Characterization of the inheritance of field-evolved resistance to diamides in the fall armyworm (Spodoptera frugiperda) (Lepidoptera: Noctuidae) population from Puerto Rico.

The fall armyworm (Spodoptera frugiperda) is one of the most destructive pests of corn. New infestations have been reported in the East Hemisphere, reaching India, China, Malaysia, and Australia, causing severe destruction to corn and other crops. In Puerto Rico, practical resistance to different mode of action compounds has been reported in cornfields. In this study, we characterized the inheritance of resistance to chlorantraniliprole and flubendiamide and identified the possible cross-resistance to cyantraniliprole and cyclaniliprole. The Puerto Rican (PR) strain showed high levels of resistance to flubendiamide (RR50 = 2,762-fold) and chlorantraniliprole (RR50 = 96-fold). The inheritance of resistance showed an autosomal inheritance for chlorantraniliprole and an X-linked inheritance for flubendiamide. The trend of the dominance of resistance demonstrated an incompletely recessive trait for H1 (♂ SUS × â™€ PR) × and an incompletely dominant trait for H2 (♀ SUS × â™‚ PR) × for flubendiamide and chlorantraniliprole. The PR strain showed no significant presence of detoxification enzymes (using synergists: PBO, DEF, DEM, and VER) to chlorantraniliprole; however, for flubendiamide the SR = 2.7 (DEM), SR = 3.2 (DEF) and SR = 7.6 (VER) indicated the role of esterases, glutathione S- transferases and ABC transporters in the metabolism of flubendiamide. The PR strain showed high and low cross-resistance to cyantraniliprole (74-fold) and cyclaniliprole (11-fold), respectively. Incomplete recessiveness might lead to the survival of heterozygous individuals when the decay of diamide residue occurs in plant tissues. These results highlight the importance of adopting diverse pest management strategies, including insecticide rotating to manage FAW populations in Puerto Rico and other continents.

Toxicity and effects on anuran tadpole metamorphosis of the anthranilic diamide insecticides chlorantraniliprole and cyantraniliprole.

The present study examined the acute and chronic toxicity attributed to commercial formulations of the anthranilic diamide insecticides chlorantraniliprole (CHLO) and cyantraniliprole (CYAN) on the neotropical amphibian species Rhinella arenarum, Rhinella fernandezae and Scinax granulatus. The median lethal concentrations obtained after 96 hr exposure (96 hr-LC50) were generally greater than 100 mg/L, except for stage 25 S. Granulatus, which were the most sensitive animals tested with a 96 hr-LC50 value of 46.78 mg/L. In subchronic exposures of R. arenarum, the 21day-LC50 were 151.4 mg/L for CHLO and >160 mg/L for CYAN, the weight gain of the tadpoles during this period not being markedly affected in both cases. Finally, when tadpoles of R. arenarum were exposed to CHLO throughout the metamorphic process, an inverted U-shaped non-monotonic dose-response relationship was observed between exposure concentrations and both % of individuals transiting between stage 39 and 42 and the time required to accomplish this. Data obtained raise the hypothesis of an effect of CHLO on the hypothalamic-pituitary-thyroid (HPT) axis, either directly or through an interaction with the stress-hormone system, as metamorphic progression from stage 39 to S42 occurs under the strict control of thyroid hormones. These observations are important as the anthranilic diamide insecticides are not currently known as endocrine disruptors. Further investigations are needed to clarify the pathways leading to these effects and examine whether environmentally-relevant aquatic concentrations of anthranilic diamides might be impacting amphibian populations in the wild.

Inhibition of X52 Corrosion in CO2-Saturated Brine by a Dialkyl-Diamide from Coffee Bagasse Oil.

This work reports the performance of a green corrosion inhibitor with double hydrocarbon chain. The evaluated inhibitor was a dialkyl-diamide from coffee bagasse oil and its electrochemical behavior was evaluated on an API-X52 steel in CO2-saturated brine at 60 °C. The electrochemical behavior was determined by measurements of open circuit potential, polarization resistance, and electrochemical impedance spectroscopy. In addition, the thermodynamic parameters of the corrosion process were obtained in the temperature range from 40 °C to 80 °C. Electrochemical studies showed that the inhibitor is capable of suppressing metal dissolution by up to 99% at 25 ppm. On the other hand, the thermodynamic parameters indicate that when adding the inhibitor, there is a strong increase in both Ea and ΔH° values, and that as time increases, they decrease until reaching similar values to those observed in the absence of the inhibitor. Furthermore, ΔS° values tend to become more negative with immersion time because of the formation of a stable film on the metal surface.

