Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

The effects of ethylene on the HCl-extractability of trace elements during soybean seed germination

Background Ethylene is capable of promoting seed germination in some plant species. Mobilization of metals such as Fe, Cu, Mn, and Zn in mature seeds takes place when seeds are germinating. However, whether ethylene is involved in the regulation of soybean seed germination and metal element mobilization during early seed germination stage remains unknown. In the present study, seeds were treated with ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and double distilled H2O (ddH(2)0) treatment was used as control. Ethylene emission, ACC synthase (ACS) expression, ACS enzyme activity and Ca, Zn, Mn, Cu and Fe content in hypocotyls were qualified to analyze the relationship between ethylene and mobilization of these elements. Results The results showed that ACS expression, ACS enzyme activity and ethylene emission peaked at 1 and 7 d after sowing. AVG inhibited ethylene production, promoted the hypocotyls length, ACS expression and its activity, concentrations of total and HCl-extractable Zn, and HCl-extractable Fe in hypocotyls, while ACC caused opposite effects. AVG and ACC treatment had no significantly effects on total and HCl-extractable Ca, Cu and HCl-extractable Mn. Total Mn concentration was promoted by AVG at 1, 3, and 5 d significantly, while ACC treatment tended to have no significantly effects on Mn concentration. Conclusion These findings suggested that ethylene is at least partly involved in the regulation of soybean seed germination. Remobilization of Zn and Fe may be negatively regulated by ethylene.

New insights into steroidogenesis in normo- and hyperandrogenic polycystic ovary syndrome patients / Novos aspectos na esteroidogênese em pacientes normo ou hiperandrogênicas com síndrome de ovários policísticos

OBJECTIVE: This study sought to examine corticosteroidogenic enzyme activities in normo- and hyperandrogenic polycystic ovary syndrome (PCOS) patients. SUBJECTS AND METHODS: This cohort study included 81 patients with biochemical hyperandrogenism and 41 patients with normal androgen levels. Enzyme activities were assessed according to the serum steroid product/precursor ratios at baseline and after adrenal stimulation. RESULTS: At baseline, in the delta 4 (Δ4) pathway, hyperandrogenic patients showed greater 17-hydroxylase and 17,20 lyase activities in converting progesterone (P4) into 17-hydroxyprogesterone (17-OHP4) and 17-hydroxypregnenolone (17-OHPE) into androstenedione (A) (p = 0.0005 and p = 0.047, respectively) compared to normoandrogenic patients. In the delta 5 (Δ5) pathway, the 17-hydroxylase and 17,20 lyase enzymes showed similar activities in both groups. Hyperandrogenic patients presented lower 21-hydroxylase, lower 11β-hydroxylase (p = 0.0001), and statistically significant increases in 3β-hydroxysteroid dehydrogenase II (3β-HSDII) activities (p < 0.0001). Following tetracosactrin stimulation, only the 17,20 lyase activity remained up-regulated in the Δ4 pathway (p < 0.0001). CONCLUSION: Hyperandrogenic patients had higher 17,20 lyase activity, both at baseline and after adrenal stimulation. Greater conversion of dehydroepiandrosterone (DHEA) into A with normal conversion of 17-OHPE to 17-OHP4 in hyperandrogenic PCOS patients indicated different levels of 3β-HSDII activity in adrenal cells, and hyperandrogenic patients had lower 11β-hydroxylase and 21-hydroxylase activities.

OBJETIVO: O objetivo deste estudo foi examinar a atividade de enzimas responsáveis pela produção de corticosteroides em pacientes normo e hiperandrogênicas com síndrome de ovários policísticos (SOP). SUJEITOS E MÉTODOS: A coorte estudada incluiu 81 pacientes com hiperandrogenismo bioquímico e 41 pacientes com níveis normais de androgênio. A atividade enzimática foi avaliada de acordo com as proporções de produto/precursor do esteroide sérico, no momento inicial do estudo e depois de estimulação adrenal. RESULTADOS: No momento inicial, na via delta 4 (Δ4), as pacientes hiperandrogênicas mostraram maior atividade da 17-hidroxilase e 17,20 liase na conversão da progesterona (P4) em 17-hidroxiprogesterona (17-OHP4) e na conversão da 17-hidroxipregnenolona (17-OHPE) em androstenediona (A) (p = 0,0005 e p = 0,047, respectivamente) em comparação com pacientes normoandrogênicas. Na via delta 5 (Δ5), a 17-hidroxilase e a 17,20 liase mostraram atividades similares nos dois grupos. As pacientes hiperandrogênicas mostraram menor atividade da 21-hidroxilase, menor atividade da 11β-hidroxilase (p = 0,0001) e aumento estatisticamente significativo na atividade da 3β-hidroxiesteroide desidrogenase II (3β-HSDII) (p < 0.0001). Após a estimulação com tetracosactrin, apenas a atividade da 17,20 liase permaneceu regulada para cima na via Δ4 (p < 0.0001). CONCLUSÃO: As pacientes hiperandrogênicas apresentaram atividade mais alta da 17,20 liase, tanto no momento inicial quanto depois da estimulação adrenal. Maior conversão da desidroepiandrosterona (DHEA) em A com conversão normal da 17-OHPE em 17-OHP4 em pacientes hiperandrogênicas com SOP indica níveis diferentes de atividade da 3β-HSDII em células da adrenal, e pacientes hiperandrogênicas apresentaram menores atividades da 11β-hidroxilase e da 21-hidroxilase.

