alpha and beta spectrin distribution during the differentiation of pyriform cells in follicles of lizard Podarcis sicula

Using alpha and beta spectrin mammalian antibodies on Western blotting, we demonstrated that lizard ovarian follicles contain two isoforms of alpha spectrin, Mr 94 and 134 kDa, and a 230 kDa beta spectrin, and that their pattern modifies in relation to pyriform cell differentiation. In fact, a positive immunoreaction is firstly evident within follicular epithelium of previtellogenic follicles when small cells differentiate into pyriform cells via intermediate cells. Later on, immunostain is present in pyriform cells and in the oocyte cortex that previously appears unstained. It is noteworthy that immunostain is also present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina and among pyriform cells, not engaged in pyriform cell differentiation. During the subsequent stages of previtellogenic phase, spectrin immunostain over the follicular epithelium and in the oocyte cortex does not change. By contrast, in vitellogenic follicles, when the follicular epithelium is constituted only by small cells, immunostain is evident at the level of the oocyte cortex and the cytoplasm of regressing pyriform cells. The present data strongly suggest that the alpha and beta spectrin pattern put in evidence during the different phases of lizard oocyte growth is related to the differentiation of small into pyriform cells, where such protein may guarantee a relationship between surface glycoproteins (Andreuccetti et al., 2001: Anat Rec 263:1-9), and the cytoskeleton distribution (Maurizii et al., 2000: Raf Mol Reprod Dev 57:159-166). Furthermore, the distribution of spectrin mRNA, similar to that observed for the protein, demonstrates that spectrin, once synthesized within pyriform cells, is transferred through intercellular bridges in the oocyte cortex, thus confirming that pyriform cells are nurse that significantly are involved in the oocyte growth. Finally, the present data demonstrate that alpha spectrin of lizard ovarian follicles has Mr quite different from those so far reported and may constitute a new group of isoforms. This important result will be the focus of future experiments. Mol. Reprod. Dev. 67: 101-107, 2004.     

Effects of simulated microgravity on the development and maturation of dissociated cortical neurons

Although a wealth of evidence supports the hypothesis that some functions of the nervous system may be altered during exposure to microgravity, the possible changes in basic neuronal physiology are not easy to assess. Indeed, few studies have examined whether microgravity affects the development of neurons in culture. In the present study, a suspension of dissociated cortical cells from rat embryos were exposed to 24 h of simulated microgravity before plating in a normal adherent culture system. Both preexposed and control cells were used after a period of 7-10 d in vitro. The vitality and the level of reactive oxygen species of cultures previously exposed did not differ from those of normal cultures. Cellular characterization by immunostaining with a specific antibody displayed normal neuronal phenotype in control cells, whereas pretreatment in simulated microgravity revealed an increase of glial fibrillary acidic protein fluorescence in the elongated stellate glial cells. Electrophysiological recording indicated that the electrical properties of neurons preexposed were comparable with those of controls. Overall, our results indicate that a short time of simulated microgravity preexposure does not affect dramatically the ability of dissociated neural cells to develop and differentiate in an adherent culture system.     

MF-8, a novel promising arylpiperazine-hydantoin based 5-HT7 receptor antagonist: In vitro drug-likeness studies and in vivo pharmacological evaluation

We report the in vitro drug-likeness studies and in vivo pharmacological evaluation for a new potent 5-HT₇ receptor antagonist MF-8 (5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-methylhydantoin). The in vitro tests showed good permeability, very good metabolic stability, low risk of drug-drug interactions and satisfying safety profile. Moreover, MF-8 showed excellent antidepressant-like activity in the forced swim test in rodents and promising anxiolytic-like activity in the four-plate test in mice. Regarding the potent affinity, high selectivity and antagonistic activity of MF-8 for the 5-HT₇ receptor as well as excellent drug – like properties in vitro and confirmed in vivo pharmacological activity, MF-8 should be considered as a very significant molecule in the search for a new class of anti-depressant drugs.     

Osservazioni sulla diffusione postglaciale dell'Abete nel versante meridionale delle Alpi

Riassunto

È stato preso in esame, mediante analisi pollinologiche di depositi torbosi, il rimboschimento postglaciale della parte meridionale della venezia Tridentina per le quote da 1000 a 1400 m. I risultati hanno permesso di stabilire che nel versante meridionale del sistema alpino l'Abete, in determinate posizioni, ha partecipato in modo considerevole — spesso imponente — alla constituzione delle foreste dopo il periodo dei Pini, fino al periodo del Faggio.

Il comportamento della sua espansione è risultato analogo a quello già descritto per il versante settentrionale; ciò vien messo in evidenza mediante un grafico the mostra schematicamente l'aspetto forestale dei due versanti nel periodo Querceto-Picea-Abies del posglaciale.     

Identification of RC-33 as a potent and selective σ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells. Part 2: g-Scale synthesis,physicochemical characterization and in vitro metabolic stability

Strong pharmacological evidences indicate that σ1 receptors are implicated in the pathophysiology of all major CNS disorders. In the last years our research group has conducted extensive studies aimed at discovering novel σ1 ligands and we recently selected (R/S)-RC-33 as a novel potent and selective σ1 receptor agonist. As continuation of our work in this field, here we report our efforts in the development of this new σ1 receptor agonist. Initially, we investigated the binding of (R) and (S) enantiomers of RC-33 to the σ1 receptor by in silico experiments. The close values of the predicted affinity of (R)-RC-33 and (S)-RC-33 for the protein evidenced the non-stereoselective binding of RC-33 to the σ1 receptor; this, in turn, supported further development and characterization of RC-33 in its racemic form. Subsequently, we set-up a scaled-up, optimized synthesis of (R/S)-RC-33 along with some compound characterization data (e.g., solubility in different media and solid state characterization by thermal analysis techniques). Finally, metabolic studies of RC-33 in different biological matrices (e.g., plasma, blood, and hepatic S9 fraction) of different species (e.g., rat, mouse, dog, and human) were performed. (R/S)-RC-33 is generally stable in all examined biological matrices, with the only exception of rat and human liver S9 fractions in the presence of NADPH. In such conditions, the compound is subjected to a relevant oxidative metabolism, with a degradation of approximately 65% in rat and 69% in human.Taken together, our results demonstrated that (R/S)-RC-33 is a highly potent, selective, metabolically stable σ1 agonist, a promising novel neuroprotective drug candidate.     

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.     

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.