Physiological quality of rice seeds as a function of different doses of the insecticide ciantraniliprole / Qualidade fisiológica de sementes de arroz em função de diferentes doses do inseticida ciantraniliprole

The seed is one of the main agricultural inputs and its quality is one of the key factors for success in rice cultivation. The objective of this work was to evaluate the physiological quality of rice seeds with different doses of the insecticide ciantraniliprole and its performance during storage of rice seeds. Rice seeds of the cultivar IRGA 424 RI treated at doses 0 (control), 10, 20, 40, 60, 80 and 100 ml of the product foreach 100 kg of seeds were used. Evaluations were performed during storage periods 0, 60, 90, 135 and 180 days after treatment. Seed quality was monitored through the germination test, cold test and emergence. Under the conditions in which the study was carried out, we can conclude that: ciantraniliprole at doses of 60, 80 and 100 ml/100kg of seeds provides greater germination when the seeds are not stored, however the dose of 60 ml/100kg showed higher percentages of germination in other storage periods. For the cold test, cyantraniliprole in all doses was superior to the control without treatment and in the doses 60 ml/100kg of seeds it was superior in the storage times 0, 135 and 180 days, already in the storage periods 60 and 90 days the dose of 60 ml/100kg did not differ from the dose of 80 ml/100kg. For the emergence of seedlings, the treatment with the insecticide at a dose of 60 ml/100kg of seeds was superior to the others, except for the storage period of 135 days.(AU)

A semente é um dos principais insumos da agricultura e sua qualidade é um dos fatores primordiais para o sucesso na cultura do arroz. O objetivo do trabalho foi avaliar a qualidade fisiológica de sementes de arroz com diferentes doses do inseticida ciantraniliprole e seu desempenho durante o armazenamento de sementes de arroz. Utilizaram-se sementes de arroz da cultivar IRGA 424 RI tratadas nas doses 0 (testemunha), 10, 20, 40, 60, 80 e 100 mldo produto para cada 100 kg de sementes. As avaliações foram realizadas durante os períodos de armazenamento 0, 60, 90,135 e 180 dias após o tratamento. A qualidade das sementes foi monitorada através do teste germinação, teste de frio e emergência. Nas condições de realização do trabalho podemos concluir que: o ciantraniliprole nas doses de 60, 80 e 100 ml/ 100kg de sementes proporciona maior germinação quando as sementes não são armazenadas, no entanto a dose de 60 ml/100kg apresentou maior percentagens de germinação nos demais períodos de armazenamento. Para o teste de frio, o ciantraniliprole em todas as doses foi superior a testemunha sem tratamento e nas doses 60 ml/100kg de sementes mostrou-se superior nas épocas de armazenamento 0, 135 e 180 dias, já nos períodos de armazenamento 60 e 90 dias a dose de 60 ml/100kg não diferiu da dose de 80 ml/100kg. Para a emergência de plântulas o tratamento com o inseticida na dose de 60 ml/100kg de sementes mostrou-se superior aos demais, exceto no período de armazenamento 135 dias.(AU)

The Cost of Resistance to Diamide Insecticide Varies With the Host Plant in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fitness costs associated with insect resistance to insecticides can be exploited to implement resistance management programs. However, most of these studies are restricted to evaluating biological traits on artificial diets. Here, we investigated the fitness cost associated with chlorantraniliprole in Spodoptera frugiperda (J.E. Smith) feeding on corn, soybean, and cotton plants. We used a near-isogenic strain of S. frugiperda resistant to chlorantraniliprole (Iso-RR), a susceptible strain (SS), and heterozygotes strains (H1 and H2) to evaluate several biological and population growth parameters. Larval survival of the Iso-RR strain was on average 90% on corn, 65% on soybean, and 57% on cotton plants. Development time of the larval stage also differed among host plants, Iso-RR strain took on average 14, 17, and 26 days to reach the pupal stage on corn, soybean, and cotton plants respectively. Net reproductive rate, intrinsic rate of population increase, and finite rate of population increase were higher for Iso-RR strain feeding on corn plants than other host plants. The relative fitness, based on the intrinsic rate of population increase, of S. frugiperda resistant strain on corn, soybean, and cotton plants were 1.04, 0.85, and 0.88, respectively. Therefore, no fitness cost was observed for S. frugiperda feeding on corn plants, but a significant fitness cost was observed when this pest fed on soybean and cotton plants. We showed that the food source influences the fitness cost of S. frugiperda resistant to diamide. Such information may help to implement resistance management strategies based on each crop.

