Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment.

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BackgroundDevelopment of the secondary palate (SP) is a complex event and abnormalities during SP development can lead to cleft palate, one of the most common birth disorders. Matrix metalloproteinases (MMPs) are required for proper SP development, although a functional role for any one MMP in SP development remains unknown. MMP-25 may have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the MMP-25 gene. We report on the gene expression profile of MMP-25 in the developing mouse SP and identify its functional role in mouse SP development.ResultsMMP-25 mRNA and protein are found at all SP developmental stages in mice, with the highest expression at embryonic day (E) 13.5. Immunohistochemistry and in situ hybridization localize MMP-25 protein and mRNA, respectively, to the apical palate shelf epithelial cells and apical mesenchyme. MMP-25 knockdown with siRNA in palatal cultures results in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium. MMP-25 mRNA and protein levels significantly decrease when cultured palate shelves are incubated in growth medium with 5 μg/mL of a TGF-β3-neutralizing antibody.ConclusionsOur findings indicate: (i) MMP-25 gene expression is highest at E12.5 and E13.5, which corresponds with increasing palate shelf growth downward alongside the tongue; (ii) MMP-25 protein and mRNA expression predominantly localize in the apical epithelium of the palate shelves, but are also found in apical areas of the mesenchyme; (iii) knockdown of MMP-25 mRNA expression impairs palate shelf fusion and results in significant medial edge epithelium remaining in contacted areas; and (iv) bio-neutralization of TGF-β3 significantly decreases MMP-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development and are the first to identify a role for a single MMP in mouse SP development.

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Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. M M R recognizes and repairs DNA mismatches and small insertion/deletion loops. Carriers of MMR gene variants have a high risk of developing colorectal, endometrial, ovarian, and other extracolonic carcinomas. We report on an ovarian cancer patient who carries a germline MSH2 c.1A>C variant which alters the translation initiation codon. Mutations affecting the MSH2 start codon have been described previously for LS-related malignancies. However, the patients often lack a clear family history indicative of LS and their tumors often fail to display microsatellite instability, a hallmark feature of LS. Therefore, the pathogenicity of start codon variants remains undefined. Loss of the MSH2 start codon has been predicted to result in a truncated protein translated from a downstream in-frame AUG that would lack the first 25 amino acids. We therefore purified recombinant MSH2(NΔ25)-MSH6 and MSH2(NΔ25)-MSH3 to examine their DNA lesion recognition and adenosine nucleotide processing functions in vitro. We found that the MSH2(NΔ25) mutant confers distinct biochemical defects on MSH2-MSH6, but does not have a significant effect on MSH2-MSH3. We confirmed that expression of the MSH2 c.1A>C cDNA results in the production of multiple protein products in human cells that may include the truncated and full-length forms of MSH2. An in vivo MMR assay revealed a slight reduction in MMR efficiency in these cells. These data suggest that mutation of the MSH2 initiation codon, while not a strong, high-risk disease allele, may have a moderate impact on disease phenotype.

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The effect of tail docking on metabolizable energy requirements and carcass characteristics was studied using 80 weaned entire Awassi (between 0·74 and 1·1 MJ/ kg M 0·75 per day). Hot and cold carcass weights were similar ( P > 0·05) for the two groups. Differences in empty body weight and fleece-free empty body weight were significant ( P < 0·05) only for the 0·443 to 0·522 MJ/kg M 0·75 per day level of ME intake. Predicted ME requirements were higher for docked lambs for an estimated LWG between 0 and 100 g/day and lower for higher LWG (125 to 225 g/day). Docking had no effect ( P > 0·05) on food conversion efficiency (FCE). Carcasses from docked lambs had significantly lower ( P < 0·001) internal plus tail fat. Pooled soft tissue excluding tail fat, for the undocked lambs contained significantly more ( P < 0·01) protein, less ( P < 0·001) fat, higher ( P < 0·01) moisture and similar ( P > 0·05) ash content.