Mitochondrial localization and ocular expression of mutant Opa3 in a mouse model of 3-methylglutaconicaciduria type III

Purpose. To investigate the developmental and ocular expression of Opa3 in a mouse model of 3-methylglutaconicaciduria type III and the effect of mutation on protein localization and mitochondrial morphology. Methods. The B6 C3-Opa3L122P mouse carrying a missense mutation in exon 2 (c.365T>C; p.L122P) of Opa3, which displays features of recessive 3-methylglutaconic aciduria type III was studied. The expression of Opa3 was determined with RT-PCR, quantitative PCR, and Western blot, in embryos (embryonic day [E]8 to postnatal day [P]0) and adult tissues, and by ocular immunohistochemistry. Mitochondria were stained using a mitochondrion-selective probe in mouse embryonic fibroblasts from Opa3−/− mutants and imaged by electron microscopy of the retinas. Results. The splice variants Opa3a and Opa3b were expressed in the lenses and the retinas in the Opa3−/− mice, with the expression of the Opa3a isoform predominant. Opa3 was expressed throughout embryonic development, with high levels of expression in the developing brain, retina, optic nerve, and lens. Opa3 localized to the mitochondria, and the L122P mutant protein did not mislocalize. Neither protein localized to the peroxisome. Opa3−/− mice displayed disrupted mitochondrial morphology in the retina. Wild-type Opa3 protein increased as the lenses aged, despite the reduction in Opa3 mRNA occurring as a part of lens differentiation. However, mutant Opa3 mRNA was upregulated in homozygous mutant lenses, suggesting a compensatory increase in expression, which may further increase Opa3 protein levels. Conclusions. Mutant Opa3 protein retains its mitochondrial localization and induces disrupted mitochondrial morphology. Opa3 accumulates in the lens. The results may reflect a slow turnover of Opa3 protein in vivo and may be important in normal lens physiology

Developmentally regulated expression of hemoglobin subunits in avascular tissues

We investigated the spatio-temporal profile of hemoglobin subunit expression in developing avascular tissues. Significant up-regulation of hemoglobin subunits was identified in microarray experiments comparing blastocyst inner cell masses with undifferentiated embryonic stem (ES) cells. Hemoglobin expression changes were confirmed using embryoid bodies (derived from in vitro differentiation of ES cells) to model very early development at pre-vascular stages of embryogenesis; i.e. prior to hematopoiesis. We also demonstrate, using RT-PCR, Western blotting and immunocytochemistry, expression of adult and fetal mouse hemoglobin subunits in the avascular ocular lens at various stages of development and maturation. Hemoglobin proteins were expressed in lens epithelial cells (cytoplasmic) and cortical lens fiber cells (nuclear and cell-surface-associated); however, a sensitive heme assay demonstrated negligible levels of heme in the developing lens postnatally. Hemoglobin expression was also observed in the developing eye in corneal endothelium and retinal ganglion cells. Gut sections showed, in addition to erythrocytes, hemoglobin protein staining in rare, individual villus epithelial cells. These results suggest a paradigm shift: hemoglobin subunits are expressed in the avascular lens and cornea and in pre-hematopoietic embryos. It is likely, therefore, that hemoglobin subunits have novel developmental roles; the absence of the heme group from the lens would indicate that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of hemoglobin subunits in the perinuclear region during lens fiber cell differentiation, when denucleation is taking place, may indicate involvement in the apoptosis-like signaling processes occurring in differentiating lens fiber cells

Cellular inhibitor of apoptosis (cIAP1) is down-regulated during retinal ganglion cell (RGC) maturation.