Contrasting susceptibility of lepidopteran pests to diamide and pyrethroid insecticides in a region of overwintering and migratory intersection.

BACKGROUND: Pesticide resistance is a growing issue worldwide, and susceptibility of pest populations should be monitored in migratory intersection regions for successful resistance management. We determined the susceptibility of eight noctuid species from the Florida Panhandle to bifenthrin (pyrethroid) and chlorantraniliprole (diamide). Larvae from field and laboratory populations were exposed to commercial insecticide formulations using the leaf-dip method in concentration-mortality bioassays. RESULTS: The field populations of Helicoverpa zea (Boddie), Spodoptera frugiperda (Smith), S. eridania (Stoll), S. exigua (Hubner) and Chloridea virescens (Fabricius) had reduced susceptibility to bifenthrin compared with the laboratory populations. Resistance ratios to bifenthrin were as high as 10 071-fold in S. exigua and 436-fold in S. frugiperda, while there was no reduced susceptibility in Agrotis ipsilon (Hufnagel). The susceptibility to chlorantraniliprole was similar between the field and laboratory populations studied, except for S. exigua that exhibited 630-fold resistance to the diamide. The probit regression equations indicated that the larval mortality of S. exigua and S. frugiperda populations was <80% with bifenthrin at the concentration equivalent to the label rate. Likewise, the estimated mortality of S. exigua larvae with chlorantraniliprole at the label rate concentration was <80%. CONCLUSIONS: The lepidopteran pest populations tested were variable in susceptibility to bifenthrin by contrast to more consistent susceptibility to chlorantraniliprole. These results help in the choice of effective insecticides for integrated pest management and resistance management in cropping systems colonized by migratory lepidopteran pests from the U.S. Gulf Coast region. © 2020 Society of Chemical Industry.

Selection and characterization of the inheritance of resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to chlorantraniliprole and cross-resistance to other diamide insecticides.

BACKGROUND: Understanding the genetic basis of insect resistance to insecticides can help to implement insecticide resistance management (IRM) strategies. In this study, we selected a strain of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistant to chlorantraniliprole using the F2 screen method, characterized the inheritance of resistance and evaluated patterns of cross-resistance to other diamide insecticides. RESULTS: The chlorantraniliprole-resistant strain (Chlorant-R) was selected from a field-collected population with an estimated allele frequency of 0.1316. The estimated median lethal concentration (LC50 ) values were 0.011 and 2.610 µg a.i. cm-2 for the susceptible (Sus) and Chlorant-R strains, respectively, resulting in a resistance ratio (RR) of 237-fold. The LC50 values of the reciprocal crosses were 0.155 and 0.164 µg a.i. cm-2 , indicating that resistance is autosomally inherited. Resistance was characterized as incompletely recessive and monogenic at concentrations close to the recommended field rates of chlorantraniliprole. Survival of the resistant strain and heterozygous larvae in maize plants treated at the field rate was ∼ 60 (similar to the susceptible strain on untreated maize) and 15%, respectively. The Chlorant-R strain showed low cross-resistance to cyantraniliprole (RR ∼ 27-fold) and very high cross-resistance to flubendiamide (RR > 42 000-fold). CONCLUSIONS: The frequency of chlorantraniliprole resistance allele was moderately high in a field-collected population of S. frugiperda. The inheritance of chlorantraniliprole resistance was characterized as autosomal, incompletely recessive and monogenic. S. frugiperda showed cross-resistance to other diamide insecticides. These results provide essential information for the implementation of IRM programs to preserve the useful life of diamide insecticides for controlling S. frugiperda in Brazil. © 2019 Society of Chemical Industry.

