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Mondejar I.,University of Murcia | Mondejar I.,Instituto Murciano Of Investigacion Biosanitaria Imib | Aviles M.,University of Murcia | Coy P.,University of Murcia | Coy P.,Instituto Murciano Of Investigacion Biosanitaria Imib
Human Reproduction | Year: 2013

Study Question: Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? Summary Answer: ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. What is Known Already: Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). Study Design , Size, Duration Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate.MATERIALS, SETTING, METHODSIn vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. Main Results and the Role of Chance: rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P < 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. Limitations, Reasons For Caution Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. Wider Implications of the Finding: s: Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed. Study Funding/Competing Interes: T(S)This work was supported by the Spanish Ministry of Science and Innovation and FEDER, Grant AGL2009-12512-C02-01-02. The authors declare no competing interest. © 2013 The Author.

Zamora A.,Instituto Murciano Of Investigacion Biosanitaria Imib | Rodriguez V.,Instituto Murciano Of Investigacion Biosanitaria Imib | Cutillas N.,Instituto Murciano Of Investigacion Biosanitaria Imib | Yellol G.S.,Instituto Murciano Of Investigacion Biosanitaria Imib | And 5 more authors.
Journal of Inorganic Biochemistry | Year: 2013

Two new steroidal 7-azaindole-based N-donor ligands 17-α-[7- azaindole-5-ethynyl]-17-β-testosterone (ET-Haza) (1) and 17-α-[7-azaindole-5-ethynyl]-19-nortestosterone (LEV-Haza) (2), and two new DNA damaging warheads with an enhanced lipophilicity [Pt(dmba)Cl(L)] (dmba = N,N-dimethylbenzylamine-κN,κC; L = ET-Haza (3) and LEV-Haza (4)) have been prepared and characterized. Values of IC50 were calculated for complexes 3 and 4 against a panel of human tumor cell lines representative of ovarian (A2780 and A2780cis) and breast cancers (T47D). At 48 h of incubation time 3 and 4 showed very low resistance factors (RF of 1) against an A2780 cell line which has acquired resistance to cisplatin, IC50 values of the new complexes towards normal human LLC-PK1 renal cells at 48 h being about double than that of cisplatin. 3 and 4 are able to react with 9-ethylguanine (9-EtG) yielding the corresponding monoadduct [Pt(dmba)(L)(9-EtG)]+ derivatives as followed by ESI-MS. Compound 3 interacts mainly with double-stranded (DS) oligonucleotides as shown by analysis with ESI-TOF-MS, being also able to displace ethidium bromide (EB) from DNA, as observed by an electrophoretic mobility study. 3 and 4 are good cathepsin B inhibitors. Theoretical calculations at the COSMO(CHCl3)/B3LYP-D/def2-TZVPPecp// B3LYP-D/def2-TZVPecp level and energy evaluations at the COSMO(CHCl 3)/PWPB95-D3/def2-TZVPPecp level of theory on compound 4 and model systems have been done. © 2013 Elsevier Inc.

Navarro-Zaragoza J.,University of Murcia | Navarro-Zaragoza J.,Instituto Murciano Of Investigacion Biosanitaria Imib | Laorden M.L.,University of Murcia | Laorden M.L.,Instituto Murciano Of Investigacion Biosanitaria Imib | And 2 more authors.
Toxicology | Year: 2014

Pharmacological evidence has accumulated showing that glucocorticoids and glucocorticoid receptor (GR) facilitate several responses to different drugs of abuse. Recent findings have attributed a prominent role to the mineralocorticoid receptor (MR) in modulating behavior during the addictive process. The purpose of this study was to investigate the effects of MR blockade on: brain stress system responses to naloxone-induced morphine withdrawal, the somatic signs of abstinence; the effects of morphine withdrawal on noradrenaline (NA) turnover in the paraventricular nucleus (PVN), c-Fos expression and tyrosine hydroxylase (TH) phosphorylated at Ser31 levels in the nucleus tractus solitarius noradrenergic cell group (NTS-A2); and finally, hypothalamus-pituitary-adrenocortical (HPA) axis activity. The role of MR signaling was assessed with i.p. pretreatment with the MR antagonist, spironolactone. Rats were implanted with two morphine (or placebo) pellets. Six days later rats were pretreated with spironolactone or vehicle 30min before naloxone. The physical signs of abstinence, NA turnover, TH activation, c-Fos expression and the HPA axis activity were measured using HPLC, immunoblotting and RIA. Spironolactone attenuated the somatic signs of withdrawal that were seen after naloxone administration to chronic morphine treated animals. On the other hand, pretreatment with spironolactone resulted in no significant modification of the increased NA turnover, TH activation, c-Fos expression or HPA axis activity that occurred during morphine withdrawal. These results suggest that somatic signs of opiate withdrawal are modulated by MR signaling. However, blockade of MR did not significantly alter the brain stress system response to morphine withdrawal. © 2014 Elsevier Ireland Ltd.

Espin R.,University of Murcia | Espin R.,Instituto Murciano Of Investigacion Biosanitaria Imib | Roca F.J.,University of Murcia | Roca F.J.,University of Washington | And 14 more authors.
DMM Disease Models and Mechanisms | Year: 2013

Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFa promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis. © 2013. Published by The Company of Biologists Ltd.

Martin F.,University of Murcia | Martin F.,Instituto Murciano Of Investigacion Biosanitaria Imib | Nunez C.,University of Murcia | Nunez C.,Instituto Murciano Of Investigacion Biosanitaria Imib | And 6 more authors.
PLoS ONE | Year: 2012

Experimental and clinical findings have shown that administration of adrenoceptor antagonists alleviated different aspects of drug withdrawal and dependence. The present study tested the hypothesis that changes in CREB activation and phosphorylated TORC1 levels in the hypothalamic paraventricular nucleus (PVN) after naloxone-precipitated morphine withdrawal as well as the HPA axis activity arises from α 1- and/or β-adrenoceptor activation. The effects of morphine dependence and withdrawal on CREB phosphorylation (pCREB), phosphorylated TORC1 (pTORC1), and HPA axis response were measured by Western-blot, immunohistochemistry and radioimmunoassay in rats pretreated with prazosin (α 1-adrenoceptor antagonist) or propranolol (β-adrenoceptor antagonist). In addition, the effects of morphine withdrawal on MHPG (the main NA metabolite at the central nervous system) and NA content and turnover were evaluated by HPLC. We found an increase in MHPG and NA turnover in morphine-withdrawn rats, which were accompanied by increased pCREB immunoreactivity and plasma corticosterone concentrations. Levels of the inactive form of TORC1 (pTORC1) were decreased during withdrawal. Prazosin but not propranolol blocked the rise in pCREB level and the decrease in pTORC1 immunoreactivity. In addition, the HPA axis response to morphine withdrawal was attenuated in prazosin-pretreated rats. Present results suggest that, during acute morphine withdrawal, NA may control the HPA axis activity through CREB activation at the PVN level. We concluded that the combined increase in CREB phosphorylation and decrease in pTORC1 levels might represent, in part, two of the mechanisms of CREB activation at the PVN during morphine withdrawal. © 2012 Martín et al.

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