RESUMEN
The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The ß-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, which induces the next molecular collapse. The aberrant WWP1 increases the ubiquitin ligase-mediated ubiquitination following severe degradation of sarcolemmal and cytoplasmic ß-dystroglycan, and an erased ß-dystroglycan in dystrophic αW fibers will lead to molecular imperfection of the dystrophin-glycoprotein complex (DGC). The DGC is a core protein of costamere that is an essential part of force transduction and protects the muscle fibers from contraction-induced damage. Caveolin-3 (Cav-3) and dystrophin bind competitively to the same site of ß-dystroglycan, and excessive Cav-3 on sarcolemma will block the interaction of dystrophin with ß-dystroglycan, which is another reason for the disruption of the DGC. It is known that fast-twitch glycolytic fibers are more sensitive and vulnerable to contraction-induced small tears than slow-twitch oxidative fibers under a variety of diseased conditions. Accordingly, the fast glycolytic αW fibers must be easy with rapid damage of sarcolemma corruption seen in chicken muscular dystrophy, but the slow oxidative fibers are able to escape from these damages.
Asunto(s)
Caveolina 3/metabolismo , Distrofias Musculares/metabolismo , Animales , PollosRESUMEN
BACKGROUND: Granulocyte colony-stimulating factor (G-CSF), a hematopoietic cytokine, was recently used to treat patients of acute myocardial infarction with beneficial effect. However, controversy exists as some patients developed re-stenosis and worsened condition post G-CSF delivery. This study presents a new disease model to study G-CSF induced cardiac thrombosis and delineate its possible mechanism. We used iron loading to mimic condition of chronic cardiac dysfunction and apply G-CSF to mice to test our hypothesis. METHODS AND RESULTS: Eleven out of fifteen iron and G-CSF treated mice (I+G) showed thrombi formation in the left ventricular chamber with impaired cardiac function. Histological analysis revealed endothelial fibrosis, increased macrophage infiltration and tissue factor expression in the I+G mice hearts. Simvastatin treatment to I+G mice attenuated their cardiac apoptosis, iron deposition, and abrogated thrombus formation by attenuating systemic inflammation and leukocytosis, which was likely due to the activation of pAKT activation. However, thrombosis in I+G mice could not be suppressed by platelet receptor inhibitor, tirofiban. CONCLUSIONS: Our disease model demonstrated that G-CSF induces cardiac thrombosis through an inflammation-thrombosis interaction and this can be attenuated via statin therapy. Present study provides a mechanism and potential therapy for G-CSF induced cardiac thrombosis.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/efectos adversos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Miocarditis/inducido químicamente , Miocarditis/tratamiento farmacológico , Trombosis/inducido químicamente , Trombosis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Células Endoteliales/patología , Fibrosis , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Hierro/farmacología , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Simvastatina/uso terapéutico , Tirofibán , Tirosina/análogos & derivados , Tirosina/uso terapéuticoRESUMEN
Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein-protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea-mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions). Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T) was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13), a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X), and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis.
Asunto(s)
Aciltransferasas/genética , Alopecia/genética , Amiloidosis/genética , Mutación , Osteoporosis/genética , Aciltransferasas/metabolismo , Envejecimiento , Alopecia/metabolismo , Alopecia/patología , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Inmunohistoquímica , Ratones , Especificidad de Órganos , Osteoporosis/metabolismo , Osteoporosis/patología , FenotipoRESUMEN
Members of the caveolin family are the main component of caveolae, and caveolin-3 is a muscle-specific protein. Caveolin-3 deficiency induces a muscular dystrophic phenotype, while its overexpression is also harmful to muscle cells. Increased caveolae were observed in chicken muscular dystrophy; however, the underlying mechanism causing the onset remains unclear. Therefore, the current study analyzes the expression of caveolin-3 and other caveola-related proteins in dystrophic chickens. Western blotting and semi-quantitative RT-PCR analysis revealed that (1) caveolin-3 is highly expressed in the damaged muscle of dystrophic chickens (7.12-fold); (2) the amount of caveolin-3 protein is regulated in posttranslational modification, since no significant increase is observed at the mRNA level (1.09-fold); and (3) the expression pattern of other caveola-related proteins is similar to that of caveolin-3. These results suggest that the accumulation of caveolin-3 protein may be associated with the causative process of chicken muscular dystrophy.
