Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation

Candida parapsilosis mitochondria contain three respiratory chains: the classical respiratory chain (CRC), a secondary parallel chain (PAR) and an “alternative” oxidative pathway (AOX). We report here the existence of similar pathways in C. albicans. To observe the capacity of each pathway to sustain yeast growth, C. albicans cells were cultured in the presence of inhibitors of these pathways. Antimycin A and KCN totally abrogated yeast growth, while rotenone did not prevent proliferation. Furthermore, rotenone promoted only partial respiratory inhibition. Lower concentrations of KCN that promote partial inhibition of respiration did not inhibit yeast growth, while partial inhibition of respiration with antimycin A did. Similarly, AOX inhibitor BHAM decreased O₂ consumption slightly but completely stunted cell growth. Reactive oxygen species production and oxidized glutathione levels were enhanced in cells treated with antimycin A or BHAM, but not rotenone or KCN. These findings suggest that oxidative stress prevents C. albicans growth.     

Inhibition of midgut protease of yellow stem borer (<Emphasis Type="Italic">Scirpophaga incertulas</Emphasis>) by cysteine protease-like inhibitor from mature jackfruit (<Emphasis Type="Italic">Artocarpus heterophyllus</Emphasis>) seed

A novel protease inhibitor was isolated and purified from the mature seeds of jackfruit (Artocarpus heterophyllus) by precipitation with ammonium sulphate, followed by DEAE-cellulose and gel filtration (Sephadex G-100) chromatography. The isolated protease inhibitor strongly inhibited papain and midgut proteases of yellow stem borer (Scipophaga incertulas) larvae, as seen by in vitro assay. The purified protease inhibitor was active over a wide range of pH with the maximum activity between pH 4 and 10. This protein was also stable up to 80°C, but the retained activity was lost at 100°C, when heated for 30 min. The molecular mass of the purified cysteine-like protease inhibitor is to be 14.50 kDa as determined by SDS-PAGE. Significant reduction in larval weight and mortality was observed, when fresh rice culms with protease inhibitor was feeded to the yellow stem borer larvae. These results may provide important information to control the yellow stem borer in rice with respect to naturally occurring insecticidal proteins. The observed differences would potentially translate into reductions in population growth of yellow stem borer, indicating a potential value of using jackfruit protease inhibitor for protecting rice plants against damage by the yellow stem borer.     

Rice (<i>Oryza sativa</i> L.) Breeding among Hassawi Landrace and Egyptian Genotypes for Stem Borer (<i>Chilo agamemnon</i> Bles.) Resistance and Related Quantitative Traits

Rice stem borer (Chilo agamemnon Bles.) is a primary insect pest of rice and is a major limiting factor to rice production. Breeding for insect-resistant crop varieties has been an economic way of integrated pest management (IPM) as it offers a viable and ecologically acceptable approach. This study was aimed to evaluate rice genotypes for their resistance against rice stem borer. Seven parental genotypes with twenty one F1 crosses were evaluated for genotypic variation in field experiments. Analysis of variance revealed significant differences for the studied traits in almost all crosses and parents. In addition, the mean squares of parents versus their crosses were signifi- cant for stem borer resistance and other associated traits. Moreover, both general combining ability (GCA) and specific combining ability (SCA) variances were highly significant for all characters studied in the F1 generation. Based on GCA, 4 genotypes (Sakha101, Gz6903-3-4-2-1, Gz9577-4-1-1 and Hassawi) exhibited highly significant negative values for stem borer resistance (–0.53, –1.06, –0.18 and –0.49, respectively) indicating they are the best combiners for stem borer resistance. Based on SCA analysis, nine cross combinations showed highly significant negative effects for stem borer resistance. Similarly, the cross Giza178 Hassawi was the best combination with significantly highest value for early maturity. In addition, seven crosses showed highly significant negative SCA for plant height trait. On the other hand, for panicle length, number of primary branches/panicle, panicle weight and 1000-grain weight, seven, four, eight and six crosses showed highly significant positive SCA, respectively. The result further revealed that the non-additive dominance genetic variance was higher than the additive variance for all evaluated traits indicating that non-additive genetic variances have a role in their inheritance. The broad-sense heritability estimates were high for all the studied traits. The stem borer resistance was significantly correlated with panicle weight and 1000-grain weight, which also showed a highly significant correlation with grain yield/plant. Thus these traits can be effectively employed in a breeding program to confer resistance against stem borer infestation in rice. It was further supported by biplot analysis, which clustered these potentially important traits into two quadrants showing their importance in any future breeding program to control stem borer infestation. This study has contributed valuable information for evaluation of genetic diversity in the local rice germplasm and its utilization in futuristic rice genetic improvement programs.     

