Natural parasitism in fruit fly (Diptera: Tephritidae) and interaction with wild hosts surrounding apple orchards adjacent to Atlantic Forest fragments in Paraná State, Brazil / Parasitismo natural de mosca-das-frutas (Diptera: Tephritidae) e interação com hospedeiros silvestres em pomares de macieira adjacentes a fragmentos de Mata Atlântica no Estado do Paraná, Brasil

The South American fruit fly, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), is an important pest in the subtropical region of Brazil. This insect has tritrophic relation between wild fruits and parasitoids and is associated with apple (Malus domestica Borkh.) orchards adjacent to the Atlantic Forest in Paraná. We thus investigated the degree of infestation of the fruit fly and natural parasitism in wild and cultivated fruits surrounding apple orchards. For this purpose, we collected fruits of Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer and Eriobotrya japonica (Thumb) in apple orchards adjacent to the Atlantic Forest located in Campo do Tenente, Lapa and Porto Amazonas counties. In total, we collected 18,289 fruits during four growing years. The occurrence of A. fraterculus depends on the susceptible period of apple fruits. A. sellowiana and P. cattleianum were considered primary fruit fly multipliers and P. guajava was secondary, all occurring after the apple harvest (IS period). The group of parasitoids with A. fraterculus was Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) and Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae) all of which are first records in the Atlantic Forest in Paraná. First record of O. bellus occurring in the State of Paraná, as well as, first record of the tritrophic association between host plant A. neosericea, parasitoids D. areolatus and O. bellus and fruit fly A. fraterculus. The host P. cattleianum stood out among the Myrtaceae species in regard to the high diversity of parasitoid species (81% of parasitoids). The total number of Figitidae species (76.5%) was higher [...].

Mosca-das-frutas sul-americana, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), é uma importante praga da região subtropical do Brasil. Este inseto tem relação tritrófico entre frutos silvestres e parasitoides e está associado a pomares de macieiras (Malus domestica Borkh.) adjacentes à Mata Atlântica no Paraná. Assim, investigamos o grau de infestação da mosca-das-frutas e o parasitismo natural em frutas silvestres e cultivadas ao redor de pomares de maçã. Para tanto, foram coletados frutos de Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer e Eriobotrya japonica (Thumb) em pomares de maçã adjacentes à Mata Atlântica localizados nos municípios de Campo do Tenente, Lapa e Porto Amazonas. No total, coletamos 18.289 frutos durante quatro anos de cultivo. A ocorrência de A. fraterculus depende do período de suscetibilidade dos frutos da maçã. A. sellowiana e P. cattleianum foram considerados multiplicadores primários de mosca-das-frutas e P. guajava foi secundário, todos ocorrendo após a colheita da maçã (período IS). Os parasitóides a associados a A. fraterculus foram Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) e Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae), todos os quais são primeiros registros na Mata Atlântica no Paraná. Primeiro registro de O. bellus ocorrendo no Estado do Paraná, assim como, primeiro registro da associação tritrófica entre o hospedeiro A. neosericea, parasitoides D. areolatus e O. bellus e mosca-das-frutas A. fraterculus. O hospedeiro P. cattleianum se destacou entre as espécies de Myrtaceae pela alta diversidade de parasitóides associados (81% dos parasitóides). O número total de espécies de Figitidae (76,5%) foi superior [...].

Natural parasitism in fruit fly (Diptera: Tephritidae) and interaction with wild hosts surrounding apple orchards adjacent to Atlantic Forest fragments in Paraná State, Brazil

Abstract The South American fruit fly, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), is an important pest in the subtropical region of Brazil. This insect has tritrophic relation between wild fruits and parasitoids and is associated with apple (Malus domestica Borkh.) orchards adjacent to the Atlantic Forest in Paraná. We thus investigated the degree of infestation of the fruit fly and natural parasitism in wild and cultivated fruits surrounding apple orchards. For this purpose, we collected fruits of Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer and Eriobotrya japonica (Thumb) in apple orchards adjacent to the Atlantic Forest located in Campo do Tenente, Lapa and Porto Amazonas counties. In total, we collected 18,289 fruits during four growing years. The occurrence of A. fraterculus depends on the susceptible period of apple fruits. A. sellowiana and P. cattleianum were considered primary fruit fly multipliers and P. guajava was secondary, all occurring after the apple harvest (IS period). The group of parasitoids with A. fraterculus was Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) and Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae) all of which are first records in the Atlantic Forest in Paraná. First record of O. bellus occurring in the State of Paraná, as well as, first record of the tritrophic association between host plant A. neosericea, parasitoids D. areolatus and O. bellus and fruit fly A. fraterculus. The host P. cattleianum stood out among the Myrtaceae species in regard to the high diversity of parasitoid species (81% of parasitoids). The total number of Figitidae species (76.5%) was higher than that of Braconidae species. The influence of climatic events in southern Brazil on wild fruit production should be further studied to understand the association of A. fraterculus with the tritrophic relationship.