Asunto(s)
Caveolina 3/genética , Caveolina 3/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Animales , Pollos , Femenino , Masculino , Distrofias Musculares/patología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Garlic (Allium sativum) has been valued in many cultures both for its health effects and as a culinary flavor enhancer. Garlic's chemical complexity is widely thought to be the source of its many health benefits, which include, but are not limited to, anti-platelet, procirculatory, anti-inflammatory, anti-apoptotic, neuro-protective, and anti-cancer effects. While a growing body of scientific evidence strongly upholds the herb's broad and potent capacity to influence health, the common mechanisms underlying these diverse effects remain disjointed and relatively poorly understood. We adopted a phenotype-driven approach to investigate the effects of garlic in a mouse model. We examined RBC indices and morphologies, spleen histochemistry, RBC half-lives and gene expression profiles, followed up by qPCR and immunoblot validation. The RBCs of garlic-fed mice register shorter half-lives than the control. But they have normal blood chemistry and RBC indices. Their spleens manifest increased heme oxygenase 1, higher levels of iron and bilirubin, and presumably higher CO, a pleiotropic gasotransmitter. Heat shock genes and those critical for erythropoiesis are elevated in spleens but not in bone marrow. The garlic-fed mice have lower plasma erythropoietin than the controls, however. Chronic exposure to CO of mice on garlic-free diet was sufficient to cause increased RBC indices but again with a lower plasma erythropoietin level than air-treated controls. Furthermore, dietary garlic supplementation and CO treatment showed additive effects on reducing plasma erythropoietin levels in mice. Thus, garlic consumption not only causes increased energy demand from the faster RBC turnover but also increases the production of CO, which in turn stimulates splenic erythropoiesis by an erythropoietin-independent mechanism, thus completing the sequence of feedback regulation for RBC metabolism. Being a pleiotropic gasotransmitter, CO may be a second messenger for garlic's other physiological effects.
Asunto(s)
Monóxido de Carbono/química , Eritrocitos/citología , Eritropoyetina/metabolismo , Ajo/metabolismo , Alimentación Animal , Animales , Médula Ósea/metabolismo , Eritrocitos/efectos de los fármacos , Eritropoyesis , Regulación de la Expresión Génica , Hemo-Oxigenasa 1/metabolismo , Ratones , Microscopía Electrónica de Rastreo/métodos , Fenotipo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS: Glucose transporter 10 (GLUT10), encoded by the SLC2A10 gene, is a member of the class III facilitative glucose transporter family. Mutations in the SLC2A10 gene cause arterial tortuosity syndrome (ATS) in humans. To further study the pathogenesis of the disease, we generated mice carrying GLUT10 mutations. METHODS AND RESULTS: Using a gene-driven N-ethyl-N-nitrosourea (ENU)-mutagenesis approach, we generated mice carrying GLUT10 mutations c.383G>A and c.449C>T, which resulted in missense mutations of glycine to glutamic acid (p.G128E) and serine to phenylalanine (p.S150F), respectively. Both mutant strains appeared normal at birth, gained weight appropriately and survived to adulthood (>18 months). Blood and urine glucose were normal. Echocardiogram and electrocardiogram were also normal and brain magnetic resonance angiography revealed normal cerebral arteries without tortuosity, stenosis/dilatation, or aneurysm. Histopathology revealed thickening and irregular vessel wall shape of large and medium size arteries characterized by markedly increased elastic fibres, both in number and size. There was also intima endothelial hypertrophy and deranged elastic fibres that resulted in disruption of internal elastic lamina in the aorta of older mice. CONCLUSION: Abnormal elastogenesis with early elastic fibre proliferation provides a clue to the pathogenesis of arterial tortuosity in human ATS. Availability of this mouse model will allow testing of the relationship between diabetes and its vascular complications, including diabetic retinopathy, nephropathy and peripheral vascular disease.
Asunto(s)
Arterias/anomalías , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Mutación , Animales , Arterias/patología , Tejido Elástico/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.