Antimicrobial Activity of 3-Phenylimidazolidine-2,4-diones and Related Compounds

The structure-activity relationships of 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were investigated by the agar dilution method using Sclerotinia sclerotiorum as a test microbe. Several compounds were tested for antimicrobial spectrum in vitro with other pathogenic microbes and for foliage protective activity in green house tests with rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. It was found that the antimicrobial activity was enhanced when the 1-position of imidazolidine ring was substituted by an alkyl group but was reduced when the 5-position was substituted by alkyl groups. Generally, 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were active against Scierotiniaceae, Corticiaceae, Dematiaceae, Polystigmataceae or Pleosporaceae. In green house tests, some of these compounds showed high protective activity against rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. Results of the green house tests on the above mentioned diseases correlate well with those of in vitro tests except in the case of kidney bean stem rot.     

Inhibitory Effect of Alcohols on Esterolytic Activity of Proteinase from Aspergillus sulphureus

(+)-Cyanofenphos was at least more than 25-fold as toxic to rice stem borer larvae as the (?)-isomer. In addition, (+)-cyanofenphos-oxon was 36-fold more potent inhibitor than the (?)-oxon toward rice stem borer larva acetylcholinesterase. The (+)-, (?)- and racemic forms of cyanofenphos were metabolized in rice stem borer larvae at almost equal rates. However, (+)-cyanofenphos produced 4- to 9-fold larger amounts of cyanofenphos-oxon in the insect body than the (?)-isomer. On the other hand, (?)-cyanofenphos-oxon was metabolized to 4-cyanophenol and its conjugate at a faster rate compared to the (+)-oxon. The difference in toxicity to rice stem borer larvae exhibited by the optical isomers of cyanofenphos is attributable to the difference in the amount and persistence of cyanofenphos-oxon isomers formed in the insect body as well as in anti-acetylcholinesterase activity of the oxon isomers.     

Difference in the time of mating activity between host-associated populations of the rice stem borer, Chilo suppressalis (Walker)

Mating activities of the rice stem borer, Chilo suppressalis, were compared between rice‐ and wateroat‐feeding populations, and two potential temporal factors that may act as reproductive barriers were examined. Seasonal data of the number of moths attracted to pheromone traps showed that the peak of emergence at the rice field was approximately 10 days earlier than that at the wateroat vegetation in the first flight season, although there was a broad overlap of emergence at the two locations. Both field observations and a laboratory experiment showed that moths from the rice field started mating earlier than those from the wateroat vegetation. However, whereas the difference was distinctive in the laboratory experiment, mating activity at the wateroat vegetation shifted significantly to an earlier time phase than that observed in the laboratory. Body size data showed that the male moths attracted to the pheromone traps at the wateroat vegetation were significantly larger than those at the rice field, suggesting that the traps at the two locations mainly attracted moths originating from different host plants. However, pheromone‐trapped males at the rice field were significantly larger than those reared from overwintering samples. These results support the idea that males from the wateroat vegetation migrate to the rice field. The differences in seasonal and temporal mating activity and their effects on development of reproductive isolation between host‐associated populations are discussed.     

Specificity of systemically released rice stem volatiles on egg parasitoid,Trichogramma japonicum Ashmead behaviour

The yellow stem borer, Scirpophaga incertulas Walker (YSB), infested rice plants emit chemicals through the surface of their infested stems. These induce attractant activity and cause arrestment responses and ovipositional stimulation in its egg parasitoid, Trichogramma japonicum Ashmead. Laboratory experiments on short‐range host searching and oviposition were performed to assess the how these crude stem extracts and their fractions influence the biological control efficiency of Trichogramma. The activity of these chemicals as long‐range attractants was confirmed through wind tunnel bioassays. Stem borer‐infested plant extracts had enhanced the parasitization rate of T. japonicum, whereas host eggs treated with the extract from undamaged stems or solvent‐treated control failed to evoke changes in the parasitoid’s behaviour. A preliminary GC‐MASS analysis indicated the presence of several hydrocarbon compounds. The analysis also revealed qualitative and quantitative differences between the chemical profiles of the infested and non‐infested plants. We hypothesized that herbivore‐induced plant chemicals are released through the stem surfaces and attract T. japonicum, even over long distances. These cues elicit parasitoid arrestment on pest‐damaged plants and subsequently lead to the successful parasitization of the stem borer.     