Resumo Mosca-das-frutas sul-americana, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), é uma importante praga da região subtropical do Brasil. Este inseto tem relação tritrófico entre frutos silvestres e parasitoides e está associado a pomares de macieiras (Malus domestica Borkh.) adjacentes à Mata Atlântica no Paraná. Assim, investigamos o grau de infestação da mosca-das-frutas e o parasitismo natural em frutas silvestres e cultivadas ao redor de pomares de maçã. Para tanto, foram coletados frutos de Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer e Eriobotrya japonica (Thumb) em pomares de maçã adjacentes à Mata Atlântica localizados nos municípios de Campo do Tenente, Lapa e Porto Amazonas. No total, coletamos 18.289 frutos durante quatro anos de cultivo. A ocorrência de A. fraterculus depende do período de suscetibilidade dos frutos da maçã. A. sellowiana e P. cattleianum foram considerados multiplicadores primários de mosca-das-frutas e P. guajava foi secundário, todos ocorrendo após a colheita da maçã (período IS). Os parasitóides a associados a A. fraterculus foram Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) e Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae), todos os quais são primeiros registros na Mata Atlântica no Paraná. Primeiro registro de O. bellus ocorrendo no Estado do Paraná, assim como, primeiro registro da associação tritrófica entre o hospedeiro A. neosericea, parasitoides D. areolatus e O. bellus e mosca-das-frutas A. fraterculus. O hospedeiro P. cattleianum se destacou entre as espécies de Myrtaceae pela alta diversidade de parasitóides associados (81% dos parasitóides). O número total de espécies de Figitidae (76,5%) foi superior ao de espécies de Braconidae. A influência de eventos climáticos no sul do Brasil na produção de frutas silvestres deve ser mais estudada para entender a associação de A. fraterculus com a relação tritrófica.

Natural parasitism in fruit fly (Diptera: Tephritidae) and interaction with wild hosts surrounding apple orchards adjacent to Atlantic Forest fragments in Paraná State, Brazil / Parasitismo natural de mosca-das-frutas (Diptera: Tephritidae) e interação com hospedeiros silvestres em pomares de macieira adjacentes a fragmentos de Mata Atlântica no Estado do Paraná, Brasil

Abstract The South American fruit fly, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), is an important pest in the subtropical region of Brazil. This insect has tritrophic relation between wild fruits and parasitoids and is associated with apple (Malus domestica Borkh.) orchards adjacent to the Atlantic Forest in Paraná. We thus investigated the degree of infestation of the fruit fly and natural parasitism in wild and cultivated fruits surrounding apple orchards. For this purpose, we collected fruits of Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer and Eriobotrya japonica (Thumb) in apple orchards adjacent to the Atlantic Forest located in Campo do Tenente, Lapa and Porto Amazonas counties. In total, we collected 18,289 fruits during four growing years. The occurrence of A. fraterculus depends on the susceptible period of apple fruits. A. sellowiana and P. cattleianum were considered primary fruit fly multipliers and P. guajava was secondary, all occurring after the apple harvest (IS period). The group of parasitoids with A. fraterculus was Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) and Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae) all of which are first records in the Atlantic Forest in Paraná. First record of O. bellus occurring in the State of Paraná, as well as, first record of the tritrophic association between host plant A. neosericea, parasitoids D. areolatus and O. bellus and fruit fly A. fraterculus. The host P. cattleianum stood out among the Myrtaceae species in regard to the high diversity of parasitoid species (81% of parasitoids). The total number of Figitidae species (76.5%) was higher than that of Braconidae species. The influence of climatic events in southern Brazil on wild fruit production should be further studied to understand the association of A. fraterculus with the tritrophic relationship.

