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1.
J Neurosci ; 33(9): 4055-65, 2013 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-23447614

RESUMEN

Invertebrate studies have highlighted a role for EH and SH3 domain Intersectin (Itsn) proteins in synaptic vesicle recycling and morphology. Mammals have two Itsn genes (Itsn1 and Itsn2), both of which can undergo alternative splicing to include DBL/PH and C2 domains not present in invertebrate Itsn proteins. To probe for specific and redundant functions of vertebrate Itsn genes, we generated Itsn1, Itsn2, and double mutant mice. While invertebrate mutants showed severe synaptic abnormalities, basal synaptic transmission and plasticity were unaffected at Schaffer CA1 synapses in mutant mice. Surprisingly, intercortical tracts-corpus callosum, ventral hippocampal, and anterior commissures-failed to cross the midline in mice lacking Itsn1, but not Itsn2. In contrast, tracts extending within hemispheres and those that decussate to more caudal brain segments appeared normal. Itsn1 mutant mice showed severe deficits in Morris water maze and contextual fear memory tasks, whereas mice lacking Itsn2 showed normal learning and memory. Thus, coincident with the acquisition of additional signaling domains, vertebrate Itsn1 has been functionally repurposed to also facilitate interhemispheric connectivity essential for high order cognitive functions.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Cognición/fisiología , Cuerpo Calloso/fisiología , Lateralidad Funcional/genética , 2-Amino-5-fosfonovalerato/farmacología , Proteínas Adaptadoras del Transporte Vesicular/genética , Análisis de Varianza , Animales , Biofisica , Mapeo Encefálico , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Señales (Psicología) , Imagen de Difusión Tensora , Embrión de Mamíferos , Antagonistas de Aminoácidos Excitadores/farmacología , Conducta Exploratoria/fisiología , Miedo , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/fisiología , Hipocampo/citología , Imagenología Tridimensional , Técnicas In Vitro , Discapacidades para el Aprendizaje/genética , Imagen por Resonancia Magnética , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/genética , Ratones , Ratones Transgénicos , Mutación/genética , Fibras Nerviosas/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Ratas , Ratas Wistar , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Vertebrados/genética , Vertebrados/metabolismo
2.
Blood ; 118(14): 3853-61, 2011 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-21666053

RESUMEN

The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.


Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Exones , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/metabolismo , Operón Lac , Leucemia/genética , Leucocitosis/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Trombocitopenia/genética
3.
Connect Tissue Res ; 54(3): 218-26, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23586515

RESUMEN

Flexor tendon healing is mediated by cell proliferation, migration, and extracellular matrix synthesis that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor tendon adhesion formation have been linked to transforming growth factor ß (TGF-ß). To elucidate the cellular and molecular events in this pathology, we implanted live flexor digitorum longus grafts from the reporter mouse Rosa26(LacZ/+) in wild-type recipients, and used histological ß-galactosidase (ß-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and reverse transcription-polymerase chain reaction to measure gene expression of TGF-ß and its receptors, extracellular matrix proteins, and matrix metalloproteinases (MMPs) and their regulators. Over the course of healing, graft cellularity and ß-gal activity progressively increased, and ß-gal-positive cells migrated out of the Rosa26(LacZ/+) graft. In addition, there was an evidence of influx of host cells (ß-gal-negative) into the gliding space and the graft, suggesting that both graft and host cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1 expression was the highest in the early phases of healing and gradually decreased thereafter, whereas Tgfb3 increased and remained upregulated later. The expression of TGF-ß receptors was also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which inflammatory MMPs were the highest early and matrix MMPs increased over time. These findings offer important insights into the complex cellular and molecular factors during flexor tendon healing.


Asunto(s)
Regulación de la Expresión Génica , Tendones/metabolismo , Tendones/patología , Adherencias Tisulares/patología , Cicatrización de Heridas/genética , Animales , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , ARN no Traducido/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Tendones/cirugía , Tendones/trasplante , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo
4.
Arthritis Rheum ; 64(11): 3649-59, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22832945

RESUMEN

OBJECTIVE: Glucocorticoid (GC) therapy is associated with increased risk of fracture in patients with rheumatoid arthritis (RA). To elucidate the cause of this increased risk, we examined the effects of chronic erosive inflammatory arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model. METHODS: Mice with established arthritis and expressing human tumor necrosis factor α (TNFα) transgene (Tg) and their wild-type (WT) littermates were continually treated with GC (prednisolone 5 mg/kg/day via subcutaneous controlled-release pellet) or placebo for 14, 28, or 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of the tibiae and lumbar vertebral bodies were assessed by micro-computed tomography, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined. RESULTS: TNF-Tg and GC treatment additively decreased mechanical strength and stiffness in both the tibiae and the vertebral bodies. GC treatment in the TNF-Tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclast activity. CONCLUSION: Our findings indicate that the concomitant decrease in bone strength and increase in bone ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low-energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy who do not have radiographic manifestations of fracture.


Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Fracturas Óseas/patología , Glucocorticoides/efectos adversos , Prednisolona/efectos adversos , Animales , Artritis Reumatoide/epidemiología , Fenómenos Biomecánicos/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Modelos Animales de Enfermedad , Fracturas Óseas/epidemiología , Glucocorticoides/administración & dosificación , Humanos , Masculino , Ratones , Ratones Transgénicos , Prednisolona/administración & dosificación , Factores de Riesgo , Espectrometría Raman , Columna Vertebral/diagnóstico por imagen , Columna Vertebral/efectos de los fármacos , Columna Vertebral/patología , Tibia/diagnóstico por imagen , Tibia/efectos de los fármacos , Tibia/patología , Transgenes/genética , Factor de Necrosis Tumoral alfa/genética , Microtomografía por Rayos X
5.
Nat Genet ; 36(7): 744-9, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15208629

RESUMEN

Faithful segregation of replicated chromosomes is essential for maintenance of genetic stability and seems to be monitored by several mitotic checkpoints. Various components of these checkpoints have been identified in mammals, but their physiological relevance is largely unknown. Here we show that mutant mice with low levels of the spindle assembly checkpoint protein BubR1 develop progressive aneuploidy along with a variety of progeroid features, including short lifespan, cachectic dwarfism, lordokyphosis, cataracts, loss of subcutaneous fat and impaired wound healing. Graded reduction of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence. Male and female mutant mice have defects in meiotic chromosome segregation and are infertile. Natural aging of wild-type mice is marked by decreased expression of BubR1 in multiple tissues, including testis and ovary. These results suggest a role for BubR1 in regulating aging and infertility.


Asunto(s)
Envejecimiento/genética , Infertilidad Femenina/genética , Infertilidad Masculina/genética , Proteínas Quinasas/fisiología , Aneuploidia , Animales , Proteínas de Ciclo Celular , Femenino , Masculino , Ratones , Ratones Mutantes , Fenotipo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas
6.
Exp Neurol ; 303: 72-79, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29337143

RESUMEN

BACKGROUND: Facial nerve paralysis is a significant cause of morbidity, affecting facial appearance, emotional expression, speech, oral competence, and vision. A more complete understanding of the complex cellular events required for successful nerve regeneration may reveal new therapeutic targets. The role of fibroblasts in regeneration, and the process by which the nerve reforms its three-dimensional structure after a transection injury, are not fully understood. The Hedgehog signaling pathway has been shown to mediate nerve sheath formation during development. We therefore sought to characterize the role of Hedgehog-responsive cells following transection of the facial nerve. METHODS: Two transgenic mouse lines with reporters for the downstream effector of Hedgehog signaling, Gli1, were used. The animals underwent a unilateral facial nerve transection injury, and the contralateral side served as a control. Facial nerves were analyzed via immunohistochemistry and immunofluorescence at predetermined time points as the facial nerve regenerated after the transection injury. RESULTS: There was a statistically significant increase in Gli1+ cells both at the site of injury and within the distal nerve segment over time. Gli1+ cells are fibroblasts within the nerve and appear to contribute to the reformation of the nerve sheath after injury. CONCLUSION: These findings describe a key signaling pathway by which fibroblasts participate in motor nerve regeneration. Fibroblasts that reside within the nerve respond to injury and may represent a novel therapeutic target in the context of facial nerve regeneration after transection injury.


Asunto(s)
Traumatismos del Nervio Facial/patología , Fibroblastos/metabolismo , Erizos/metabolismo , Regeneración Nerviosa/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Antígenos/metabolismo , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Citometría de Flujo , Galactósidos/genética , Galactósidos/metabolismo , Regulación de la Expresión Génica/genética , Erizos/genética , Indoles/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Proteoglicanos/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Transducción de Señal/genética , Proteína con Dedos de Zinc GLI1/genética
7.
Exp Biol Med (Maywood) ; 232(1): 70-80, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17202587

