Chromosome evolution in Cophomantini (Amphibia, Anura, Hylinae)

The hylid tribe Cophomantini is a diverse clade of Neotropical treefrogs composed of the genera Aplastodiscus, Boana, Bokermannohyla, Hyloscirtus, and Myersiohyla. The phylogenetic relationships of Cophomantini have been comprehensively reviewed in the literature, providing a suitable framework for the study of chromosome evolution. Employing different banding techniques, we studied the chromosomes of 25 species of Boana and 3 of Hyloscirtus; thus providing, for the first time, data for Hyloscirtus and for 15 species of Boana. Most species showed karyotypes with 2n = 2x = 24 chromosomes; some species of the B. albopunctata group have 2n = 2x = 22, and H. alytolylax has 2n = 2x = 20. Karyotypes are all bi-armed in most species presented, with the exception of H. larinopygion (FN = 46) and H. alytolylax (FN = 38), with karyotypes that have a single pair of small telocentric chromosomes. In most species of Boana, NORs are observed in a single pair of chromosomes, mostly in the small chromosomes, although in some species of the B. albopunctata, B. pulchella, and B. semilineata groups, this marker occurs on the larger pairs 8, 1, and 7, respectively. In Hyloscirtus, NOR position differs in the three studied species: H. alytolylax (4p), H. palmeri (4q), and H. larinopygion (1p). Heterochromatin is a variable marker that could provide valuable evidence, but it would be necesserary to understand the molecular composition of the C-bands that are observed in different species in order to test its putative homology. In H. alytolylax, a centromeric DAPI+ band was observed on one homologue of chromosome pair 2. The band was present in males but absent in females, providing evidence for an XX/XY sex determining system in this species. We review and discuss the importance of the different chromosome markers (NOR position, C-bands, and DAPI/CMA3 patterns) for their impact on the taxonomy and karyotype evolution in Cophomantini

Pseudo-immunolabelling with the avidin-biotin-peroxidase complex (ABC) due to the presence of endogenous biotin in retinal Muller cells of goldfish and salamander

Immunodetection techniques are dependent on enzyme-protein conjugates for the visualisation of antigen-antibody complexes. One of the most widely used is the avidin-biotin-peroxidase complex (ABC) method. The present study demonstrates that direct treatment of goldfish and salamander retinal sections with ABC, followed by an incubation with the chromogenic substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) and H2O2, manifested a punctate staining pattern across the neural retinae, presumably through binding of avidin to endogenous biotin. Incubation with a primary antiserum against biotin followed by immunoprocessing with the peroxidase--anti-peroxidase (PAP) method showed a pattern similar to the punctuate framework as detected with solo ABC-treated sections. Moreover, the ABC-DAB/H2O2 mediated pattern corresponded to the spatial orientation of Müller cells as identified by GFAP immunostaining. These findings indicate the presence of endogenous biotin in Müller cells and calls for caution in the application of the ABC method in immunotechniques in retinal researc

VEGF-A induced hyperpermeability of blood-retinal barrier endothelium in vivo is predominantly associated with pinocytotic vesicular transport and not with formation of fenestrations. Vascular endothelial growth factor-A

PURPOSE: In tissues outside the brain, vascular endothelial growth factor-A (VEGF) causes vascular hyper-permeability by opening of inter-endothelial junctions and induction of fenestrations and vesiculo-vacuolar organelles (VVOs). In preliminary studies, we observed that in blood-retinal barrier endothelium, other cellular mechanisms may underlie increased permeability caused by VEGF. This was further investigated in material of an in vivo experimental model of VEGF-induced retinopathy. METHODS: Two monkeys received 4 intravitreal injections of 0.5 microg VEGF in one eye and PBS in the other eye prior to sacrifice at day 9. One monkey received 12 injections of 1.25 microg VEGF in one eye and PBS in the other eye prior to sacrifice at day 24. As a control, an untreated eye of a fourth monkey was studied. RESULTS: In the high-dose VEGF-injected eye, fluorescein angiography showed intense retinal micro-vascular leakage. This leakage was also demonstrated by immunohistochemistry demonstrating extravasation of endogenous fibrinogen and IgG. However, in these leaky blood vessels the number of pinocytotic vesicles (caveolae) at the endothelial luminal membrane were significantly higher and, only in the VEGF-injected eyes, these pinocytotic vesicles transported plasma IgG. By electron microscopy, no fenestrations or VVOs were found in the endothelial cells of the VEGF-injected eyes. CONCLUSION: We conclude that increased vascular permeability for plasma proteins induced by VEGF in blood-retinal barrier endothelium is predominantly caused by a mechanism involving active trans-endothelial transport via pinocytotic vesicles and not by formation of endothelial fenestrations or VVO