Resumo Mosca-das-frutas sul-americana, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), é uma importante praga da região subtropical do Brasil. Este inseto tem relação tritrófico entre frutos silvestres e parasitoides e está associado a pomares de macieiras (Malus domestica Borkh.) adjacentes à Mata Atlântica no Paraná. Assim, investigamos o grau de infestação da mosca-das-frutas e o parasitismo natural em frutas silvestres e cultivadas ao redor de pomares de maçã. Para tanto, foram coletados frutos de Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer e Eriobotrya japonica (Thumb) em pomares de maçã adjacentes à Mata Atlântica localizados nos municípios de Campo do Tenente, Lapa e Porto Amazonas. No total, coletamos 18.289 frutos durante quatro anos de cultivo. A ocorrência de A. fraterculus depende do período de suscetibilidade dos frutos da maçã. A. sellowiana e P. cattleianum foram considerados multiplicadores primários de mosca-das-frutas e P. guajava foi secundário, todos ocorrendo após a colheita da maçã (período IS). Os parasitóides a associados a A. fraterculus foram Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) e Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae), todos os quais são primeiros registros na Mata Atlântica no Paraná. Primeiro registro de O. bellus ocorrendo no Estado do Paraná, assim como, primeiro registro da associação tritrófica entre o hospedeiro A. neosericea, parasitoides D. areolatus e O. bellus e mosca-das-frutas A. fraterculus. O hospedeiro P. cattleianum se destacou entre as espécies de Myrtaceae pela alta diversidade de parasitóides associados (81% dos parasitóides). O número total de espécies de Figitidae (76,5%) foi superior ao de espécies de Braconidae. A influência de eventos climáticos no sul do Brasil na produção de frutas silvestres deve ser mais estudada para entender a associação de A. fraterculus com a relação tritrófica.

Epidermal growth factor receptor regulates fibrinolytic pathway elements in cervical cancer: functional and prognostic implications

Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.

Faunistic analyses of fruit fly species (Diptera: Tephritidae) in orchards surrounded by Atlantic Forest fragments in the metropolitan region of Curitiba, Paraná state, Brazil / Análise faunística de moscas-das-frutas (Diptera: Tephritidae) em pomares rodeados por fragmentos da Mata Atlântica na região metropolitana de Curitiba, Paraná, Brasil

Abstract The objective of this trial was to characterize the assemblage structure of fruit flies (Diptera: Tephritidae) by determining the diversity and faunal indices. Fruit flies were collected for ten years between 2003 and 2015 in six municipalities of the Metropolitan Region of Curitiba, Paraná. The orchards were divided into three crop protection strategies: organic, conventional, and integrated pest management. The characteristic of each community was determined using the Shannon-Wiener, Margalef, and Pielou's indexes. The frequency, constancy, and dominance indexes were also determined. Biological material was analyzed using the explanatory variables: site, crop, plant protection strategy, and susceptibility period. A total of 8,089 fruit flies were collected, of which 4,681 were females. The species Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene and A. pickeli Lima were recorded for the first time in Paraná State, the occurrence of three other species was recorded for the first time in the Metropolitan Region of Curitiba (A. montei Lima, A. obliqua Macquart, and A. sororcula Zucchi), in addition A. fraterculus (Wiedemann), A. grandis (Macquart) and Ceratitis capitata (Wiedemann). A. fraterculus accounted for 98.5% of the collected species. Only A. fraterculus and A. sororcula were found in fruits. Fruit flies occurred predominantly in peach trees cultivated under organic conditions. The Shannon index indicated that the municipality of Cerro Azul had the highest diversity and richness whereas apple orchards had the lowest diversity. Crops cultivated under organic conditions exhibited the highest diversity whereas those cultivated under conventional conditions had the highest evenness index. The largest specimens number was collected during the period of crop susceptibility.

Resumo O objetivo deste estudo foi caracterizar agrupamento de espécies de moscas-das-frutas (Diptera: Tephritidae) pela determinação da diversidade e dos índices faunísticos. As moscas-das-frutas foram coletadas por dez anos entre 2003 e 2015 em seis municípios da Região Metropolitana de Curitiba, no Paraná. Os pomares foram divididos em três estratégias de proteção de culturas: orgânica, convencional e integrada. A característica de cada comunidade foi determinada usando os índices de Shannon-Wiener, Margalef e Pielou. Os índices de freqüência, constância e dominância também foram determinados. O material biológico foi analisado utilizando as variáveis explicativas: local, cultura, estratégia de proteção e período de susceptibilidade. Foram coletadas 8.089 moscas-das-frutas, sendo 4.681 fêmeas. As espécies Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene e A. pickeli Lima foram registradas pela primeira vez no Estado do Paraná, e pela primeira vez, na Região Metropolitana de Curitiba, as espécies A. montei Lima, A. obliqua Macquart e A. sororcula Zucchi), além de A. fraterculus (Wiedemann), A. grandis (Macquart) e Ceratitis capitata (Wiedemann). A. fraterculus foi responsável por 98,5% das espécies coletadas. Apenas A. fraterculus e A. sororcula foram encontrados em frutos. As moscas-das-frutas ocorreram predominantemente em pessegueiros cultivados sob condições orgânicas. O índice de Shannon indicou que o município de Cerro Azul teve a maior diversidade e riqueza, enquanto que a cultura macieira teve a menor diversidade. As culturas sob condição orgânica exibiram a maior diversidade, enquanto as cultivadas em condições convencionais apresentaram o maior índice de uniformidade. O maior número de espécimes foi coletado durante o período de suscetibilidade à cultura.