RESUMEN

Chronic caloric restriction (CCR) prevents mammary tumorigenesis in rodents, but a protective effect for intermittent caloric restriction (ICR) is less well documented. We recently reported that ICR reduced mammary tumor (MT) incidence of mouse mammary tumor virus-transforming growth factor (MMTV-TGF)-alpha mice to a greater extent than did CCR. Here, we repeated this protocol and obtained serum and tissue samples. Ad libitum (AL) MMTV-TGF-alpha mice were fed AIN-93M diet. Beginning at 10 weeks of age, ICR mice received isocaloric AIN-93M-mod diet (2-fold increases in protein, fat, vitamins, and minerals) at 50% of ad libitum for 3 weeks followed by 3 weeks refeeding with AIN-93M diet. CCR mice were pair-fed AIN-93M:AIN-93M-mod (2:1) matching intakes for restriction/refeeding cycles. Mice were sacrificed for MT size, at 79 (end of 12th restriction) or at 80 (1 week after 12th refeeding) weeks of age. AL and ICR-80 mice had heavier body weights than ICR-79 and CCR mice (P < 0.0001). Cumulative food intakes of ICR and CCR mice were reduced 12% and 15% versus AL mice (P < 0.0001). However, ICR mice consumed significantly (P < 0.0001) more food than did AL mice during refeeding. MT incidence was 84%, 13%, and 27% for AL, ICR, and CCR mice, respectively. MT weight (P < 0.0011) and number (P < 0.01) were higher for AL mice compared with ICR and CCR mice. AL and ICR-80 mice had similar serum IGF-I levels, but only AL values were higher than those of ICR-79 and CCR mice (P < 0.0017). ICR mice had more MT DNA breaks compared with AL and CCR mice, suggesting enhanced apoptosis (P < 0.02). AL mice had higher mammary fat pad ObR and ObRb leptin receptor mRNA expression than did ICR and CCR mice (P < 0.001), but there was no effect on MTs. These results confirm that ICR prevents development of MTs to a greater extent than does CCR, although "overeating" during refeeding may compromise this protection.


Asunto(s)
Restricción Calórica , Ingestión de Energía , Neoplasias Mamarias Animales/prevención & control , Animales , Peso Corporal , Roturas del ADN , Dieta , Femenino , Expresión Génica , Incidencia , Factor I del Crecimiento Similar a la Insulina/análisis , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , ARN Mensajero/análisis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Leptina , Factor de Crecimiento Transformador alfa/genética
8.
J Natl Cancer Inst ; 94(22): 1704-11, 2002 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-12441326

RESUMEN

BACKGROUND: Obesity is a risk factor for breast cancer in postmenopausal women. As body weight and fat mass increase, circulating leptin increases. Leptin is an adipocyte-derived cytokine that acts through the long form of its receptor, termed OB-Rb. To investigate whether leptin is associated with breast cancer, we determined the expression of OB-Rb in human breast epithelial HBL100 cells and human breast carcinoma-derived T-47D cells, determined whether leptin influenced the proliferation of these cells, and evaluated the structure of mammary tissue in genetically obese leptin-deficient Lep(ob)Lep(ob) and leptin receptor-deficient Lepr(db)Lepr(db) mice. METHODS: Cell numbers and cell colony formation by HBL100 and T-47D cells were determined by anchorage-dependent and anchorage-independent growth assays. OB-Rb expression was examined by reverse transcription-polymerase chain reaction and immunoblot analyses. Expression of leptin signaling pathway components was evaluated with immunoblot and electrophoretic mobility shift assays. Mammary gland development in lean and obese mice was investigated in whole-mount studies. All statistical tests were two-sided. RESULTS: Leptin enhanced anchorage-dependent proliferation by 138% (95% confidence interval [CI] = 108% to 169%) in T-47D cells and 50% (95% CI = 38% to 60%) in HBL100 cells. In both cell lines, OB-Rb was expressed, and leptin increased the expression of phosphorylated signal transducers and activators of transcription 3 (STAT3), phosphorylated extracellular signal-regulated kinase (ERK), and transcript activator protein 1 (AP-1). However, leptin increased anchorage-independent cell growth only in the breast cancer cell line (by 81% [95% CI = 62% to 101%] compared with untreated cells). Obese Lep(ob)Lep(ob) and Lepr(db)Lepr(db) mice had minimal epithelial development in the mature mammary gland compared with their lean counterparts. CONCLUSIONS: Leptin appears to be able to control the proliferation of both normal and malignant breast epithelial cells. Consequently, the leptin pathway should be further studied as a target for interventions to treat or prevent breast cancer.


