Tissue interaction between the retinal pigment epithelium and the choroid triggers retinal regeneration of the newt Cynops pyrrhogaster

Complete retinal regeneration in adult animals occurs only in certain urodele amphibians, in which the retinal pigmented epithelial cells (RPE) undergo transdifferentiation to produce all cell types constituting the neural retina. A similar mechanism also appears to be involved in retinal regeneration in the embryonic stage of some other species, but the nature of this mechanism has not yet been elucidated. The organ culture model of retinal regeneration is a useful experimental system and we previously reported RPE transdifferentiation of the newt under this condition. Here, we show that cultured RPE cells proliferate and differentiate into neurons when cultured with the choroid attached to the RPE, but they did not exhibit any morphological changes when cultured alone following removal of the choroid. This finding indicates that the tissue interactions between the RPE and the choroid are essential for the former to proliferate. This tissue interaction appears to be mediated by diffusible factors, because the choroid could affect RPE cells even when the two tissues were separated by a membrane filter. RPE transdifferentiation under the organotypic culture condition was abolished by a MEK (ERK kinase) inhibitor, U0126, but was partially suppressed by an FGF receptor inhibitor, SU5402, suggesting that FGF signaling pathway has a central role in the transdifferentiation. While IGF-1 alone had no effect on isolated RPE, combination of FGF-2 and IGF-1 stimulated RPE cell transdifferentiation similar to the results obtained in organ-cultured RPE and choroid. RT-PCR revealed that gene expression of both FGF-2 and IGF-1 is up-regulated following removal of the retina. Thus, we show for the first time that the choroid plays an essential role in newt retinal regeneration, opening a new avenue for understanding the molecular mechanisms underlying retinal regeneration.     

Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex

PAP-1 has been identified by us as a Pim-1-binding protein and has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). We have then shown that PAP-1 plays a role in pre-mRNA splicing. Because four causative genes for adRP, including PAP-1, Prp31, Prp8, and Prp3, encode proteins that function as splicing factors or splicing-modulating factors, we investigated the interaction of PAP-1 with Prp3p and Prp31p in this study. The results showed that PAP-1 interacted with Prp3p but not Prp31p in human cells and yeast, and that the basic region of PAP-1 and the C-terminal region of Prp3p, regions beside spots found in adRP mutations, were needed for binding. Furthermore, both Prp3p and a part of PAP-1 were found to be components of the U4/U6.U5-tri-snRNP complex, one form of the spliceosome, in Ba/F3 and K562 cells by analysis of sucrose density gradients, suggesting that PAP-1 is weakly associated with the spliceosome. These results also suggest that splicing factors implicated in adRP contribute alone or mutually to proper splicing in the retina and that loss of their functions leads to onset of adRP.     

Effects of voltage-gated Na+ channel toxins from Tityus serrulatus venom on rat arterial blood pressure and plasma catecholamines

Scorpion toxins interact with ionic channels of excitable cells, leading to a massive release of neurotransmitters. Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: alpha- and beta-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, we compared the effects of these toxins on mean arterial pressure (MAP) and catecholamines release in rats. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). All experiments were performed on conscious unrestrained rats previously catheterised. The toxins (15 and 30 microg/kg) and TsV (50 and 100 microg/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5-3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity.     

Blastocladiella emersonii expresses a centrin similar to Chlamydomonas reinhardtii isoform not found in late-diverging fungi

Centrins are members of the calcium-binding EF-hand protein superfamily which can be divided into two subfamilies, probably associated with different functions: one related to Chlamydomonas reinhardtii centrin, CrCenp, and the other, represented by Saccharomyces cerevisiae isoform, ScCdc31p. ESTs encoding the two isoforms (BeCen1 and BeCen3) from the chytridiomycete Blastocladiella emersonii were isolated, and expression of the CrCenp-type centrin, BeCen1, was analyzed throughout the fungus life cycle. Becen1 mRNA levels increase transiently during sporulation and protein levels present a similar pattern. Immunolocalization studies seem to localize BeCen1 at the basal body zone and in the cytoplasm surrounding the nuclear cap, a zoospore organelle.     

Karyologic diversification and phylogenetic relationships of the genus<Emphasis Type="Italic">Thalpomys</Emphasis> (Rodentia,Sigmodontinae)

We describe the karyotype ofThalpomys species, from different Brazilian localities of the Cerrado.Thalpomys cerradensis Herskovitz, 1990 showed 2n=36, FN=34 andT. lasiotis Thomas, 1916 2n=38, FN=38. Comparisons of G-band karyotypes showed evident inter-specific homologies indicating that their chromosome complements could be derived from one another by two presumed rearrangements. Both species showed pericentromeric C-band regions in almost all chromosomes but a comparison with CMA3/DA/DAPI staining indicated that the molecular content of heterochromatic regions was different.T. lasiotis specimens from two different localities differed in the morphology of the X chromosome due to the presence of a short heterochromatic arm. These chromosome types are apparently fixed in each population rather than maintained as a polymorphic variation. Phylogenetic analyses supported the monophyly of the genusThalpomys but was not capable of elucidating its phylogenetic relationship to other Akodontini rodents. These analyses also showed inter-individual variation inT. lasiotis, even within a given population. Phylogenetic analyses placedT. lasiotis specimens with different karyotypes in different monophyletic branches. Molecular and karyologic data confirmed the identity of the genusThalpomys.     

Synthesis and anti-inflammatory activity of new arylidene-thiazolidine-2,4-diones as PPARγ ligands

Eight new 5-arylidene-3-benzyl-thiazolidine-2,4-diones with halide groups on their benzyl rings were synthesized and assayed in vivo to investigate their anti-inflammatory activities. These compounds showed considerable biological efficacy when compared to rosiglitazone, a potent and well-known agonist of PPARγ, which was used as a reference drug. This suggests that the substituted 5-arylidene and 3-benzylidene groups play important roles in the anti-inflammatory properties of this class of compounds. Docking studies with these compounds indicated that they exhibit specific interactions with key residues located in the site of the PPARγ structure, which corroborates the hypothesis that these molecules are potential ligands of PPARγ. In addition, competition binding assays showed that four of these compounds bound directly to the ligand-binding domain of PPARγ, with reduced affinity when compared to rosiglitazone. An important trend was observed between the docking scores and the anti-inflammatory activities of this set of molecules. The analysis of the docking results, which takes into account the hydrophilic and hydrophobic interactions between the ligands and the target, explained why the 3-(2-bromo-benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-dione compound had the best activity and the best docking score. Almost all of the stronger hydrophilic interactions occurred between the substituted 5-arylidene group of this compound and the residues of the binding site.     

COMPARATIVE STUDY ON THE PHOTOSYNTHETIC PROPERTIES OF PRASIOLA (CHLOROPHYCEAE) AND NOSTOC (CYANOPHYCEAE) FROM ANTARCTIC AND NON‐ANTARCTIC SITES1

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kütz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100 μmol photons · m^?2 · s^?1, respectively. The difference seemed to reflect the presence of high amounts of UV‐absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30°C–35°C and 20°C–25°C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration‐induced quenching of PSII fluorescence. Re‐reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20°C–25°C and at a light intensity of 700 μmol photons ? m^?2 ? s^?1. Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.