Lopesia davillae (Diptera, Cecidomyiidae), a new species of gall midge from Brazil associated with Davilla rugosa (Dilleniaceae) / Lopesia davillae (Diptera, Cecidomyiidae), uma nova espécie de cecidomiídeo do Brazil associada à Davilla rugosa (Dilleniaceae)

Abstract Lopesia davillae, a new species of gall midge associated with the reproductive structures of Davilla rugosa (Dilleniaceae), is described and illustrated (larva, pupa, male, female, and damage) based on material collected in Rio de Janeiro, Brazil. This new species is compared with its congeneric species. This is the first record of Lopesia on Dilleniaceae.

Resumo Lopesia davillae, uma nova espécie de Cecidomyiidae associada a estruturas reprodutivas de Davilla rugosa (Dilleniaceae), é descrita e ilustrada (larva, pupa, macho, fêmea e dano) com base em material coletado no estado do Rio de Janeiro, Brasil. A nova espécie é comparada com as outras espécies conhecidas do mesmo gênero. Este é o primeiro registro de Lopesia em Dilleniaceae.

Faunistic analyses of fruit fly species (Diptera: Tephritidae) in orchards surrounded by Atlantic Forest fragments in the metropolitan region of Curitiba, Paraná state, Brazil

Abstract The objective of this trial was to characterize the assemblage structure of fruit flies (Diptera: Tephritidae) by determining the diversity and faunal indices. Fruit flies were collected for ten years between 2003 and 2015 in six municipalities of the Metropolitan Region of Curitiba, Paraná. The orchards were divided into three crop protection strategies: organic, conventional, and integrated pest management. The characteristic of each community was determined using the Shannon-Wiener, Margalef, and Pielous indexes. The frequency, constancy, and dominance indexes were also determined. Biological material was analyzed using the explanatory variables: site, crop, plant protection strategy, and susceptibility period. A total of 8,089 fruit flies were collected, of which 4,681 were females. The species Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene and A. pickeli Lima were recorded for the first time in Paraná State, the occurrence of three other species was recorded for the first time in the Metropolitan Region of Curitiba (A. montei Lima, A. obliqua Macquart, and A. sororcula Zucchi), in addition A. fraterculus (Wiedemann), A. grandis (Macquart) and Ceratitis capitata (Wiedemann). A. fraterculus accounted for 98.5% of the collected species. Only A. fraterculus and A. sororcula were found in fruits. Fruit flies occurred predominantly in peach trees cultivated under organic conditions. The Shannon index indicated that the municipality of Cerro Azul had the highest diversity and richness whereas apple orchards had the lowest diversity. Crops cultivated under organic conditions exhibited the highest diversity whereas those cultivated under conventional conditions had the highest evenness index. The largest specimens number was collected during the period of crop susceptibility.