Asunto(s)
Neoplasias de la Mama/química , Mama/química , Células Epiteliales/química , Leptina/análisis , Obesidad/metabolismo , Receptores de Superficie Celular/análisis , Animales , Western Blotting , Mama/citología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/química , División Celular , Línea Celular , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leptina/biosíntesis , Ratones , Ratones Obesos , Obesidad/complicaciones , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Leptina , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/metabolismo , Células Tumorales Cultivadas
9.
Bone Marrow Res ; 2016: 3152065, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27057356

RESUMEN

The procedure for aspiration of bone marrow from the femur of patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA) may vary from an OR (operating room) to OR based on the surgeon's skill and may lead to varied extent of clotting of the marrow and this, in turn, presents difficulty in the isolation of mesenchymal stem cells (MSCs) from such clotted bone marrow. We present a simple detailed protocol for aspirating bone marrow from such patients, isolation, and characterization of MSCs from the aspirated bone marrow specimens and show that the bone marrow presented no clotting or exhibited minimal clotting. This represents an economical source and convenient source of MSCs from bone marrow for use in regenerative medicine. Also, we presented the detailed protocol and showed that the MSCs derived from such bone marrow specimens exhibited MSCs characteristics and generated micromass cartilages, the recipe for regenerative medicine for osteoarthritis. The protocols we presented can be used as standard operating procedures (SOPs) by researchers and clinicians.

10.
Oncogene ; 22(43): 6679-89, 2003 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-14555981

RESUMEN

Mutations within members of the EGF/ErbB receptor family frequently release the oncogenic potential of these receptors, resulting in the activation of downstream signaling events independent of ligand regulatory constraints. We previously have demonstrated that the signal transduction events originating from S3-v-ErbB, a ligand-independent, oncogenic EGF receptor mutant, are qualitatively distinct from the ligand-dependent mitogenic signaling pathways associated with the wild-type EGF receptor. Specifically, expression of S3-v-ErbB in primary fibroblasts results in anchorage-independent growth, increased invasive potential, and the formation of a transformation-specific phosphoprotein signaling complex, all in a Ras-independent manner. Here we demonstrate the transformation-specific interaction between two components of this complex: the adaptor protein Grb2 and the cytoskeletal regulatory protein caldesmon. This interaction is mediated via both the amino-terminal SH3 and central SH2 domains of Grb2, and the amino-terminal (myosin-binding) domain of caldesmon. Expression of a dominant-negative Grb2 deletion mutant, which lacks the carboxy-terminal SH3 domain, in fibroblasts expressing S3-v-ErbB results in a reduction in phosphoprotein complex formation, the loss of anchorage-independent growth, and a reduction in invasive potential. Together, these results demonstrate a Ras-independent role for Grb2 in modulating cytoskeletal function during ligand-independent EGF receptor-mediated transformation, and provide further support for the hypothesis that ligand-independent oncogenic signaling is qualitatively distinct from ligand-dependent mitogenic signaling by the EGF receptor.


Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Citoesqueleto/metabolismo , Receptores ErbB/metabolismo , Proteínas/metabolismo , Actinas/química , Agar/química , Animales , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/metabolismo , Línea Celular Transformada , Transformación Celular Neoplásica , Células Cultivadas , Embrión de Pollo , Fibroblastos/metabolismo , Proteína Adaptadora GRB2 , Glutatión Transferasa/metabolismo , Ligandos , Microscopía Fluorescente , Modelos Biológicos , Mutación , Invasividad Neoplásica , Fosforilación , Fosfotirosina/química , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/genética , Transducción de Señal , Tirosina/metabolismo , Dominios Homologos src
11.
Exp Biol Med (Maywood) ; 229(2): 182-93, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14734797

RESUMEN

Being overweight is a risk factor for postmenopausal breast cancer and is associated with an increased incidence and shortened latency of spontaneous and chemically induced mammary tumors in rodents. However, leptin-deficient obese Lep(ob)Lep(ob) female mice have reduced incidences of spontaneous and oncogene-induced mammary tumors. Of interest, leptin enhances the proliferation of human breast cancer cell lines in which leptin receptors are expressed, which suggests that leptin signaling plays a role in tumor development. We evaluated oncogene-induced mammary tumor development in obese MMTV-TGF-alpha/Lepr(db)Lepr(db) mice that exhibit a defect in OB-Rb, which is considered to be the major signaling isoform of the leptin receptor. Lepr and MMTV-TGF-alpha mice were crossed, and the offspring were genotyped for oncogene expression and the determination of Lepr status. Lean MMTV-TGF-alpha/Lepr(+)Lepr(+) (homozygous) and MMTV-TGF-alpha/Lepr(+)Lepr(db) (heterozygous) mice and obese MMTV-TGF-alpha/Lepr(db)Lepr(db) mice were monitored until age 104 weeks. Body weights of MMTV-TGF-alpha/ Lepr(db)Lepr(db) mice were significantly heavier than those of the lean groups. No mammary tumors were detected in MMTV-TGF-alpha/Lepr(db)Lepr(db) mice, whereas the incidence of mammary tumors in MMTV-TGF-alpha/Lepr(+)Lepr(+) and MMTV-TGF-alpha/ Lepr(+)Lepr(db) mice was 69% and 82%, respectively. Examination of mammary tissue whole mounts indicated an absence of duct formation and branching for MMTV-TGF-alpha/Lepr(db)Lepr(db) mice. Both age at mammary tumor detection and tumor burden (tumors/mouse and tumor weights) were similar for the lean genotypes. Serum leptin levels of MMTV-TGF-alpha/Lepr(db)Lepr(db) mice were 12-20-fold higher than levels of lean mice. Thus, despite elevated serum leptin levels, leptin receptor-deficient MMTV-TGF-alpha/Lepr(db)Lepr(db) mice do not develop mammary tumors. This study provides additional evidence that leptin and its cognate receptor may be involved in mammary tumorigenesis.


