Manganese superoxide dismutase (SOD2) converts superoxide to oxygen plus hydrogen peroxide and serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in humans has been associated with several chronic diseases, including ovarian cancer and type I diabetes, and SOD2 overexpression appears to suppress malignancy in cultured cells. We have produced a line of SOD2 knockout mice (SOD2m1BCM/SOD2m1BcM) that survive up to 3 weeks of age and exhibit several novel pathologic phenotypes including severe anemia, degeneration of neurons in the basal ganglia and brainstem, and progressive motor disturbances characterized by weakness, rapid fatigue, and circling behavior. In addition, SOD2m1BCM/SOD2m1BCM mice older than 7 days exhibit extensive mitochondrial injury within degenerating neurons and cardiac myocytes. Approximately 10% of SOD2m1BCM/SOD2m1BCM mice exhibit markedly enlarged and dilated hearts. These observations indicate that SOD2 deficiency causes increased susceptibility to oxidative mitochondrial injury in central nervous system neurons, cardiac myocytes, and other metabolically active tissues after postnatal exposure to ambient oxygen concentrations. Our SOD2-deficient mice differ from a recently described model in which homozygotes die within the first 5 days of life with severe cardiomyopathy and do not exhibit motor disturbances, central nervous system injury, or ultrastructural evidence of mitochondrial injury.Superoxide radicals produced as by-products of metabolic oxidation can cause extensive cellular injury, and several different superoxide dismutases (SODs) have evolved to inactivate both intracellular and extracellular superoxide (1-7). Two closely related SODs containing either manganese or iron as cofactors are produced in most bacterial species, whereas most eukaryotic species contain at least two different intracellular SODs: (i) manganese superoxide (Mn SOD/SOD2) localized within the mitochondrial matrix and (ii) copper-and zinc-containing SOD1 (Cu/Zn SOD1/SOD1) localized predominantly in cytoplasmic and nuclear compartments (8).Another copper-and zinc-containing SOD found predominantly in extracellular compartments (EC SOD/SOD3) has recently been described (9). Although Mn SOD is located within the mitochondrial matrix, the SOD2 gene encoding Mn SOD is located within nuclear chromosomal DNA (10).Yeast and bacterial mutants devoid of all SOD activities exhibit hypersensitivity to oxygen and redox compounds such as paraquat, and they survive ambient oxygen by using predominantly anaerobic metabolic pathways to minimize superoxide production (2, 11-13). SODl-deficient mutants of Drosophila melanogaster are viable, but exhibit oxygen and paraquat sensitivity, decreased lifespan, and female infertility (14). Although other potent antioxidants such as glutathione, ascorbate, and tocopherols are present to varying degrees within eukaryotic and prokaryotic cells, none of these inactivates superoxide as rapidly or effectively as SODs.Several recent studies have sugg...
Paget's disease of bone (PDB) is the second most common bone disease and is characterized by focal bone lesions which contain large numbers of abnormal osteoclasts (OCLs) and very active normal osteoblasts in a highly osteoclastogenic marrow microenvironment. The etiology of PDB is not well understood and both environmental and genetic causes have been implicated in its pathogenesis. Mutations in the SQSTM1/p62 gene have been identified in up to 30% of Paget's patients. To determine if p62 mutation is sufficient to induce PDB, we generated mice harboring a mutation causing a P-to-L (proline-to-leucine) substitution at residue 394 (the murine equivalent of human p62(P392L), the most common PDB-associated mutation). Bone marrow cultures from p62(P394L) mice formed increased numbers of OCLs in response to receptor activator of NF-kappaB ligand (RANKL), tumor necrosis factor alpha (TNF-alpha) or 1alpha,25-(OH)(2)D(3), similar to PDB patients. However, purified p62(P394L) OCL precursors depleted of stromal cells were no longer hyper-responsive to 1alpha,25-(OH)(2)D(3), suggesting effects of the p62(P394L) mutation on the marrow microenvironment in addition to direct effects on OCLs. Co-cultures of purified p62(P394L) stromal cells with either wild-type (WT) or p62(P394L) OCL precursors formed more OCLs than co-cultures containing WT stromal cells due to increased RANKL production by the mutant stromal cells. However, despite the enhanced osteoclastogenic potential of both OCL precursors and marrow stromal cells, the p62(P394L) mice had histologically normal bones. These results indicate that this PDB-associated p62 mutation is not sufficient to induce PDB and suggest that additional factors acting together with p62 mutation are necessary for the development of PDB in vivo.
We describe an infant with del(17) (p11.2p12) whose deleted chromosome was inherited from a mosaic mother. The child had manifestations consistent with Smith-Magenis syndrome. The mother appeared to be of normal intelligence and she had minimal findings of Smith-Magenis syndrome. Separation of chromosome 17 homologues in somatic cell hybrids and molecular studies confirmed the cytogenetic diagnoses and the fact that the mother was mosaic. Furthermore, molecular analysis demonstrated novel breakpoints in this family, with the deletion extending into and completely encompassing the markers duplicated in Charcot-Marie-Tooth (CMT) disease. Although this Smith-Magenis syndrome patient is completely deleted for the CMT region, her electrophysiological findings are different from those found in CMT. This is the only reported case of Smith-Magenis syndrome with transmission from a partially affected mosaic mother. Transmission of interstitial deletions from mosaic parents may be more common than thought; therefore, parental chromosomes should be examined when interstitial deletions are identified.
We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 doublemutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthineguanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. An increasing volume of data supports a model of action of these three mammalian MutS homologues. In humans, the MSH2 protein forms heterodimeric complexes with both MSH6 and MSH3. These heterodimers are known as MutSalpha (MSH2-MSH6) and MutSbeta (MSH2-MSH3) (13,25). In combination with other DMR proteins, MutSalpha functions in the repair of single base mismatches and small I/D heterologies, whereas MutSbeta repairs larger I/D heterologies. Thus, MSH2 is critical for the repair of all mismatched lesions in mammals, whereas MSH6 and MSH3 modulate the function of MSH2 by providing specificity for different lesion types (44).Mutations in these DMR genes lead to genomic instability in bacteria, yeast, and mammals by affecting a number of cellular processes. Msh2 and Msh6 mutations lead to an increase in the accumulation of spontaneous mutations, also known as a mutator phenotype, due to the lack of repair of mismatches and I/D heterologies that arise as errors during replication (5, 12, 18). Overexpression of MSH3 also leads to a mutator phenotype in mammalian cells due to the sequestration of the available MSH2 into MutSbeta heterodimers, leading to a functional loss of MutSalpha activity (9,22).Mutations in Msh2, Msh6, and Msh3 also result in instability of the size of simple sequence repeats or microsatellites (24,26,37,41). This instability is thought to occur due to the lack of repair of DNA polymerase slippage products that arise during the replication of these highly repetitive sequences. Microsatellite instability in certain human tumors was a critical clue to the identification of the involvement of DMR genes in cancer and has since been used to demonstrate their involvement in familial cancer syndromes as well as in sporadic tumors (1,12,18,28,37,43). DMR acts as a block to mitotic homeologous recombination, presumably by recognizing and binding mismatches and I/D heterologies that arise in the heteroduplex intermediates of recombination between diverged substrates. Esc...
ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate-resistant acid phosphatase (TRAP)–ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP-ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone-resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF-κB, Erk, and Akt signaling compared with wild-type (WT) littermates. This increased bone-resorbing capacity per OCL was associated with increased levels of p-Pyk2 and p-Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF-α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF-α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease. © 2011 American Society for Bone and Mineral Research.
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