Resumo O objetivo deste estudo foi caracterizar agrupamento de espécies de moscas-das-frutas (Diptera: Tephritidae) pela determinação da diversidade e dos índices faunísticos. As moscas-das-frutas foram coletadas por dez anos entre 2003 e 2015 em seis municípios da Região Metropolitana de Curitiba, no Paraná. Os pomares foram divididos em três estratégias de proteção de culturas: orgânica, convencional e integrada. A característica de cada comunidade foi determinada usando os índices de Shannon-Wiener, Margalef e Pielou. Os índices de freqüência, constância e dominância também foram determinados. O material biológico foi analisado utilizando as variáveis explicativas: local, cultura, estratégia de proteção e período de susceptibilidade. Foram coletadas 8.089 moscas-das-frutas, sendo 4.681 fêmeas. As espécies Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene e A. pickeli Lima foram registradas pela primeira vez no Estado do Paraná, e pela primeira vez, na Região Metropolitana de Curitiba, as espécies A. montei Lima, A. obliqua Macquart e A. sororcula Zucchi), além de A. fraterculus (Wiedemann), A. grandis (Macquart) e Ceratitis capitata (Wiedemann). A. fraterculus foi responsável por 98,5% das espécies coletadas. Apenas A. fraterculus e A. sororcula foram encontrados em frutos. As moscas-das-frutas ocorreram predominantemente em pessegueiros cultivados sob condições orgânicas. O índice de Shannon indicou que o município de Cerro Azul teve a maior diversidade e riqueza, enquanto que a cultura macieira teve a menor diversidade. As culturas sob condição orgânica exibiram a maior diversidade, enquanto as cultivadas em condições convencionais apresentaram o maior índice de uniformidade. O maior número de espécimes foi coletado durante o período de suscetibilidade à cultura.

Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Produção de biomassa, teor e composição do óleo essencial de Mentha x piperita L. em resposta a fontes e doses de nitrogênio / Biomass production, essential oil yield and composition of Mentha x piperita L. according to nitrogen sources and doses

A adubação nitrogenada influencia o teor e a qualidade dos óleos essenciais de plantas aromáticas. O objetivo deste trabalho foi avaliar a influência de diferentes fontes e doses de nitrogênio na produção de biomassa e teor do óleo essencial de M. x piperita. O delineamento experimental foi de blocos casualizados em esquema fatorial 2 x 3 (duas fontes de nitrogênio e três doses). A aplicação nitrogenada de cobertura foi realizada 30 dias após o transplante das mudas. O óleo essencial foi extraído pelo processo de hidrodestilação em aparelho graduado Clevenger e sua composição química foi analisada por cromatografia gasosa acoplada à espectrometria de massa. As fontes e doses de nitrogênio não influenciaram significativamente a biomassa de folhas e ramos, assim como a produtividade e o teor do óleo essencial. No entanto, a fonte de nitrogênio pode alterar significativamente a composição do óleo essencial de M. x piperita. O teor de mentol e neomentol foi menor quando 40 kg ha-1 de uréia foi aplicada, em comparação com a mesma dose de sulfato de amônio. Os teores de mentona foram influenciados apenas pela fonte de nitrogênio, onde níveis mais elevados foram observados quando o sulfato de amônio foi aplicado. Nenhuma alteração nos teores de mentofurano foi encontrada para as diferentes fontes e doses de nitrogênio. Considerando a grande importância econômica de mentol presente no óleo essencial de menta, os resultados obtidos indicaram que a utilização de 20 kg ha-1 de nitrogênio é suficiente para garantir a boa produtividade de óleo essencial e alto teor de mentol, e que altas doses de uréia devem ser evitadas.

Nitrogen fertilization affects the essential oil yield and composition of aromatic plants. The objective of this work was to evaluate the effect of nitrogen sources and doses on biomass production and essential oil yield and composition of M. x piperita L. The experimental design was in randomized blocks with four replications and the treatments in a 2 x 3 factorial design, corresponding to two nitrogen sources and three nitrogen doses. The nitrogen topdressing was applied 30 days after planting. The essential oil was obtained by hydrodestilation using a Clevenger apparatus, and the composition was analyzed by gas chromatography and coupled to a mass spectrometry. The nitrogen sources and doses did not significantly affect the leaf and stem biomass, as well as the essential oil yield and productivity. However, the nitrogen source can significantly change the essential oil composition of M. x piperita. The level of menthol and neomenthol was lower when 40 kg ha-1 of urea was applied compared to the same dose of ammonium sulphate. Menthone levels were affected only by the nitrogen source, where higher levels were observed when ammonium sulphate was applied. No changes on menthofurane levels were found for different nitrogen sources and doses. Considering the great economic importance of menthol from mint essential oils, the obtained results indicated that the use of 20 kg ha-1 of nitrogen is enough to guarantee satisfactory essential oil yield and great menthol levels and that high doses of urea should be avoided.

