural analysis presented here points to a high flexibility of the loop that includes the peroxidatic cysteine which facilitates Cys52 to form an inter-chain disulfide bond with the resolving cysteine (Cys173), thereby preventing over-oxidation which would inactivate the enzyme. Analysis of the electrostatic surface potentials of both LmTXN and LmTXNPx unveils the structural elements at the basis of functionally relevant interaction between the two proteins. Finally, the structural analysis of TXNPx allows us to identify the position of the epitopes that make the protein antigenic and therefore potentially suitable to be used in an anti-leishmanial polyprotein vaccine.