Effect of seed treatment with insecticides on the control of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in soybean / Efeito do tratamento de sementes com inseticidas no controle de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) na cultura da soja

Fall armyworm is one of the main insect pests in Brazil. Thus, the present work aimed to evaluate the seed treatment effect on the control of Spodoptera frugiperda in initial infestations of soybean crops. The experimental design was completely randomized with four replicates of six insecticide treatments applied through seed treatment: imidacloprid plus thiodicarb at the dose of 52.5 plus 105 g a.i. (active ingredient) 100 kg-1 of seed (Cropstar® 0.350 L 100 kg-1 of seed); thiamethoxam at 105 g a.i. 100 kg-1 of seed (Cruiser 350 FS® 0.3 L 100 kg-1 of seed); chlorantraniliprole at 62.5 g a.i. 100 kg-1 of seed (Dermacor® 0.1 L 100 kg-1 of seed); cyantraniliprole at 120 g a.i. 100 kg-1 of seed (Fortenza 600 FS® 0.2 L 100 kg-1 of seed); fipronil plus pyraclostrobin and thiophanate-methyl 50 + 5 + 45 g a.i. 100 kg-1 of seed (Standak Top® 0.2 L 100 kg-1 of seed), and a control treatment. The experiment was carried out in a greenhouse. Diamide insecticides (chlorantraniliprole and cyantraniliprole) presented the best results among all treatments, with lower consumption of the treated leaves by the caterpillars and greater control efficacy of this insect. We verified that seed treatment is a viable alternative for controlling S. frugiperda at the beginning of crop development, when the caterpillar presents the behavior of cutting the seedlings and/or the consumption of leaf area, causing a reduction in the plant population and a consequent yield loss.(AU)

A lagarta-do-cartucho é um dos principais insetos-praga no Brasil. Assim, o presente trabalho teve por objetivo avaliar o efeito do tratamento de sementes no controle de Spodoptera frugiperda nas infestações iniciais da cultura da soja. O delineamento experimental foi inteiramente casualizado, com quatro amostras replicadas de seis tratamentos inseticidas aplicados via tratamento de sementes: imidacloprida mais tiodicarbe na dose de 52,5 mais 105 g i.a. (ingrediente ativo) 100 kg-1 de sementes (Cropstar® 0,350 L 100 kg-1 de sementes); tiametoxam a 105 g i.a. 100 kg-1 de sementes (Cruiser 350 FS® 0,3 L 100 kg-1 de sementes); clorantraniliprole a 62,5 g i.a. 100 kg-1 de sementes (Dermacor® 0,1 L 100 kg-1 de sementes); ciantraniliprole a 120 g i.a. 100 kg-1 de sementes (Fortenza 600 FS® 0,2 L 100 kg-1 de sementes); fipronil mais piraclostrobina e tio-fanato-metílico 50 + 5 + 45 g i.a. 100 kg-1 de sementes (Standak Top® 0,2 L 100 kg-1 de sementes) e um tratamento controle. O experimento foi conduzido em uma vegetação. Dentre todos os tratamentos, os inseticidas do grupo químico das diamidas (clorantraniliprole e ciantraniliprole) apresentaram os melhores resultados, com consumo inferior pelas lagartas das folhas tratadas e maior eficiência de controle deste inseto. Foi constatado que o tratamento de sementes é uma alternativa viável para o controle de S. frugiperda no início do desenvolvimento da cultura, quando a lagarta apresenta o comportamento de cortar as plântulas e/ou consumir área foliar, ocasionando uma redução da população de plantas e uma consequente perda de produtividade.(AU)

Effect of seed treatment with insecticides on the control of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in soybean / Efeito do tratamento de sementes com inseticidas no controle de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) na cultura da soja

Fall armyworm is one of the main insect pests in Brazil. Thus, the present work aimed to evaluate the seed treatment effect on the control of Spodoptera frugiperda in initial infestations of soybean crops. The experimental design was completely randomized with four replicates of six insecticide treatments applied through seed treatment: imidacloprid plus thiodicarb at the dose of 52.5 plus 105 g a.i. (active ingredient) 100 kg-1 of seed (Cropstar® 0.350 L 100 kg-1 of seed); thiamethoxam at 105 g a.i. 100 kg-1 of seed (Cruiser 350 FS® 0.3 L 100 kg-1 of seed); chlorantraniliprole at 62.5 g a.i. 100 kg-1 of seed (Dermacor® 0.1 L 100 kg-1 of seed); cyantraniliprole at 120 g a.i. 100 kg-1 of seed (Fortenza 600 FS® 0.2 L 100 kg-1 of seed); fipronil plus pyraclostrobin and thiophanate-methyl 50 + 5 + 45 g a.i. 100 kg-1 of seed (Standak Top® 0.2 L 100 kg-1 of seed), and a control treatment. The experiment was carried out in a greenhouse. Diamide insecticides (chlorantraniliprole and cyantraniliprole) presented the best results among all treatments, with lower consumption of the treated leaves by the caterpillars and greater control efficacy of this insect. We verified that seed treatment is a viable alternative for controlling S. frugiperda at the beginning of crop development, when the caterpillar presents the behavior of cutting the seedlings and/or the consumption of leaf area, causing a reduction in the plant population and a consequent yield loss.(AU)