Diversity of 16S rRNA genes from bacteria of sugarcane rhizosphere soil

Sugarcane is an important agricultural product of Brazil, with a total production of more than 500 million tons. Knowledge of the bacterial community associated with agricultural crops and the soil status is a decisive step towards understanding how microorganisms influence crop productivity. However, most studies aim to isolate endophytic or rhizosphere bacteria associated with the plant by culture-dependent approaches. Culture-independent approaches allow a more comprehensive view of entire bacterial communities in the environment. In the present study, we have used this approach to assess the bacterial community in the rhizosphere soil of sugarcane at different times and under different nitrogen fertilization conditions. At the high taxonomic level, few differences between samples were observed, with the phylum Proteobacteria (29.6 percent) predominating, followed by Acidobacteria (23.4 percent), Bacteroidetes (12.1 percent), Firmicutes (10.2 percent), and Actinobacteria (5.6 percent). The exception was the Verrucomicrobia phylum whose prevalence in N-fertilized soils was approximately 0.7 percent and increased to 5.2 percent in the non-fertilized soil, suggesting that this group may be an indicator of nitrogen availability in soils. However, at low taxonomic levels a higher diversity was found associated with plants receiving nitrogen fertilizer. Bacillus was the most predominant genus, accounting for 19.7 percent of all genera observed. Classically reported nitrogen-fixing and/or plant growth-promoting bacterial genera, such as Azospirillum, Rhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia were also found although at a lower prevalence.

Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.

Desenvolvimento vegetativo de Mentha campestris Schur e produção de mentol em diferentes espaçamentos de plantio e épocas de colheita / Vegetative development of Mentha campestris Schur and menthol production in different row spaces and harvest times

A produção de óleos essenciais nas plantas aromáticas é influenciada por fatores bióticos e abióticos. A demanda por esses produtos tem aumentado, sendo os óleos essenciais do gênero Mentha de grande interesse nas indústrias farmacêutica, de cosméticos, alimentícia e agrícola, principalmente em função do composto mentol. Esse trabalho teve como objetivo avaliar o efeito de três espaçamentos de plantio (0,60 x 0,15 m; 0,60 x 0,30 m e 0,60 x 0,45 m) e duas épocas de colheita (60 e 90 dias após o plantio) na espécie Mentha campestris Schur. O experimento foi conduzido no Centro de Estações Experimentais do Canguiri-UFPR, em Pinhais-PR, no período de janeiro a abril de 2008. O delineamento utilizado foi o de blocos ao acaso em esquema de parcelas subdivididas. Houve diferença significativa para todas as variáveis analisadas. As massas secas de folhas, ramos e total foram maiores que na primeira época. Para a biomassa seca de folhas foram observados maiores valores no menor espaçamento de plantio. O rendimento de óleo essencial foi maior na segunda época de colheita e nos espaçamentos maiores. A produtividade do óleo também foi maior na segunda época de colheita, porém no espaçamento mais adensado. Pode-se concluir como recomendação para M. campestris Schur o espaçamento 0,60 x 0,15 m e colheita aos 90 dias, por terem atingido maior biomassa, rendimento de óleo essencial e produtividade de mentol por hectare.

Essential oil production in aromatic plants is influenced by biotic and abiotic factors. The demand for these products has increased, and essential oils from the genus Mentha have been of great interest for pharmaceutical, cosmetic, food and agronomic industries, especially because of the compound menthol. This study aimed to evaluate the effect of three row spaces (0.60 x 0.15 m; 0.60 x 0.30 m and 0.60 x 0.45 m) and two harvest times (60 and 90 days after planting) on the species Mentha campestris Schur. The experiment was carried out at Canguiri Experimental Stations-UFPR, in Pinhais, Paraná State , Brazil, between January and April 2008. The experimental design was in randomized blocks in split-plot. There was significant difference for all variables. In the second harvest time, leaf, branch and total dry yields were higher than those in the first time. For leaf dry mass, the highest values were observed in the narrowest space. Essential oil yield was higher in the second harvest and in wider rows. Essential oil productivity was also higher in the second harvest time, but in the narrowest row. The space 0.60 x 0.15 m and harvest at 90 days can be recommended due to their higher biomass, essential oil yield and menthol productivity per hectare.

Increased expression of tissue factor and protease-activated receptor-1 does not correlate with thrombosis in human lung adenocarcinoma

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. Moreover, TF may promote angiogenesis and tumor growth. In addition to TF, thrombin seems to play a relevant role in tumor biology, mainly through activation of protease-activated receptor-1 (PAR-1). In the present study, we prospectively studied 39 lung adenocarcinoma patients in relation to the tumor expression levels of TF and PAR-1 and their correlation with thrombosis outcome and survival. Immunohistochemical analysis showed TF positivity in 22 patients (56 percent), most of them in advanced stages (III and IV). Expression of PAR-1 was found in 15 patients (39 percent), most of them also in advanced stages (III and IV). Remarkably, no correlation was observed between the expression of TF or PAR-1 and risk for thrombosis development. On the other hand, patients who were positive for TF or PAR-1 tended to have decreased long-term survival. We conclude that immunolocalization of either TF or PAR-1 in lung adenocarcinoma may predict a poor prognosis although lacking correlation with thrombosis outcome.