A malate-oxaloacetate shuttle linking cytosolic fatty acid α-oxidation to the mitochondrial electron transport chain in uncoupled fresh potato slices

The possible existence of a malonate-sensitive dicarboxylate-mediated electron shuttle between microsomal NAD-linked fatty acid α-oxidation and the mitochondrial electron transport chain in uncoupled fresh potato slices was investigated. Uncoupled slice respiration is inhibited by benzylmalonate and butylmalonate, inhibitors of dicarboxylate transport into mitochondria. Uncoupled slice respiration is also inhibited by rotenone, an indication of intramitochondrial NADH oxidation. Since fatty acid α-oxidation per se is rotenone insensitive, rotenone and benzylmalonate inhibition of the oxidation of carboxyl-labeled myristate in slices points to a dicarboxylic acid shuttle linking microsomal fatty acid a-oxidation with intramitochondrial NADH dehydrogenase.
Malonute inhibits both respiration and ¹⁴CO², release from carboxyl-labeled myristate in fresh uncoupled slices, as do inhibitors of dicarboxylate transport. Mitochondrial studies show that malonate inhibits malate oxidation but not malate dehydrogenase per se. Furthermore, malonate inhibits malate transport more severely than malate oxidation. Accordingly, mulonate inhibition of uncoupled slice respiration in the absence of tricarboxylic acid cycle activity is attributed to its interference with mitochondrial malate transport, and its consequent curtailment of a putative malate-OAA shuttle linked to cytosolic NAD-mediated fatty acid α-oxidation.     

Effect of Dopamine, Dimethoxyphenylethylamine, Papaverine, and Related Compounds on Mitochondrial Respiration and Complex I Activity

Abstract: We report the effect of papaverine, tetrahydropapaverine, laudanosine, dimethoxyphenylethylamine, dopamine, and its metabolites on mitochondrial respiration and activities of the enzymes in the electron transfer complexes, as mitochondrial toxins may be implicated in the etiology and the pathogenesis of Parkinson's disease. Papaverine was the most potent inhibitor of complex I and NADH-linked mitochondrial respiration among the compounds tested next to rotenone. Tetrahydropapaverine, dimethoxyphenylethylamine, and laudanosine also inhibited NADH-linked mitochondrial respiration and complex I activity in this order. Dopamine and its metabolites showed either no inhibition or only very weak inhibition. Compounds with dimethoxy residues in the phenyl ring were associated with more potent inhibition of complex I than those without. Our results warrant further studies on these and some related compounds as candidate neurotoxins causing Parkinson's disease.     

EFFECTS OF BT RICE ON THE FOOD CONSUMPTION,GROWTH AND SURVIVAL OF CHILO SUPPRESSAUS LARVAE UNDER DIFFERENT TEMPERATURES*

Abstract The Bt transgenic rice line, i.e. KMD1 containing a synthetic cry 1 Ab gene from Bacillus thuringiensis Berliner was tested in the laboratory for evaluating its effects on larval food consumption, growth and survival of striped stem borer (SSB), Chilo suppressalis (Walker) under different temperatures. The food consumption and body weight growth as well as the survival of both 3rd and 5th instar larvae fed on Bt rice were significantly reduced, as compared with those on the control. The temperature showed no marked effect on both food consumption and body weight growth of larvae fed on Bt rice stems since the onset of 3rd instar, conversely had significant effects as since the initial stage of 5th instar. In contrast, the temperature did not distinctly influence the survival of both 3rd and 5th instar larvae. There was a linearly positive relationship between the corrected mortality of larvae and the accumulative amount of Bt rice tissues ingested by larvae.     

Control of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging

We compared NAD-dependent state 4 and state 3 respiration, NADH oxidation and Complex I specific activity in liver mitochondria from 4- and 30-month-old rats. All the activities examined were significantly decreased with aging. In both groups of animals, the flux control coefficients measured by rotenone titration indicated that Complex I is largely rate controlling upon NADH aerobic oxidation while, in state 3 respiration, it shares the control with other steps in the pathway. Moreover, we observed a trend wherein flux control coefficients of Complex I became higher with age. This indication was strengthened by examining the rotenone inhibition thresholds showing that Complex I becomes more rate controlling, over all the examined activities, during aging. Our results point out that age-related alterations of the mitochondrial functions are also present in tissues considered less prone to accumulate mitochondrial DNA mutations.     

Control of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging

We compared NAD-dependent state 4 and state 3 respiration, NADH oxidation and Complex I specific activity in liver mitochondria from 4- and 30-month-old rats. All the activities examined were significantly decreased with aging. In both groups of animals, the flux control coefficients measured by rotenone titration indicated that Complex I is largely rate controlling upon NADH aerobic oxidation while, in state 3 respiration, it shares the control with other steps in the pathway. Moreover, we observed a trend wherein flux control coefficients of Complex I became higher with age. This indication was strengthened by examining the rotenone inhibition thresholds showing that Complex I becomes more rate controlling, over all the examined activities, during aging. Our results point out that age-related alterations of the mitochondrial functions are also present in tissues considered less prone to accumulate mitochondrial DNA mutations.     

Cytosol-mitochondria transfer of reducing equivalents by a lactate shuttle in heterotrophic Euglena.