Asunto(s)
Pollos/genética , Distrofia Muscular Animal/genética , Enfermedades de las Aves de Corral/genética , Ubiquitina-Proteína Ligasas/genética , Secuencia de Aminoácidos , Animales , Expresión Génica , Datos de Secuencia Molecular , Mutación MissenseRESUMEN
Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.
Asunto(s)
Hígado Graso/metabolismo , Complejos Multienzimáticos/genética , Miocardio/metabolismo , Mutación Puntual , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Secuencia de Bases , Western Blotting , Carnitina/análogos & derivados , Carnitina/metabolismo , Mapeo Cromosómico , Etilnitrosourea/toxicidad , Hígado Graso/inducido químicamente , Hígado Graso/patología , Femenino , Fibrosis , Trastornos del Metabolismo de los Lípidos/inducido químicamente , Trastornos del Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Trifuncional Mitocondrial , Subunidad alfa de la Proteína Trifuncional Mitocondrial , Subunidad beta de la Proteína Trifuncional Mitocondrial , Complejos Multienzimáticos/metabolismo , Mutagénesis/efectos de los fármacos , Miocardio/patología , Reacción en Cadena de la Polimerasa , Embarazo , Espectrometría de Masas en TándemRESUMEN
Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive hereditary neurodegenerative disease of humans. So far, no responsible gene has been cloned or mapped to any chromosome. For chromosome mapping and positional cloning of the responsible gene, establishment of an animal model would be useful. Here we describe a new mouse model for INAD, named inad mouse. In this mouse, the phenotype is inherited in an autosomal recessive manner, symptoms occur in the infantile period, and the mouse dies before sexual maturity. Axonal dystrophic change appearing as spheroid bodies in central and peripheral nervous system was observed. These features more closely resembled human INAD than did those of the gad mouse, the traditional mouse model for INAD. Linkage analysis linked the inad gene to mouse Chromosome 1, with the highest LOD score (=128.6) at the D1Mit45 marker, and haplotype study localized the inad gene to a 7.5-Mb region between D1Mit84 and D1Mit25. In this linkage area some 60 genes exist: Mutation of one of these 60 genes is likely responsible for the inad mouse phenotype. Our preliminary mutation analysis in 15 genes examining the nucleotide sequence of exons of these genes did not find any sequence difference between inad mouse and C57BL/6 mouse.
Asunto(s)
Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Modelos Animales de Enfermedad , Distrofias Neuroaxonales/genética , Animales , Encéfalo/patología , Análisis Mutacional de ADN , Genes Recesivos , Escala de Lod , Ratones , Músculo Esquelético/patología , Distrofias Neuroaxonales/patología , LinajeRESUMEN
Despite intensive studies of muscular dystrophy of chicken, the responsible gene has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to chromosome 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11-24.3, where the beta-1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) genes are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease.
Asunto(s)
Pollos , Proteínas Asociadas a la Distrofina , GTP Fosfohidrolasas/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas Musculares/genética , Distrofia Muscular Animal/genética , Enfermedades de las Aves de Corral/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Clonación Molecular , GTP Fosfohidrolasas/química , Humanos , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas Musculares/química , Polimorfismo Genético , Proteoglicanos/química , Sindecano-2RESUMEN
The gracile axonal dystrophy (gad) mice are known to have a deletion within the gene encoding ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) and show hereditary sensory deterioration and motor paresis. Expression of Uch-L1 is reported to be almost limited to the nervous system and testis. To understand whether Uch-L1, one of the major ubiquitin carboxy-terminal hydrolase (UCH) isozymes in the testis, affects spermatogenesis and other UCH isozymes (Uch-L3, L4 and L5) expression in the testis, we compared the testis between gad, hetero and wild type mice by histological, immunohistochemical analyses and RT-PCR. Histological analysis in 25-week-old gad mice showed shrinking of seminiferous tubules, decreasing total number of cells and enlargement of remaining cells in seminiferous tubules. By immunohistochemistry, a significant decrease (p < 0.05) in the number of proliferating cell nuclear antigen (PCNA) positive cells was observed. Expression of other UCH isozyme mRNAs was not apparently affected by Uch-L1 deficiency in 25-week-old gad mice. This study is the first report on the testis of gad mutant mouse.
