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211.
Floriana Volpicelli Massimiliano Caiazzo Bruno Moncharmont Umberto di Porzio Luca Colucci-D’Amato 《PloS one》2014,9(10)
Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival. 相似文献
212.
Benedetta Apollonio Maria Teresa Sabrina Bertilaccio Umberto Restuccia Pamela Ranghetti Federica Barbaglio Paolo Ghia Federico Caligaris-Cappio Cristina Scielzo 《Journal of visualized experiments : JoVE》2014,(92)
The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease’s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as “spots” on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells. 相似文献
213.
The somatic recombination of different germline-encoded gene segments constitutes a principal source of antibody diversity. In order to investigate the diversity in recombined gene segments encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii, a VH library was constructed by 5'-RACE (rapid amplification of cDNA ends) using RNA isolated from the spleen of an individual specimen. Analysis of cDNA sequences of 45 rearranged VH/D/JH segments revealed specific features, such as: high number of repeats, up to 8 bp long, and palindromic sequences, especially in CDRs (complementary determining regions); occurrence of the RGYW consensus, known as mutational hot spot, higher than in other species. Sixty-four percent of single base substitutions was found within this motif. In addition, the usage of serine codons showed a clear bias for AGY in CDRs, particularly in CDR2, and for TCN in FRs (framework regions). In CDRs, the frequency of non-synonymous changes was higher than that of synonymous changes. Diversity generated by insertions/deletions occurred more often than in other species; inserted bases were often repeats of adjacent bases. In particular the CDR2 showed the highest length variability as compared to other species. Alignment of VH sequences indicated that also the gene conversion mechanism may contribute to generating diversity. These data indicate a CDR mutability higher than in other species and provide some insights into the hypermutational events that may also occur in teleosts. 相似文献
214.
Sharon L. Ripp Jalal A. Aram Christopher J. Bowman Gary Chmielewski Umberto Conte David M. Cross Hongying Gao Elise M. Lewis Jian Lin Ping Liu Haran T. Schlamm 《Birth defects research. Part B, Developmental and reproductive toxicology》2012,95(1):89-94
Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4–8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography‐tandem mass spectrometry. The mean plasma Cmax and AUC0‐24 values were consistent with single‐dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone‐to‐plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3‐ to 1.8‐fold. Brain concentrations were low after single dose, with brain‐to‐plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single‐dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues. 相似文献
215.
F Noulin C Borlon P van den Eede L Boel CM Verfaillie U D'Alessandro A Erhart 《PloS one》2012,7(7):e40798
The development of a system for the continuous culture of Plasmodium vivax in vitro would benefit from the use of reticulocytes derived from differentiated hematopoietic stem cells (HCS). At present, the need to use both fresh reticulocytes and fresh P. vivax isolates represents a major obstacle towards this goal, particularly for laboratories located in non-endemic countries. Here, we describe a new method for the cryopreservation of HSC-derived reticulocytes to be used for both P. falciparum and P. vivax invasion tests. Cryopreserved P. falciparum and P. vivax isolates could invade both fresh and cryopreserved HSC-derived reticulocytes with similar efficiency. This new technique allows the storage of HSC-derived reticulocytes which can be used for later invasion tests and represents an important step towards the establishment of a continuous P. vivax culture. 相似文献
216.
Thomas C. Lindsay Musa Jawara Umberto D’Alessandro Margaret Pinder Steven W. Lindsay 《PloS one》2012,7(11)
African pit latrines produce prodigious numbers of the latrine fly, Chrysomya putoria, a putative vector of diarrhoeal pathogens. We set out to develop a simple, low-cost odour-baited trap for collecting C. putoria in the field. A series of field experiments was carried out in The Gambia to assess the catching-efficiency of different trap designs. The basic trap was a transparent 3L polypropylene box baited with 50 g of fish, with a white opaque lid with circular entrance holes. We tested variations of the number, diameter, position and shape of the entrance holes, the height of the trap above ground, degree of transparency of the box, its shape, volume, colour, and the attractiveness of gridded surfaces on or under the trap. Traps were rotated between positions on different sampling occasions using a Latin Square design. The optimal trapping features were incorporated into a final trap that was tested against commercially available traps. Features of the trap that increased the number of flies caught included: larger entrance holes (compared with smaller ones, p<0.001), using conical collars inside the holes (compared with without collars, p = 0.01), entrance holes on the top of the trap (compared with the side or bottom, p<0.001), traps placed on the ground (compared with above ground, p<0.001), the box having transparent sides (compared with being opaque, p<0.001), and with no wire grids nearby (compared with those with grids, p = 0.03). This trap collected similar numbers of C. putoria to other common traps for blow flies. The optimum trap design was a transparent box, with a white plastic lid on top, perforated with 10 conical entrance holes, placed on the ground. Our simple trap provides a cheap, low-maintenance and effective method of sampling C. putoria in the field. 相似文献
217.