Apoptosis, is the main type of cell death that occurs in ageing and neurodegenerative disease, such as glaucoma. This study therefore characterises the expression profile of caspases (pro-apoptosis) and inhibitors of apoptosis (IAPs; anti-apoptosis) during maturation of the Brown Norway rat retina between 6 weeks and >24 weeks and also examines concomitant changes in expression of tumor necrosis factor receptor associated factor 2 (TRAF2). The expression profiles of caspases (initiator caspases 8, 9 and effector caspases 6, 7, 3) and inhibitors of apoptosis (IAPs) (Neuronal IAP), cellular IAP1 and 2 (cIAP1/2), X-chromosome linked IAP (XIAP), Survivin, Bruce and Livin) were examined in retinae from 6 weeks and >24 weeks old BN rats using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, Western blotting and immunofluoroscence analysis. Caspase expression was not altered significantly during the study interval. IAP expression showed a general reduction during maturation of BN retina, which was statistically significant for cIAP1. cIAP1 reduction was confirmed by Western blotting and immunoflouroscence and was restricted to cells in the retinal ganglion cell layer (RGCL). Accumulation of TRAF2 was observed in the RGCL accompanying the down-regulation of cIAP1 observed. Our results suggest that cells in the mature RGCL may have a greater susceptibility to cell death compared to their younger counterparts and this may be due in part to a reduction in activation of survival pathways involving IAPs and TRAFs

Goethean pedagogy A case in innovative science education and implications for work based learning

Purpose The purpose here is to highl ight the profound learning associated with the Goethean methodology in the Holistic Science MSc at Schumacher College, Devon, U.K. This is prese n t ed as a case study in profound pedagogy and as an exploration of the implications for workplace learning. Some c omparison s are also made with reflective practice . Design/ methodology/ approach Background is provided on Goethe?s ? way of science ? and Barfield?s ?participation? . S tudents were also interviewed about their learning and reflect on their experiences an d challenges in learning the Goethean methodology, particularly regarding perceptions and participation, on their altered modes of thinking and feelings about learning, as well as on an ?immediate? , corporeal and potentially co - operative mode of knowing in a ?community of practice?, which can be extrapolated to the workplace. Findings The profound ness of the student experience and personal transformation presented in the interviews reveals that Goethean methodology has a place alongside the more specific analytical knowledge focu s of Universities. Wh ile t he method has challenges in reconciling existing modes of knowing with the new approach, the students are able to see and intuit the wholeness and 2 dynamism of phenomena more easily , and t hey gain a differe nt perspective and learn to participate more full y in the world . Individuality/ value The article asks that this template for educational practice be considered more widely relevant to today?s educational landscape in better providing skills and prepa ring students for the workplace in a world of ?super - complexity?

"I found that I've been free all along": Knowledge, meaning, creativity and holistic science

no abstract availabl

Science playtime

A cold Friday in December in Dublin…as it often is, the wind is biting, blowing in from the North East off the Irish Sea, across Dublin Bay and up the River Liffey. Blustering its busy way between the buildings of Trinity College. It’s 9:45 am. I’m heading towards the old Botany lecture theatre-a very traditional space…wooden benches for seats that seem to trap the history here within them. How many lecturers’ words have fallen on sleepy students’ deaf ears in this place

Goethean pedagogy a case in innovative science education and implications for work based learning

Purpose The purpose here is to highlight the profound learning associated with the Goethean methodology in the Holistic Science MSc at Schumacher College, Devon, U.K. This is presented as a case study in profound pedagogy and as an exploration of the implications for workplace learning. Some comparisons are also made with reflective practice. Design/ methodology/ approach Background is provided on Goethe’s ‘way of science’ and Barfield’s ‘participation’. Students were also interviewed about their learning and reflect on their experiences and challenges in learning the Goethean methodology, particularly regarding perceptions and participation, on their altered modes of thinking and feelings about learning, as well as on an ‘immediate’, corporeal and potentially co-operative mode of knowing in a ‘community of practice’, which can be extrapolated to the workplace. Findings The profoundness of the student experience and personal transformation presented in the interviews reveals that Goethean methodology has a place alongside the more specific analytical knowledge focus of Universities. While the method has challenges in reconciling existing modes of knowing with the new approach, the students are able to see and intuit the wholeness and dynamism of phenomena more easily, and they gain a different perspective and learn to participate more fully in the world. Individuality/ value The article asks that this template for educational practice be considered more widely relevant to today’s educational landscape in better providing skills and preparing students for the workplace in a world of ‘supercomplexit