Asunto(s)
Leptina/metabolismo , Neoplasias Mamarias Experimentales/genética , Obesidad/genética , Oncogenes/genética , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/fisiología , Animales , Peso Corporal , Modelos Animales de Enfermedad , Femenino , Leptina/sangre , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Obesidad/patología , Receptores de Leptina , Análisis de Supervivencia , Factor de Crecimiento Transformador alfa/genética
12.
Reprod Fertil Dev ; 14(7-8): 407-12, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12617783

RESUMEN

Sandhoff disease is a human lysosomal storage disease. In a knockout mouse model of Sandhoff disease, which lacks the beta-subunit of beta-hexosaminidase A (Hex A, alphabeta subunits) and B (Hex B, betabeta subunits), the mutant homozygous mice (Hexb(-/-)) are healthy until 15 weeks of age when they develop neurodegenerative symptoms. This study was designed to analyse the fertility profile of male and female Hexb(-/-) mice. Mating behaviour of Hexb(-/-) mice was assessed at different ages. The ovarian function of Hexb(-/-) females was determined by superovulation studies. The quality of spermatozoa and ova was assessed by an in vitro fertilization (IVF) procedure. Hexb(-/-) mice were fertile at a young age. Males were fertile up to the age of 69.3 +/- 6.3 days (mean +/- SD) and females were fertile up to the age of 56-63 days. Since both the Hexb (-/-) sexes showed fertility, the results indicate that Hex A and Hex B (major isozymes of beta-hexosaminidase) may not be required for sperm-ovum interactions, in contrast to the widely accepted belief. On the other hand, young adult Hexb(-/-) males showed a reduction in mating behaviour at the age of 84.8 +/- 2.2 days and an absence of mating behaviour at 94.2 +/- 2.0 days. Spermatozoa from Hexb(-/-) mice (aged 109.2 +/- 1.8 days) showed a lower IVF rate. Among Hexb (-/-) females aged 85.6 +/- 2.1 days, no mice became pregnant although they were positive for a vaginal plug when caged with fertile males. The number of ova recovered from Hexb(-/-) females (aged 111.0 +/- 3.1 days) and the IVF rate of ova were lower than those of controls. In conclusion, Hex A and Hex B may not be required for sperm-ovum interactions. Mice lacking Hex A and Hex B activities develop infertility at a young adult age in an age-dependent manner.


Asunto(s)
Modelos Animales de Enfermedad , Infertilidad/etiología , Enfermedad de Sandhoff/complicaciones , Envejecimiento , Animales , Femenino , Fertilización In Vitro , Hexosaminidasa A , Hexosaminidasa B , Infertilidad/genética , Masculino , Ratones , Ratones Noqueados , Óvulo/fisiología , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Enfermedad de Sandhoff/genética , Superovulación , beta-N-Acetilhexosaminidasas/deficiencia , beta-N-Acetilhexosaminidasas/genética
13.
Indian J Physiol Pharmacol ; 47(2): 147-56, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15255617