Akhmedov D De Marchi U Wollheim CB Wiederkehr A 《Biochimica et biophysica acta》2012,1823(10):1815-1824
Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells. 相似文献
218.
Rastelli G Costantino L Gamberini MC Del Corso A Mura U Petrash JM Ferrari AM Pacchioni S 《Bioorganic & medicinal chemistry》2002,10(5):1427-1436
The relative binding affinities to human aldose reductase (ALR2) of three new 7-hydroxy-2-benzyl-4H-1-benzopyran-4-one inhibitors were predicted by free energy perturbation (FEP) simulations. Molecular substitutions were specifically designed to investigate the role of hydrogen bonding at the active site of ALR2. Starting from the lead inhibitor 7-hydroxy-2-(4'-hydroxybenzyl)-4H-1-benzopyran-4-one, the 4'-hydroxyl was mutated to methyl and to trifluoromethyl, and an hydroxyl at position 8 was additionally introduced. Once synthesized and tested as inhibitors of ALR2, the compounds displayed variations of K(i) that were in qualitative to quantitative agreement with the calculated relative free energies of binding. The results, discussed in terms of balance between free energies of solvation and free energies of binding to ALR2, elucidate the importance of hydrogen bonding with Thr113 and with Trp111 and cofactor, and provide a rationale to the observed differences in binding affinities. 相似文献
219.
Traverso N Menini S Odetti P Pronzato MA Cottalasso D Marinari UM 《Free radical biology & medicine》2002,32(4):350-359
This study assesses whether the HNE accumulation we formerly observed in liver microsomes and mitochondria of BB/Wor diabetic rats depends on an increased rate of lipoperoxidation or on impairment of enzymatic removal. There are three main HNE metabolizing enzymes: glutathione-S-transferase (GST), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). In this study we show that GST and ALDH activities are reduced in liver microsomes and mitochondria of diabetic rats; in contrast, ADH activity remains unchanged. The role of each enzyme in HNE removal was evaluated by using enzymatic inhibitors. The roles of both GST and ALDH were markedly reduced in diabetic rats, while ADH-mediated consumption was significantly increased. However, the higher level of lipohydroperoxides in diabetic liver indicated more marked lipoperoxidation. We therefore think that HNE accumulation in diabetic liver may depend on both mechanisms: increased lipoperoxidation and decreased enzymatic removal. We suggest that glycoxidation and/or hyperglycemic pseudohypoxia may be involved in the enzymatic impairment observed. Moreover, since HNE exerts toxic effects on enzymes, HNE accumulation, deficiency of HNE removal, and production of reactive oxygen species can generate vicious circles able to amplify the damage. 相似文献
220.
De Marchi U Szabò I Cereghetti GM Hoxha P Craigen WJ Zoratti M 《Biochimica et biophysica acta》2008,1777(11):1438-1448
Patch-clamp experiments on swollen mitochondria of human, mouse and rat origins have revealed activity by an approximately 400 pS (in 150 mM KCl), voltage-dependent and anion-selective channel. This channel is located in the inner membrane, as shown by experiments with mitochondria from cells expressing a fluorescent mitochondrial tag protein and by the co-presence of the 107 pS channel and of the permeability transition pore (PTP). The frequency of appearance was inversely related to the presence of the PTP. This and the comparison of its electrophysiological characteristics with those of the PTP indicate that it is closely related to the latter, possibly corresponding to a monomeric unit whose dimer constitutes the full PTP. The channel is similar but not identical to isolated-and-reconstituted mitochondrial porin, and it is present also in mitochondria from cells lacking porin isoforms. Its identification with porin is therefore to be excluded. It most likely coincides instead with the "maxi-chloride channel" characterized in the plasma membrane of various cell types. 相似文献