Bloqueio meiótico como alternativa para aumentar a produção de embriões clone por transferência nuclear

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2016.

Phosphodiesterase 6 generates intracellular cGMP microdomains in the native endothelium

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains

Phosphodiesterase 6 generates intracellular cGMP microdomains in the native endothelium

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains

Progress in understanding of the molecular basis underlying functional diversification of cyclic di-nucleotide turnover proteins

Cyclic di-GMP was the first cyclic di-nucleotide second messenger described, presaging the discovery of additional cyclic di-nucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, to include both enzymatically active and inactive members with a subset involved in synthesis and degradation of other cyclic di-nucleotides. Here we summarize current knowledge of sequence and structural varitions that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL and HD-GYP domain proteins.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

ABSTRACT: BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no ?-carotene was observed, variable amounts of a ?-ring derived xanthophyll, lutein, was detected; with greater accumulation of ?-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.