RESUMEN

Gap junctions are made of connexin (Cx) molecules and provide communications between adjacent cells through which small molecules (<1kDa) move between cytoplasms. The connexin gene family consists of at least 17 members. Gap junctions play important roles in ovarian and testicular function. Connexin 43 (Cx43) knockout mouse is a model for developmentally impaired fetal gonads. The aim of the current study was twofold: (a) to analyze mRNA expression pattern of multiple members of connexin gene family in normal fetal gonads; (b) to investigate any alteration of mRNA expression of connexins in developmentally impaired fetal gonads. The study was conducted in normal gonads obtained from 17.5 dpc wildtype (Cx43+/+) and heterozygote (Cx43+/-) fetuses and in developmentally impaired gonads obtained from 17.5 dpc knockout (Cx43-/-) fetuses. The mRNA expression pattern of connexins (Cx26, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, Cx43, Cx45, Cx46 and Cx50) was analyzed by RT-PCR. The mRNA transcripts for Cx32 and Cx50 were absent in fetal testis of all the genotypes, and the transcripts for Cx26, Cx30.3, Cx31.1, Cx32, Cx40, Cx46 and Cx50 were absent in fetal ovary of all the genotypes. The transcripts for Cx43 showed expression in Cx43+/+ and Cx43+/- gonads and were absent in Cx43-/- gonads, as expected. Additionally, the mRNA transcripts for 8 more connexins (Cx26, Cx30.3, Cx31, Cx31.1, Cx37, Cx40, Cx45 and Cx46) showed expression in Cx43+/+ and Cx43+/- fetal testes, but the transcripts for only 4 connexins (Cx26, Cx37, Cx40 and Cx45) showed expression in Cx43-/- fetal testis. In fetal ovary, the mRNA transcripts for 3 more connexins (Cx31, Cx37 and Cx45) were expressed in all the genotypes. In summary, from the mRNA expression analysis of 11 members of connexin, gene family in 17.5 dpc fetal gonads, besides the expression of Cx43, 8 additional connexins were expressed in normal fetal testis but only 4 connexins were expressed in developmentally impaired testis. The fetal ovary showed the expression of 3 additional connexins besides the expression of Cx43. In developmentally impaired fetal ovary, only Cx43 was not expressed as expected but other three connexins were expresed. The study may be useful in interpreting human testis defects in infertility cases.


Asunto(s)
Conexinas/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Gónadas/anomalías , Gónadas/metabolismo , ARN Mensajero/biosíntesis , Animales , Conexina 26 , Conexinas/genética , Femenino , Feto/anomalías , Feto/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , ARN Mensajero/genética
14.
Bone ; 60: 148-61, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24316420

RESUMEN

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.


Asunto(s)
Enfermedades Óseas Metabólicas/complicaciones , Enfermedades Óseas Metabólicas/patología , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Columna Vertebral/anomalías , Factores de Transcripción/metabolismo , Animales , Fenómenos Biomecánicos , Enfermedades Óseas Metabólicas/diagnóstico por imagen , Enfermedades Óseas Metabólicas/genética , Colágeno/genética , Colágeno/ultraestructura , Femenino , Marcación de Gen , Sitios Genéticos/genética , Proteínas Hedgehog/genética , Humanos , Disco Intervertebral/diagnóstico por imagen , Disco Intervertebral/patología , Cifosis/congénito , Cifosis/diagnóstico por imagen , Cifosis/genética , Cifosis/patología , Lordosis/congénito , Lordosis/diagnóstico por imagen , Lordosis/genética , Lordosis/patología , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/patología , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Ratones , Mutación/genética , Osteogénesis , Proto-Oncogenes , Receptor de Hormona Paratiroídea Tipo 1/genética , Columna Vertebral/diagnóstico por imagen , Columna Vertebral/patología , Tendones/diagnóstico por imagen , Tendones/patología , Tendones/ultraestructura , Vértebras Torácicas/diagnóstico por imagen , Vértebras Torácicas/patología , Microtomografía por Rayos X
15.
J Tissue Eng ; 4: 2041731413492741, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23762501

RESUMEN

To understand scar and adhesion formation during postsurgical period of intrasynovial tendon graft healing, a murine model of flexor digitorum longus tendon graft repair was developed, by utilizing flexor digitorum longus tendon allograft from donor Rosa26/+ mouse, and the healing process at days 3, 7, 14, 21, 28, and 35 post surgery of host wild-type mouse was followed. Using X-gal staining, ß-galactosidase positive cells of allograft origin were detectable in tissue sections of grafted tendon post surgery. Graft healing was assessed for the cellular density, scar and adhesion formation, and their interaction with surrounding tissue. From histological analysis, it was evident that the healing of intrasynovial flexor digitorum longus tendon graft takes place in an interactive environment of donor graft, host tendon, and host surrounding tissue. A total of 32 genes, analyzed by RNA analysis, expressed during healing process. Particularly, Alk1, Postn, Tnc, Tppp3, and Mkx will be further investigated for therapeutical value in reducing scars and adhesions.

16.
Arthritis ; 2013: 154812, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24324885

RESUMEN

This review summarizes the genetic alterations and knockdown approaches published in the literature to assess the role of key proteoglycans and glycoproteins in the structural development, function, and repair of tendon, ligament, and enthesis. The information was collected from (i) genetically altered mice, (ii) in vitro knockdown studies, (iii) genetic variants predisposition to injury, and (iv) human genetic diseases. The genes reviewed are for small leucine-rich proteoglycans (lumican, fibromodulin, biglycan, decorin, and asporin); dermatan sulfate epimerase (Dse) that alters structure of glycosaminoglycan and hence the function of small leucine-rich proteoglycans by converting glucuronic to iduronic acid; matricellular proteins (thrombospondin 2, secreted phosphoprotein 1 (Spp1), secreted protein acidic and rich in cysteine (Sparc), periostin, and tenascin X) including human tenascin C variants; and others, such as tenomodulin, leukocyte cell derived chemotaxin 1 (chondromodulin-I, ChM-I), CD44 antigen (Cd44), lubricin (Prg4), and aggrecan degrading gene, a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5 (Adamts5). Understanding these genes represents drug targets for disrupting pathological mechanisms that lead to tendinopathy, ligamentopathy, enthesopathy, enthesitis and tendon/ligament injury, that is, osteoarthritis and ankylosing spondylitis.