Contribution to the discussions on the origin of the cerrado biome: Brazilian savanna / Contribuição às discussões sobre a origem do bioma cerrado: savana brasileira

Theories that attempt to explain the origin of the cerrado biome are mostly based on the isolated action of three major factors: climate, fire and soil. Another factor that has been mentioned is that of human interference. We hypothesise that the evolutionary origin of this biome resulted from the complex interaction of climate, fire and soil, with climate being the triggering agent of this assumed interaction. Fire, as well as acid and dystrophic soils, would be factors involved in the selection of savanna species throughout climatic events, during the Tertiary and the Quaternary, e.g. Pliocene and Pleistocene. The genesis of the physiognomies that would give rise to cerrado sensu lato, rather than forest formations, could have occurred due to the strong pressure exerted by the reduction in water availability, and the selection of the species adapted to the new conditions imposed by the environment. The characteristics of cerrado sensu lato soil, originated from edaphic impoverishment caused by lixiviation and successive past fires, would remain, even after hydric availability increased following the Pleistocene glaciations.

Teorias que tentam explicar a origem do bioma cerrado baseiam-se, principalmente, na ação isolada de três fatores principais: o clima, o fogo e o solo. Outro fator mencionado ainda é a interferência humana. Quanto à origem evolutiva desse bioma, o presente estudo propõe uma hipótese que considerou a complexa interação dos três primeiros fatores, mencionados acima, considerando o clima como elemento desencadeador dessa possível interação. O fogo e solos ácidos e distróficos teriam atuado como fatores de seleção de espécies savânicas, ao longo das oscilações climáticas durante o terciário e o quaternário, e.g. Plioceno e Pleistoceno. A gênese das fisionomias que passariam a compor o chamado cerrado sensu lato, em detrimento de formações florestais, teria resultado da forte pressão exercida pela redução da disponibilidade hídrica e da seleção de espécies adaptadas às novas condições impostas pelo ambiente. As características do solo, que passariam a manter o cerrado sensu lato, oriundas do empobrecimento edáfico pela lixiviação e sucessivos incêndios pretéritos, permaneceriam, mesmo com o restabelecimento de uma maior disponibilidade hídrica, após as glaciações do Pleistoceno.

Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein

Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.

Ecotoxicological assessment of water and sediment of the Corumbataí River, SP, Brazil

The Corumbataí River drains an economically important area which is mainly represented by the municipalities of Piracicaba and Rio Claro. In view of the impacts caused by the discharge of industrial waste and domestic sewage into the Piracicaba River, the Corumbataí has become increasingly significant as a source of water for the municipality of Piracicaba. However, chemical, physical, and microbiological analyses carried out prior to the present study had already indicated a decline in the quality of the Corumbataí waters. This study aimed to assess, through water and sediment samples, both acute and chronic toxicity to Daphnia magna and Daphnia similis, and to analyze acid-volatile sulfide (AVS) and simultaneously extracted metal (SEM) in the sediment. Resulting data were intended to be a contribution to future projects for the management and recuperation of this system. To that aim, water and sediment were collected at seven Corumbataí sampling stations in November 2003 and March 2004. Acute toxicity to D. similis was detected in water and sediment samples from the Piracicaba station, located at the mouth of the Corumbataí River. Chronic toxicity was identified in the water or sediment samples of all stations, with the exception of Analândia Montante (upstream), at the head of the river. This was found to affect survival, growth, and fecundity of the test-organisms. The AVS and SEM analyses showed the bioavailability of the metals, thus explaining toxicity found in bioassaying samples of water and sediment. The use of two test-organism species made it possible to obtain a better assessment of the condition of both water and sediment samples of the Corumbataí River.