A lagarta-do-cartucho é um dos principais insetos-praga no Brasil. Assim, o presente trabalho teve por objetivo avaliar o efeito do tratamento de sementes no controle de Spodoptera frugiperda nas infestações iniciais da cultura da soja. O delineamento experimental foi inteiramente casualizado, com quatro amostras replicadas de seis tratamentos inseticidas aplicados via tratamento de sementes: imidacloprida mais tiodicarbe na dose de 52,5 mais 105 g i.a. (ingrediente ativo) 100 kg-1 de sementes (Cropstar® 0,350 L 100 kg-1 de sementes); tiametoxam a 105 g i.a. 100 kg-1 de sementes (Cruiser 350 FS® 0,3 L 100 kg-1 de sementes); clorantraniliprole a 62,5 g i.a. 100 kg-1 de sementes (Dermacor® 0,1 L 100 kg-1 de sementes); ciantraniliprole a 120 g i.a. 100 kg-1 de sementes (Fortenza 600 FS® 0,2 L 100 kg-1 de sementes); fipronil mais piraclostrobina e tio-fanato-metílico 50 + 5 + 45 g i.a. 100 kg-1 de sementes (Standak Top® 0,2 L 100 kg-1 de sementes) e um tratamento controle. O experimento foi conduzido em uma vegetação. Dentre todos os tratamentos, os inseticidas do grupo químico das diamidas (clorantraniliprole e ciantraniliprole) apresentaram os melhores resultados, com consumo inferior pelas lagartas das folhas tratadas e maior eficiência de controle deste inseto. Foi constatado que o tratamento de sementes é uma alternativa viável para o controle de S. frugiperda no início do desenvolvimento da cultura, quando a lagarta apresenta o comportamento de cortar as plântulas e/ou consumir área foliar, ocasionando uma redução da população de plantas e uma consequente perda de produtividade.(AU)

Redox modulation of plant developmental regulators from the class I TCP transcription factor family.

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants.

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide

To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.

Sterol analogues with diamide side chains interfere with the intracellular localization of viral glycoproteins.

The need to develop novel antiviral agents encouraged us to assess the antiviral activity of synthetic sterol analogues with a diamide side chains. Cytotoxicity and antiviral activity of a family of azasterol previously synthesized was evaluated against herpes simplex virus 1 (HSV-1) (KOS and B2006) and vesicular stomatitis virus (VSV). This family of compounds was extended by the synthesis of novel analogs using an Ugi multicomponent reaction and their ability to inhibit viral multiplication was also evaluated. The results show that some of the compounds tested exert an antiviral activity. Besides, the effect of the azasterols on the intracellular localization of viral glycoproteins was examined. Strikingly, alteration on the glycoprotein D (gD) of HSV-1 fluorescence pattern was observed with both the antiherpetic compounds and the inactive azasterols.

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide.

To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function.

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.

Synthesis, gastroprotective effect and cytotoxicity of new amino acid diterpene monoamides and diamides.

