Radical-induced oxidation of trans-resveratrol

trans-Resveratrol (RVT) (3,5,4'-trihydroxystilbene), a polyphenolic constituent of red wine, is thought to be beneficial in reducing the incidence of cardiovascular diseases, partly via its antioxidant properties. However, the mechanism of action by which trans-resveratrol displays its antioxidant effect has not been totally unravelled. This study aimed at establishing a comprehensive scheme of the reaction mechanisms of the direct scavenging of HO(*) and O(2)(*-) radicals generated by water gamma radiolysis. Aerated aqueous solutions of trans-RVT (from 10 to 100μmolL(-1)) were irradiated with increasing radiation doses (from 25 to 400Gy) and further analyzed by UV-visible absorption spectrophotometry for detection of trans-RVT oxidation products. Separation and quantification of RVT and its four oxidation products previously identified by mass spectrometry, i.e., piceatannol (PCT), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,5-dihydroxybenzaldehyde (3,5-DHB) and para-hydroxybenzaldehyde (PHB), were performed by HPLC/UV-visible spectrophotometry. Determination of the radiolytic yields of trans-RVT consumption and oxidation product formation has allowed us to establish balance between trans-RVT disappearance and the sum of oxidation products formation. Under our conditions, O(2)(-) radicals seemed to poorly initiate oxidation of trans-RVT, whereas the latter, whatever its initial concentration, quantitatively reacted with HO() radicals, via a dismutation mechanism. Two reaction pathways involving HO()-induced trans-RVT primary radicals have been proposed to explain the formation of the oxidation end-products of trans-RVT.     

Cdc6 is required for meiotic spindle assembly in Xenopus oocytes

During the maturation of Xenopus oocytes, Cdc6 expression is necessary to establish replication competence to support early embryonic DNA replication. However, Cdc6 is expressed before the completion of MI at a time when its function as a replication factor is not required, suggesting additional roles for Cdc6 in meiosis. Confocal immunofluorescence microscopy revealed that Cdc6 protein was distributed around the spindle precursor at the time of germinal vesicle breakdown (GVBD) and localized to the margin of the nascent spindle early in prometaphase. Cdc6 subsequently localized to spindle poles in late prometaphase, where it remained until metaphase arrest. Microinjection of antisense oligonucleotides specific for Cdc6 mRNA disrupted spindle assembly, resulting in defects, including delayed spindle assembly, misoriented and unattached anaphase spindles, monasters, multiple spindles, microtubule aggregates associated with condensed chromosomes, or the absence of recognizable spindle-like structures, depending on the level of residual Cdc6 expression. Furthermore, Cdc6 co-localized with γ-tubulin in centrosomes during interphase in all somatic cells analyzed and associated with spindle poles in mitotic COS cells. Our data suggest a role for Cdc6 in spindle formation in addition to its role as a DNA replication factor.Key words: Cdc6, spindle assembly, Xenopus, oocytes, pre-RC proteins     

A comparison of the host preference of monarch butterflies (Danaus plexippus) for milkweed (Asclepias syriaca) over dog-strangler vine (Vincetoxicum rossicum)

Observations in the field indicate that monarch butterflies will oviposit on dog‐strangler vine, an invasive introduced species in the same family as milkweed (Asclepias spp.), the principal larval host of monarchs. The potential impact of this behaviour depends on the strength of the preference of monarch adults to oviposit on these two hosts and the relative ability of larvae to survive on each. We determined the preference for milkweed vs. dog‐strangler vine of ovipositing adults and first instar larvae in choice and no‐choice tests. We also compared the ability of larvae to consume, develop, and survive on either host. In the presence of both hosts, adults exhibited a strong preference to oviposit on milkweed over dog‐strangler vine (mean 80.7 eggs compared to 0.4 eggs over 48 h, respectively). In the absence of milkweed, adults ceased oviposition (mean 0.9 eggs in 48 h), but resumed oviposition when the dog‐strangler vine was replaced with milkweed (mean 99.1 eggs in 48 h). Given a choice between hosts over 24 h, 92% of larvae moved to milkweed leaves and consumed 3.94 cm² of milkweed leaves compared to 2% of larvae that moved to dog‐strangler vine and consumed negligible amounts of leaf material (0.01 cm²). Without a choice, larvae on dog‐strangler vine never consumed more than mean 0.02 cm² larva^?1 in a 24‐h period, did not develop beyond the first instar, and died within 96 h. We obtained no data in support of an effect of the presence of dog‐strangler vine on monarch butterfly populations.     

Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.     

An evaluation of the mechanisms developmentally involved on cellular and extracellular glycosaminoglycans accumulation in chick embryo skin fibroblasts.

1. Ammonium chloride, a lysosomotropic amine known to inhibit lysosomal function, was administered to 7-day cultured and 14-day chick embryo skin fibroblasts to evaluate the relationship between synthesis, degradation and uptake of glycosaminoglycans (GAG). 2. Following amine treatment, the amount of 3H-glucosamine and 35SO4 labelled cellular GAG increased, was more at 14 days than at 7 days. Hyaluronic acid (HA) incorporation was mainly interested at 7 days and that of sulphated GAG at 14 days. 3. The extracellular accumulation declined proportionally to the cellular increase of undegraded GAG. HA was mainly affected at 7 days and sulphated GAG at 14 days. 4. The amine did not change 3H-HA uptake and it was unable to inhibit its degradation. 5. The products of degradation of uptaken 3H-HA were retained inside the cell. Those released by degradation of newly synthesized GAG flowed out of the cell.     

Characterization of the biochemical,physiological, and medicinal properties of Streptomyces hygroscopicus ACTMS-9H isolated from the Amazon (Brazil)

Applied Microbiology and Biotechnology - Actinomycetes are known to produce numerous secondary bioactive metabolites of pharmaceutical interest. The purpose of this study was to isolate,...     

Studies in mice, hamsters, and rats demonstrate that repression of hepatic apoA-I expression by taurocholic acid in mice is not mediated by the farnesoid-X-receptor

It is claimed that apoA-I expression is repressed in mice by cholic acid (CA) and its taurine conjugate, taurocholic acid (TCA) via farnesoid X receptor (FXR) activation. We measured apoA-I expression in mice, hamsters, and rats treated with highly potent and selective synthetic FXR agonists or with TCA. All of the synthetic agonists bound to FXR with high affinity in a scintillation proximity assay. However, TCA did not compete with the radioligand up to the highest concentration used (100 μM). The C-site regulatory region of apoA-I, through which FXR has been reported to regulate its expression, is completely conserved across the species investigated. In both male and female human apoA-I-transgenic mice, we reproduced the previously reported strong inhibition of human apoA-I expression upon treatment with the typical supraphysiological dose of TCA used in such studies. However, in contrast to some previous reports, TCA did not repress murine apoA-I expression in the same mice. Also, more-potent and -selective FXR agonists did not affect human or murine apoA-I expression in this model. In LDL receptor-deficient mice and Golden Syrian hamsters, selective FXR agonists did not affect apoA-I expression, whereas in Wistar rats, some even increased apoA-I expression. In conclusion, selective FXR agonists do not repress apoA-I expression in rodents. Repression of human apoA-I expression by TCA in transgenic mice is probably mediated through FXR-independent mechanisms.     

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A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.     

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.