The tissue distribution of microfibrils reacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associated microfibrils

Elastic tissue, when viewed in the electron microscope, consists of an amorphous component that is immunoreactive with anti-tropoelastin (TE) antibodies and microfibrils, that react with monospecific antibodies against a 31 kDa microfibrillar glycoprotein constituent, called MAGP. A detailed study of the tissue distribution of microfibrils and of the two elastic tissue antibodies has been carried out, using single and double-labeled immunogold techniques in high resolution electron microscopy. Microfibrils similar in appearance to those associated with elastic tissue and immunoreactive with the anti-MAGP antibody, have been demonstrated in many tissues in the absence of amorphous elastic tissue. In the majority of these tissues, specific anti-TE antibody localization was demonstrated in the immediate vicinity of the microfibrils, or alternatively, the microfibrils were shown to be in direct continuity with microfibrils of similar morphology, which were associated with material immunoreactive with anti-TE antibody. The diameter of these microfibrils varied between 8 nm and 16 nm. They were unbranched structures of indefinite length, with a tubular profile on cross section and periodic staining in longitudinal section. In some tissues, notably in the ciliary zonule and in the mesangial region of the renal glomerulus, microfibrils of similar morphology were demonstrated which were immunoreactive with anti-MAGP antibody, but which were unrelated to amorphous elastic tissue and with which anti-TE antibody localization could not be demonstrated. The evidence available supports the conclusion that all these microfibrils are members of a single class of structures, which are widely distributed in the tissues and which are secreted by a range of cell types. Attention is directed to the close relationship between these microfibrils and the basement membrane of the glomerulus, of uterine smooth muscle, of the basal cells of the epidermis and of the reticulum cells of the spleen.     

A Distinct Smoothened Mutation Causes Severe Cerebellar Developmental Defects and Medulloblastoma in a Novel Transgenic Mouse Model

Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common pediatric brain malignancy. About 25 to 30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened (Smo) is a key transducer of the Shh pathway. Activating mutations in Smo that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the developmental and oncogenic effects of two closely positioned point mutations in Smo, we characterized NeuroD2-SmoA2 mice and compared them to NeuroD2-SmoA1 mice. While both SmoA1 and SmoA2 transgenes cause medulloblastoma with similar frequencies and timing, SmoA2 mice have severe aberrations in cerebellar development, whereas SmoA1 mice are largely normal during development. Intriguingly, neurologic function, as measured by specific tests, is normal in the SmoA2 mice despite extensive cerebellar dysplasia. We demonstrate how two nearly contiguous point mutations in the same domain of the encoded Smo protein can produce striking phenotypic differences in cerebellar development and organization in mice.     

Diversity Patterns of Bornean Butterfly Assemblages

Borneo contains a diverse rainforest butterfly community, but its forests are under threat from logging and ENSO- (El Niño Southern Oscillation) induced fires. Contrasts in butterfly assemblage structure were examined in nine 450 ha landscapes in logged forest, primary unburned continuous and isolated forest, and forest affected by surface fires during the 1997/98 ENSO event. Temporally the effect of the 1997/98 ENSO event was followed in a single burned landscape from 1997 to 2004. In total, 517 species were present in 190 sampling sites. There was a five-fold difference in species richness among landscapes, with highest richness in continuous landscapes and lowest richness in burned landscapes. Richness was also higher in logged forest than proximate unlogged forest. Temporally, species richness dropped dramatically from 1997 to 1998, but afterwards increased remaining, however, substantially lower than pre-ENSO (1997) sampling. Sites in burned landscapes were distinct from other sites in terms of vegetation structure with the slash-and-burn area the most dissimilar to other landscapes. There was much less structure among unburned landscapes. The pattern of butterfly community composition was similar to that of vegetation structure with the community from the slash-and-burn area the most distinct. However, there was much less overlap among sites from different landscapes. Temporally, 1998 possessed the most distinct assemblage when compared to assemblages from other years. The community composition was, however, slowly returning to a pre-disturbance composition. Variance in community composition explained by environmental and spatial factors differed substantially among landscapes. The spatial fraction was the only explanatory component in recently burned landscapes and a proximate small unburned isolate, but explained no variation in logged landscapes. The environmental fraction explained substantial amounts of variation in logged landscapes and the slash-and-burn area. When all landscapes were pooled high proportions of variation in butterfly community composition were explained by both geographic distance between sites and environmental variables. In contrast when only unburned landscapes were considered, most variation was explained by the geographic distance among them. Despite differences among landscapes there was a general pattern of relatively sharp decline in similarity at short distances that levels out over greater distances, a result that agrees with previous studies on other tropical species assemblages.     

A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.