17.
PLoS One ; 7(12): e51411, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23251524

RESUMEN

Flexor tendon injuries are among the most challenging problems for hand surgeons and tissue engineers alike. Not only do flexor tendon injuries heal with poor mechanical strength, they can also form debilitating adhesions that may permanently impair hand function. While TGF-ß1 is a necessary factor for regaining tendon strength, it is associated with scar and adhesion formation in the flexor tendons and other tissues as well as fibrotic diseases. The pleiotropic effects of TGF-ß1 on tendon cells and tissue have not been characterized in detail. The goal of the present study was to identify the targets through which the effects of TGF-ß1 on tendon healing could be altered. To accomplish this, we treated flexor tendon tenocytes cultured in pinned collagen gels with 1, 10 or 100 ng/mL of TGF-ß1 and measured gel contraction and gene expression using RT-PCR up to 48 hours after treatment. Specifically, we studied the effects of TGF-ß1 on the expression of collagens, fibronectin, proteoglycans, MMPs, MMP inhibitors, and the neotendon transcription factors, Scleraxis and Mohawk. Area contraction of the gels was not dose-dependent with the TGF-ß1 concentrations tested. We observed dose-dependent downregulation of MMP-16 (MT3-MMP) and decorin, and upregulation of biglycan, collagen V, collagen XII, PAI-1, Scleraxis, and Mohawk by TGF-ß1. Inter-gene analyses were also performed to further characterize the expression of ECM and MMP genes in the tenocyte-seeded collagen gels. These analyses illustrate that TGF-ß1 tilts the balance of gene expression in favor of ECM synthesis rather than the matrix-remodeling MMPs, a possible means by which TGF-ß1 promotes adhesion formation.


Asunto(s)
Perfilación de la Expresión Génica , Pleiotropía Genética/efectos de los fármacos , Modelos Biológicos , Tendones/metabolismo , Tendones/patología , Factor de Crecimiento Transformador beta1/farmacología , Cicatrización de Heridas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biglicano/metabolismo , Colágeno/genética , Colágeno/metabolismo , Decorina/genética , Decorina/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Geles/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Metalotioneína 3 , Ratones , Ratones Endogámicos C57BL , Inhibidor 1 de Activador Plasminogénico/metabolismo , Tendones/efectos de los fármacos , Tendones/crecimiento & desarrollo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Cicatrización de Heridas/efectos de los fármacos
18.
Hum Reprod ; 20(4): 881-93, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15705627

RESUMEN

BACKGROUND: Hrb is an HIV-1 Rev-binding/interacting protein and is a cofactor for Rev export pathway. Hrb interacts with Eps15 homology (EH) domain-containing proteins and is a component of EH network and functions in vesicle sorting. Earlier, we reported that Hrb-deficient male mice are infertile and that they show oligozoospermia. Their sperm lack acrosomes and present globozoospermia. The aim of this study was: (i) to investigate the additional defects in spermatogenesis in Hrb-deficient mice; and (ii) to investigate the effect of acrosomelessness on spermatid differentiation in Hrb-deficient mice. METHODS: Hrb(-/-) testes, epididymides, spermatids and sperm were analyzed by histology and electron microscopy. Centrioles were analyzed in spermatids and sperm by indirect immunofluorescence technique. RESULTS: Hrb(-/-) male mice exhibited multiple anomalies during meiosis and spermiogenesis that produced developmentally impaired sperm with unshaped or deformed nuclei, loss in cell polarity, intracellular flagellar coiling, multinucleation, supernumerary centrioles and multiflagellation. A total of 13.0% Hrb(-/-) sperm showed macrocephaly. The Hrb(-/-) sperm exhibited variation in head size and shape, disarranged cellular organelles, nuclear and cytoplasmic vacuolization, mitochondrial loss or scattering and no forward motility. CONCLUSIONS: These aberrations, in Hrb(-/-) mouse spermatids and sperm, are reminiscent of human familial male infertility with oligoasthenoteratozoospermia syndrome. The Hrb-deficient mouse may be useful in understanding familial oligoasthenoteratozoospermia syndrome.