Pectinase production by Penicillium viridicatum RFC3 by solid state fermentation using agricultural wastes and agro-industrial by-products

Pectin lyase and polygalacturonase production by newly isolated Penicillium viridicatum strain Rfc3 was carried out by means of solid fermentation using orange bagasse, corn tegument, wheat bran and mango and banana peels as carbon sources. The maximal activity value of polygalacturonase (Pg) (30 U.g-1) was obtained using wheat bran as carbon source while maximal pectin lyase (Pl) (2000 U.g-1) activity value was obtained in medium composed of orange bagasse. Mixtures of banana or mango peels with sugar cane bagasse resulted in increased Pg and Pl production compared to fermentations in which this residue was not used. The mixture of orange bagasse and wheat bran (50 percent) increased the production of Pg and Pl to 55 U.g-1 and 3540 U.g-1 respectively. Fractions of Pg and Pl, isolated by gel filtration in Sephadex G50, presented optimum activity at pH 5.0 and 10.5 respectively. Maximal activity of Pg and Pl fractions was determined at 55oC and 50oC respectively. Pg was stable in neutral pH range and at 40oC whereas Pl was stable in acidic pH and at 35oC, for 1h.

Screening and genetic improvement of pectinolytic fungi for degumming of textil e fibers

Aiming at contributing to technological improvements in plant fiber processing methods, this paper reports research work on the obtainment of more efficient pectinase-producing fungi strains. More specifically, this work reports the analysis of 18 strains of filamentous fungi, with the purpose of obtaining enzymes for textile fibers degumming. The strains were evaluated for production of pectinolytic enzymes under several growth conditions (culture medium and growth temperature). Production of pectinases was measured by an enzymatic index (EI) in solid pectin medium. Among the tested strains, Penicillium chrysogeum IFO 4626 (Q 176) showed the best performance. Genetic improvement of this strain was carried out to increase its pectinase production, while keeping cellulase activity down to a negligible level, since cellulases are known to decrease the resistance of the fiber. Variability was induced through several cycles of mutation and selection by exposing conidea to ultra-violet light (UV). We selected 39 out of 390 isolated colonies. Resulting mutants produced nine times more pectin lyase (PL) than the original strain in terms of PL specific activity, and five times more in terms of PL activity (i.e.mmoles liberated per minute of reaction per mL of medium). Periodically, mutant performance was evaluated in solid pectin medium. Genetic stability was maintained for four years after isolation.

Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer

Penicillium griseoroseum foi cultivado em biorreatores em meio mineral suplementado com extrato de levedura e sacarose. As influências das concentrações do inóculo e da fonte de carbono, da aeração e do pH do meio de cultivo sobre a produção de pectina liase (PL), bem como a capacidade de P. griseoroseum em produzir PL quando cultivado em caldo de cana diluído foram avaliadas. A concentração do inóculo não influenciou significativamente a produção de PL. O cultivo do fungo em biorreatores não aerados favoreceu a produção da PL em detrimento aos biorreatores com injeção de ar. Maior produção de PL foi obtida com o cultivo de P. griseoroseum em meio com pH 6,3 - 7,2, adicionado de 60 mM de sacarose. Quando cultivado em caldo de cana diluído, 25per center (v/v), sem suplementação com extrato de levedura, a atividade máxima de PL alcançada foi igual as das condições citadas acima.

Deficiencia da 17,20-desmolase (lyase) e pseudo-hermafroditismo masculino entre indios do rio Curipi. / Deficiency of 17,20-desmolase (lyase) and male pseudohermaphroditism in Indians of the Curipi river

O autor relata a ocorrencia de 5 casos de pseudo-hermafroditismo masculino, devidos a defeito de sintese da testosterona entre os indios do rio Curipi, no Territorio do Amapa.Estuda as dosagens hormonais da testosterona (T), androstenediona (delta), do sulfato de deidroepiandrosterona (DHEA-S), da progesterona (P) e do estradiol (E2) basais e apos estimulo com gonadotropina corionica (HCG).Observou valores diminuidos da testosterona, da androstenediona e do sulfato de deidroepiandrosterona, valores altos da progesterona e da 17-alfa-hidroxiprogesterona, o que indica defeito de sintese da testosterona devido a falta parcial da enzima 17,20-desmolase