Asunto(s)
Eliminación de Gen , Túbulos Seminíferos/patología , Tioléster Hidrolasas/genética , Animales , Atrofia , Inmunohistoquímica , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Mutantes , Antígeno Nuclear de Célula en Proliferación/análisis , Túbulos Seminíferos/citología , Túbulos Seminíferos/enzimología , Tioléster Hidrolasas/metabolismo , Ubiquitina TiolesterasaRESUMEN
Caveolin, a 20-24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co-immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma.
Asunto(s)
Caveolinas/metabolismo , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Miocardio/metabolismo , Útero/citología , Animales , Caveolina 1 , Caveolina 3 , Distrofina/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Macaca fascicularis , Microscopía Confocal , Músculo Esquelético/citología , Músculo Liso/citología , Miocardio/citología , Sarcolema/metabolismo , Sarcolema/ultraestructura , Útero/metabolismoRESUMEN
Accumulation of unfolded and malfolded proteins causes endoplasmic reticulum (ER) stress, stimulating unfolded protein response (UPR) and c-Jun N-terminal kinase (JNK) activation and activating caspase-12 located on the ER. Little is known about the relationship between the ER stress and polyglutamine [poly(Q)] aggregates. Poly(Q)72 repeats [poly(Q)(72)] induced the stimulation of ER stress signals such as JNK activation, upregulation of Grp78/Bip and caspase-12 activation in C2C5 cells. We prepared antiserum against the cleavage site of mouse caspase-12 at D(318) (anti-m12D318), and showed that poly(Q)(72) with perinuclear aggregates, cytoplasmic inclusions and nuclear inclusions stimulated JNK activation and anti-m12D318 immunoreactivity, but poly(Q)(72) with dispersed aggregates and small nuclear aggregates showed a significantly less effect. Poly(Q)(72) and poly(Q)(11) dispersed in cytoplasm did not. Anti-m12D318-positive cells showed apoptotic features. Unlike anti-m8D387 immunoreactivity, the anti-m12D318 immunoreactivity was not coaggregated with poly(Q). Ac-IETD-fmk (caspase-8 inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) did not prevent the anti-m12D318 immunoreactivity induced by poly(Q)(72) aggregates. Anti-m12D318 immunoreactivity was detected in caspase-8(-/-) and caspase-3(-/-) mouse embryonic fibroblasts expressing poly(Q)(72) aggregates. Thus, caspase-12 was activated by poly(Q)(72) aggregates via a pathway independent of caspase-8 and caspase-3 activation, and caspase-12 activation was closely associated with poly(Q) aggregate-mediated cell death. Stimulation of ER stress signals may be involved in the pathogenesis of neurodegenerative disorders with poly(Q) expansion.
Asunto(s)
Caspasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico , Péptidos/metabolismo , Animales , Apoptosis , Células COS , Proteínas Portadoras/metabolismo , Caspasa 12 , Caspasa 8 , Caspasa 9 , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Genes jun , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Chaperonas Moleculares/metabolismo , Transducción de Señal/fisiologíaRESUMEN
It has been reported that light exposure to one eye induces functional lateralization, which can be inverted by exposing the opposite eye to the light. However, the anatomical basis of the functional inversion by the light has not been shown. To address this issue, we labeled cells in the dorsolateral anterior thalamus (DLA) using retrograde fluorescent tracers injected into visual Wulst, counted the labeled cell number, and compared the anatomical asymmetry of DLA between the left eye occluded and the right eye occluded chickens. We found that a rostral part of DLA (DLAda) and a lateral/ventral part of DLA differentially projected to the visual cortex ipsilaterally and contralaterally, respectively. These regions showed anatomical asymmetry that was inverted by the light. An antibody against a nicotinic acetylcholine receptor subunit more intensively and widely stained the side of DLA receiving the light stimulation and the cell labeled by the tracers co-localized with the immunoreactive neuropil. These results indicated that the light experience induced the anatomical lateralization of thalamofugal visual pathway.