Asunto(s)
Acrosoma/fisiología , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Infertilidad Masculina/fisiopatología , Ratones Mutantes , Oligospermia/fisiopatología , Acrosoma/patología , Acrosoma/ultraestructura , Proteínas Adaptadoras del Transporte Vesicular , Animales , Centriolos/patología , Centriolos/fisiología , Centriolos/ultraestructura , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Masculino , Ratones , Microscopía Electrónica , Oligospermia/genética , Oligospermia/patología , Espermátides/patología , Espermátides/fisiología , Espermátides/ultraestructura , Espermatogénesis/fisiología
19.
Nutr Cancer ; 50(2): 174-80, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15623464

RESUMEN

Obesity is a risk factor for postmenopausal breast cancer and is associated with shortened latency and/or increased mammary tumor (MT) incidence in animals. Elevated body weight is usually associated with hormone-responsive tumors. In agreement with these data we previously showed that latency of hormone-responsive MTs in MMTV-TGF-alpha mice with diet-induced obesity was significantly shortened. Here, we used the same protocol to determine the impact of diet-induced obesity on estrogen receptor-negative MT development in MMTV-neu (strain 202) mice. Mice were fed a low-fat diet (n=20) or a high-fat diet (n=54) from 10 wk of age. Body weight at 19 wk of age was used to assign high-fat mice to obesity-prone, overweight, and obesity-resistant groups. Mice were euthanized due to MT size or at 85 wk of age. Final body weights of obesity-prone mice were heaviest, and those of obesity-resistant and low-fat groups were similar. Fat pad weights were heaviest in obesity-prone mice followed by overweight and obesity-resistant groups, and lightest in low-fat mice. Serum IGF-I levels were similar for low-fat and high-fat mice, whereas leptin was higher in high-fat mice (P <0.0001). MT latency, incidence, metastasis, and burden were similar for all groups. These findings support that obesity is not a risk factor for development of estrogen-negative breast cancer.


Asunto(s)
Neoplasias Mamarias Experimentales/etiología , Virus del Tumor Mamario del Ratón , Obesidad/complicaciones , Tejido Adiposo/patología , Animales , Dieta , Ingestión de Energía , Femenino , Genes erbB-2 , Factor I del Crecimiento Similar a la Insulina/análisis , Leptina/sangre , Neoplasias Mamarias Experimentales/sangre , Neoplasias Mamarias Experimentales/patología , Ratones , Factor de Crecimiento Transformador alfa/fisiología
20.
Breast Cancer Res Treat ; 77(3): 205-15, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12602920

RESUMEN

Elevated body weight is a risk factor for postmenopausal breast cancer and is associated with increased incidence of spontaneous and chemically induced mammary tumors (MTs) in rodents. In this study, genetically obese Lep(ob)Lep(ob) female mice that overexpress human TGF-alpha (transforming growth factor-alpha) were used to assess the role of body weight on oncogene-induced MT development in comparison to lean counterparts. MMTV (mouse mammary tumor virus)-TGF-alpha and Lep strain mice were crossed to produce TGF-alpha/Lep(+)Lep(+) (homozygous lean), TGF-alpha/Lep(+)Lep(ob) (heterozygous lean) and TGF-alpha/Lep(ob)Lep(ob) (homozygous obese) genotypes. Body weights were determined weekly and mice palpated for the presence of MTs until 104 weeks of age. Despite their significantly higher body weight, obese TGF-alpha/Lep(ob)Lep(ob) mice failed to develop MTs. MTs were detected between 48 and 104 weeks of age for 26/39 TGF-alpha/Lep(+)Lep(ob) mice and for 19/38 TGF-alpha/Lep(+)Lep(+) mice between 67 and 104 weeks of age. Although MT incidence was not statistically different between the lean groups, age of MT detection tended to be younger for TGF-alpha/Lep(+)Lep(ob) mice (p < 0.09). There were significant effects of both genotype and MTs on final body weight, that is, TGF-alpha/Lep(+)Lep(ob) mice weighed more than homozygous lean mice, and mice with MTs weighed more than those without MTs. TGF-alpha/Lep(ob)Lep(ob) mice are not a good model to evaluate the effect of body weight on MT development possibly due to leptin deficiency. However, the finding that increased body weight is associated with increased oncogene-induced MT development within the normal weight range provides experimental support for the role of body weight in breast cancer.


Asunto(s)
Neoplasias Mamarias Experimentales/genética , Obesidad/genética , Animales , Peso Corporal , Cartilla de ADN , Modelos Animales de Enfermedad , Femenino , Leptina/genética , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos C57BL/genética , Ratones Obesos/genética , Reacción en Cadena de la Polimerasa , Análisis de Supervivencia , Factor de Crecimiento Transformador alfa/genética
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