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Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1ß, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1ß transcription, but unlike Rec8, Smc1ß is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women's fertility depends on the longevity of cohesin proteins that established cohesion in utero.

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BACKGROUND: Forest residues represent an abundant and sustainable source of biomass which could be used as a biorefinery feedstock. Due to the heterogeneity of forest residues, such as hog fuel and bark, one of the expected challenges is to obtain an accurate material balance of these feedstocks. Current compositional analytical methods have been standardised for more homogenous feedstocks such as white wood and agricultural residues. The described work assessed the accuracy of existing and modified methods on a variety of forest residues both before and after a typical pretreatment process. RESULTS: When "traditional" pulp and paper methods were used, the total amount of material that could be quantified in each of the six softwood-derived residues ranged from 88% to 96%. It was apparent that the extractives present in the substrate were most influential in limiting the accuracy of a more representative material balance. This was particularly evident when trying to determine the lignin content, due to the incomplete removal of the extractives, even after a two stage water-ethanol extraction. Residual extractives likely precipitated with the acid insoluble lignin during analysis, contributing to an overestimation of the lignin content. Despite the minor dissolution of hemicellulosic sugars, extraction with mild alkali removed most of the extractives from the bark and improved the raw material mass closure to 95% in comparison to the 88% value obtained after water-ethanol extraction. After pretreatment, the extent of extractive removal and their reaction/precipitation with lignin was heavily dependent on the pretreatment conditions used. The selective removal of extractives and their quantification after a pretreatment proved to be even more challenging. Regardless of the amount of extractives that were originally present, the analytical methods could be refined to provide reproducible quantification of the carbohydrates present in both the starting material and after pretreatment. CONCLUSION: Despite the challenges resulting from the heterogeneity of the initial biomass substrates a reasonable summative mass closure could be obtained before and after steam pretreatment. However, method revision and optimisation was required, particularly the effective removal of extractives, to ensure that representative and reproducible values for the major lignin and carbohydrate components.

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Dopamine, serotonin and glutamate play a role in the pathophysiology of schizophrenia. In the brain a functional crosstalk between the serotonin receptor 5-HT(2A) and the metabotropic glutamate receptor mGlu(2) has been demonstrated. Such a crosstalk may be mediated indirectly through neuronal networks or directly by receptor oligomerization. A direct link of the 5-HT(2A)-mGlu(2) heterocomplex formation to receptor function, i.e. to intracellular signaling, has not been fully demonstrated yet. Here we confirm the formation of 5-HT(2A)-mGlu(2) heterocomplexes using quantitative Snap/Clip-tag based HTRF methods. Additionally, mGlu(2) formed complexes with 5-HT(2B) and mGlu(5) but not 5-HT(2C) indicating that complex formation is not specific to the 5-HT(2A)-mGlu(2) pair. We studied the functional consequences of the 5-HT(2A)-mGlu(2) heterocomplex addressing cellular signaling pathways. Co-expression of receptors in HEK-293 cells had no relevant effects on signaling mediated by the individual receptors when mGlu(2) agonists, antagonists and PAMs, or 5-HT(2A) hallucinogenic and non-hallucinogenic agonists and antagonists were used. Hallucinogenic 5-HT(2A) agonists induced signaling through G(q/11), but not G(i) and thus did not lead to modulation of intracellular cAMP levels. In membranes of the medial prefrontal cortex [(3)H]-LY341495 binding competition of mGlu(2/3) agonist LY354740 was not influenced by 2,5-dimethoxy-4-iodoamphetamine (DOI). Taken together, the formation of GPCR heterocomplexes does not necessarily translate into second messenger effects. These results do not put into question the well-documented functional cross-talk of the two receptors in the brain, but do challenge the biological relevance of the 5-HT(2A)-mGlu(2) heterocomplex.

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Cytochrome P4502C19 (CYP2C19) is an important drug-metabolizing enzyme involved in the biotransformation of, for example, proton pump inhibitors and antidepressants. Several in vivo studies have shown that the CYP2C19 activity is inhibited by oral contraceptives, which can cause important drug interactions. The underlying molecular mechanism has been suggested to be competitive inhibition. However, the results presented here indicate that estradiol derivatives down-regulate CYP2C19 expression via estrogen receptor (ER) α, which interacts with the newly identified ER-binding half site [estrogen response element (ERE)] at the position -151/-147 in the CYP2C19 promoter. In gene reporter experiments in Huh-7 hepatoma cells, the activity of the luciferase construct carrying a 1.6-kb long CYP2C19 promoter fragment cotransfected with ERα was down-regulated upon treatment with 17ß-estradiol (EE) or 17α-ethinylestradiol (ETE) at half-maximum concentrations of 10(-7) and 10(-8) M, respectively. Mutations introduced into the ERE half site -151/-147 significantly inhibited these ligand-dependent effects. Electrophoretic mobility shift assays and quantitative chromatin immunoprecipitation experiments revealed that estrogen receptor α binds to this element. A significant suppression of CYP2C19 transcription by female sex steroids was confirmed by reverse transcription polymerase chain reaction after hormonal treatment of human hepatocytes. Inhibition experiments using a stable human embryonic kidney 293 CYP2C19 cell line revealed competitive inhibition at much higher concentrations of EE and ETE compared with those required for transcriptional inhibition. These results indicate that both EE and ETE inhibit CYP2C19 expression via an ERα-dependent regulatory pathway, thus providing a new insight into the molecular mechanism behind the inhibitory effect of oral contraceptives on CYP2C19 activity.

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