To assess the expression and physiological role of the mitochondrial NAD(+)-independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the d- and l-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three- to fourfold from latency to the stationary phase. Intracellular levels of D- and L-lactate were high (5-40 mm) from the start of the culture and increased (20-80 mm) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactate-grown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle).     

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC₅₀ values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles.     

Nutritional studies of the rice stem borer,Chilo suppressalis Walker,and its mass rearing

The nutritional requirements of the rice stem borer,Chilo suppressalis Walker were studied by rearing on synthetic food media in relation to the constituents of the rice plant. The larvae required amino acids, vitamins, carbohydrates, cholesterol, minerals and water extract of the rice plant for their growth. Among amino acids, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane and valine were indispensable for the growth, and these amino acids could not be synthesized in the larval body. Among vitamines, thiamine, riboflavin, nicotinic acid, pyridoxine, Ca-pantothenate, folic acid, biotin, cholin chloride and ascorbic acid were essential for the growth. Cholesterol is the only essential nutrient among fat-soluble factors. Fructose, glucose and sucrose showed high nutritive values among carbohydrates. Addition of the water extract of the rice plant to the media improved the development of the insects, but the excess amounts of it resulted inhibition of the growth. Two aromatic acids, benzoic and salicylic acids, were isolated as the growth inhibitory substance. p-methyl acetophenone isolated from the rice plant seemed to play an important role for the attraction of the insect. Mass rearing of the rice stem borer in a laboratory was carried out on two kinds of foods: living rice plant seedlings and synthetic food media. The rearing the insects on rice seedlings is easy if rice seeds are available. The rearing on synthetic food media is modified to simplify from these for the nutritional study. The rearing practices, merit and demerit of both methods are discussed.     

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.     

Antiviral and Antitumor Antibiotics. XIV. Effects of Ascochlorin and Other Respiration Inhibitors on Multiplication of Newcastle Disease Virus in Cultured Cells

Antiviral activity on Newcastle disease virus was examined with some respiration inhibitors including ascochlorin, rotenone, antimycin A₃, piericidin A, dicoumarol, 2, 4-dinitrophenol, pentachlorophenol, and fatty acids. Of the chemicals tested, ascochlorin and rotenone showed significant inhibitory effect on the viral growth in cultured cells as determined by the plate and tube assay methods. Dose response of ascochlorin and rotenone was observed in rate and final yield of hemagglutinin synthesis. Ratio of infectivity for hemagglutinin was nearly equal at any test concentrations of ascochlorin. Thus, the possibility of inhibition of virus maturation by the antibiotic was excluded. Ascochlorin had no activity on virus particles and on viral adsorption onto host cells.     

In vitro insect-feeding bioassay to determine the resistance of transgenic rice plants transformed with insect resistance genes against striped stem borer (Chilo suppressalis)

Summary To determine the degree of insect resistance in transgenic plants, different bioassays are used which typically use either whole plant or small pieces of leaves or stems of transgenic plants, following culture under greenhouse conditions. An in vitro insect-feeding bioassay is presented which permits the infestation of transgenic plantlets with newly hatched larvae from the striped stem borer. The bioassay consists of the germination of rice seeds in vitro using Murashige and Skoog medium in test tubes, and then infestation of each 3–4 cm long seedling with one neonate larva obtained from surfacesterilized eggs of Chilo suppressalis. The infested in vitro plantlets are kept in culture rooms at 25°C for several days and then the seedling damage and the growth of the larvae are analyzed. Senia (japonica variety) homozygous transgenic rice plants were used for these experiments. The plants were transformed with either the cry1B or the maize proteinase inhibitor (mpi) genes. Both genes confer resistance to Chilo suppressalis. With non-transformed plants the larvae grew and developed normally, feeding on the small rice plantlets. In contrast, with cry1B plants, the neonate larvae died during the first days of the infestation. These plantlets recovered completely and developed similarly to the non-infested control plants. With transgenic plants transformed with the mpi gene, the neonate larvae did not die but grew more slowly compared with the controls. Thus, this in vitro insect-feeding bioassay is a rapid and easy method to detect the resistance of cry and mpi transgenic plants to stem borers such as Chilo suppressalis.     

Synthesis,larvicidal activity,and SAR studies of new benzoylphenylureas containing oxime ether and oxime ester group

A series of new structural benzoylphenylureas (BPUs) containing oxime ether and oxime ester group were designed and synthesized. The larvicidal activities against Oriental armyworm and mosquito of these benzoylphenylureas were evaluated and the result of bioassay displayed specific structure–activity relationship (SAR). Most of the compounds exhibited excellent larvicidal activities against Oriental armyworm and mosquito. Interestingly, some compounds showed different structure–activity relationship towards diamondback moth, beet armyworm, and corn borer although three tested insects all belong to the same insect order.     

Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.