O rio Corumbataí drena uma área de importância econômica representada principalmente pelos municípios de Piracicaba e Rio Claro. Face aos impactos causados pelos lançamentos de efluentes industriais e domésticos no rio Piracicaba, o rio Corumbataí assumiu importância para o abastecimento do município de Piracicaba. Entretanto, análises químicas, físicas e microbiológicas realizadas no rio Corumbataí anteriormente a este estudo, indicaram a queda da qualidade de suas águas. Os objetivos deste estudo foram a avaliação da toxicidade aguda e da toxicidade crônica das amostras de água e sedimento, para Daphnia magna e Daphnia similis, e a análise do sulfeto volatilizável por acidificação (SVA) e dos metais simultaneamente extraídos do sedimento (MSE), no sentido de fornecer dados que possam contribuir com projetos futuros de manejo e recuperação desse sistema. Para tanto, água e sedimento provenientes de sete estações de coleta do rio Corumbataí foram coletados em novembro de 2003 e março de 2004. Foi detectada toxicidade aguda para D. similis das amostras de água e sedimento da estação Piracicaba, na foz do rio Corumbataí. A toxicidade crônica foi identificada na água ou no sedimento de todas as estações de coleta, exceto Analândia Montante (nascente do rio), influenciando a sobrevivência, crescimento e fecundidade dos organismos-teste. As análises do SVA e MSE revelaram a biodisponibilidade dos metais, explicando a toxicidade das amostras de água e de sedimento encontrada nos bioensaios. A adoção de duas espécies de organismos-teste possibilitou uma melhor avaliação dos compartimentos amostrados.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

Morphometric Analysis of Nucleus and Nucleolar Organizer Regions (NORs) in Tongue Squamous Cell Carcinoma (SCC) / Análisis Morfométrico de Núcleos y Regiones del Organizador Nucleolar (NORs) en Carcinomas de Células Escamosas de la Lengua (SCC)

The tongue is one of the most likely places for the primary squamous cell carcinoma (SCC) to arise in the western world.This work intends to do a morphometric evaluation of the cellular nucleus, as well as a morphometric analysis of the nucleolar organizer regions (NORs). The biopsy specimen was removed from patients with tongue SCC, staged T2N0M0, before they underwent treatment. They were, then, subject to hemiglossectomy and supraomohyoid neck dissection (SOHND) and presented different clinical evolution. According to that, they were subdivided in three groups: Group A, control group, formed by patients without tongue neoplasm (the histopathologic specimen was obtained from the Department of Pathology at Hospital de Base and Faculdade de Medicina de Sao José do Rio Preto - FAMERP-HB); Group B, T2N0M0+ hemiglossectomy + SOHND, survival of only 36 months; Group C, T2N0M0 + hemiglossectomy + SOHND, survival of 5 years. The morphometric analysis included the study of larger diameter, smaller diameter, medium diameter, the relation between larger/smaller diameters, perimeter, area, volume, the relation between volume/area, idiosyncrasy, shape coefficient and the nuclear contour index of the tumor cells from patients with tongue SCC, as well as the same measurements of the nucleolar organizer regions. The analysis was aleatory and double-blind. All the morphometric data were compiled and statistically evaluated by the Kruskall-Wallis test and by the ANOVA test, and a 5% alpha error rate was adopted.

La lengua es probablemente, el lugar en donde más frecuentemente surge el carcinoma primario de células escamosas (SCC), en el mundo occidental. Este estudio propone realizar la evaluación morfométrica del núcleo celular, así como el análisis morfométrico de las regiones de los organizadores nucleolares (NORs). La muestra de biopsia fue removida de pacientes con SCC de lengua, etapificado en T2N0M0, antes de recibir tratamiento. Los pacientes fueron sometidos a una hemiglosectomía y una disección supraomohiodea del cuello (SOHND) y presentaron diferente evolución clínica. De acuerdo a esto, fueron subdividos en tres grupos: Grupo A, grupo control formado por pacientes sin neoplasia de lengua (la muestra fue obtenida del Departamento de Patología del Hospital Base de la Facultad de Medicina de Sao José do Rio Preto - FAMERP-HB, Brasil); Grupo B, T2N0M0 + hemiglosectomía + SOHND, sobrevida de 36 meses; Grupo C, T2N0M0 + hemiglosectomía + SOHND, sobrevida 5 años. El estudio morfométrico incluyó los análisis del diámetro mayor, diámetro menor, diámetro medio, la relación entre el diámetro mayor/menor, perímetro, área, volumen, relación volumen/área, idiosincrasia, coeficiente de forma y el índice de contorno nuclear de células tumorales en pacientes con SCC de lengua, así también las mismas mediciones de las regiones nucleolares organizadas. El análisis fue aleatorio y doble ciego. Todos los datos morfométricos fueron recopilados y evaluados estadísticamente con el test de Kruskall-Wallis y el Test ANO VA, con un valor alfa de un 5%.