Following our studies on the gastroprotective effect and cytotoxicity of terpene derivatives, new amides were prepared from the diterpene 8(17)-labden-15,19-dioic acid (junicedric acid) and its 8(9)-en isomer with C-protected amino acids (amino acid esters). The new compounds were evaluated for their gastroprotective effect in the ethanol/HCl-induced gastric lesions model in mice, as well as for cytotoxicity using the following human cell lines: normal lung fibroblasts (MRC-5), gastric adenocarcinoma cells (AGS) and liver hepatocellular carcinoma (Hep G2). A dose-response experiment showed that at 25 mg/kg the C-15 leucyl and C-15,19-dileucylester amides of junicedric acid reduced gastric lesions by about 65.6 and 49.6%, respectively, with an effect comparable to lansoprazole at 20 mg/kg (79.3% lesion reduction). The comparison of the gastroprotective effect of 18 new amino acid ester amides was carried out at a single oral dose of 25 mg/kg. Several compounds presented a strong gastroprotective effect, reducing gastric lesions in the 70.9-87.8% range. The diprolyl derivative of junicedric acid, the most active product of this study (87.8% lesion reduction at 25 mg/kg) presented a cytotoxicity value comparable with that of the reference compound lansoprazole. The structure-activity relationships are discussed.

Plant growth inhibitory activity of N, N´-di-(4-R-phenyl)-alkanediamides / Actividad inhibitoria de crecimiento de plantas de N, N’-di-(4-R-phenyl)-alcanodiamidas

Diamides are a class of metabolites that occurring in some Meliaceae plants, in Aglaia spp for example, with an ample body of biological activities, being insecticidal and herbicidal two of the most important. In our program of search for botanical pesticides, a series of N,N´-di-(4-R-phenyl)-alkanediamides was evaluated for its herbicidal activity. Many of the analogues tested exhibited moderate to good herbicidal activity both pre-emergence and post-emergence and have been found to inhibit energetic metabolism of pre-emergence weeds. The structure-activity relationships were probed by substitution on the benzene ring. Among the variations investigated, it was found that maximal herbicidal activity was obtained by substitution of –F, -CN and -Br at the aromatic portion and by n=2 of the aliphatic long chain. This last number of carbons (n=2) substitution was the key for the inhibitory activity.

Diamidas son una clase de metabolitos que estan presentes en plantas perteneciente a la familia de la Meliaceas, en Aglaia por ejemplo, poseen un amplio cuerpo de actividades biologicas, siendo la insecticida y la herbicida dos de las mas importantes. En nuestro programa para la busqueda de pesticidas botanicos, una serie de N,N’-di-(4-R-phenyl)-alkanodiamidas se evaluo para su actividad herbicida. Muchos de los analogos exhibieron desde buenas a moderadas actividades, tanto como pre-emergentes como post-emergentes y ademas se encontro que inhiben el metabolismo pre-emergente energetico de malezas. La relacion estructura-actividad fue probada por sustitución sobre al anillo aromatico. Entre las variaciones investigadas, se encontro que la maxima actividad herbicida se obtuvo por sustitución de F, CN y Br en la porcion aromatica y por n=2 del largo de la cadena alifatica. Este ultimo numero de carbonos de sustitución (n=2) fue clave para la actividad inhibitoria.

Sensitivities of the alternative respiratory components of potato tuber mitochondria to thiol reagents and Ca2+.

Plant mitochondria differ from those of mammals, since they incorporate an alternative electron transport pathway, which branches at ubiquinol to an alternative oxidase (AOX), characteristically inhibited by salicylhydroxamic acid (SHAM). Another feature of plant mitochondria is that besides complex I (EC 1.6.5.3) they possess alternative NAD(P)H-dehydrogenases insensitive to rotenone. Many stress conditions are known to alter the expression of the alternative electron transport pathway in plant mitochondria. In the present study we investigated the effects of some thiol reagents and Ca(2+) on potato mitochondrial respiratory chain presenting different activities of the alternative respiratory components AOX and external NADH dehydrogenase, a condition induced by previous treatment of potato tubers (Solanum tuberosum L., cv. Bintje) to cold stress. The results showed that Ca(2+) presented an inhibitory effect on AOX pathway in potato mitochondria energized with NADH or succinate, which was only now observed when the cytochrome pathway was inhibited by cyanide. When the cytochrome pathway was functional, Ca(2+) stimulated the external NADH dehydrogenase. Diamide was a potent AOX inhibitor and this effect was only now observed when the cytochrome pathway was inactive, as was the case for the calcium ion. Mersalyl inhibited the externally located NADH dehydrogenase and had no effect on AOX activity. The results may represent an important function of Ca(2+) on the alternative mitochondrial enzymes NADH-DH(ext) and AOX.

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore.

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.

Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy.

We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200 nm. Under conditions of oxidative stress the diameters changed to 400 nm. This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry.