Absence of Dp71 in mdx3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS.

In muscle, the absence of dystrophin alters the dystrophin-associated protein complex (DAPC), which is involved in the clustering and anchoring of signaling proteins and ion and water channels. Here we show that mice spermatozoa express only dystrophin Dp71 and utrophin Up71. The purpose of this study was to explore the effect of the absence of Dp71 on the morphology and membrane distribution of members of the DAPC, ion channels and signaling proteins of spermatozoa obtained from dystrophic mutant mdx3cv mice. Our work indicates that although the absence of Dp71 results in a dramatic decrease in beta-dystroglycan, it induces membrane redistribution and an increase in the total level of alpha-syntrophin, voltage-dependent Na+ (micro1) and K+ (Kv1.1) channels and neural nitric oxide synthase (nNOS). The short utrophin (Up71) was upregulated and redistributed in the spermatozoa of mdx3cv mice. A significant increase in abnormal flagella morphology was observed in the absence of Dp71, which was partially corrected when the plasma membrane was eliminated by detergent treatment. Our observations point to a new phenotype associated with the absence of Dp71. Abnormal flagellar structure and altered distribution of ion channels and signaling proteins may be responsible for the fertility problems of mdx3cv mice

Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed

Utrophins compensate for Dp71 absence in mdx3cv in adhered platelets.

International audiencePlatelet adhesion is a critical step due to its hemostatic role in stopping bleeding after vascular damage. Short dystrophins are the most abundant dmd gene products in nonmuscle tissues, and in association with cytoskeleton proteins contribute to their intrinsic function; while utrophins are dystrophin-homologous related family proteins with structural and functional similarities. We previously demonstrated the presence of Dp71 isoforms, utrophins, and various dystrophin-associated proteins and their participation in cytoskeleton re-organization, filopodia and lamellipodia extension, and in centralizing cytoplasmic granules during the adhesion process of human platelets. To evaluate the morphologic changes and actin-based structures of mdx platelets during the adhesion process, we compared the topographic distribution of Dp71d/Dp71Delta110 and dystrophin-associated protein in adhered platelets from dystrophic mdx mouse. By confocal microscopy, we showed that absence of Dp71 isoforms in platelets from this animal model disrupted dystrophin-associated protein expression and distribution without modifying the platelet morphology displayed during the glass-adhesion process. By immunoprecipitation assays, we proved that up-regulated utrophins were associated with dystrophin-associated proteins to conform the dystrophin-associated protein complex corresponding to utrophins, which might compensate for Dp71 absence in mdx platelets

Carga financiera del cuidado familiar del enfermo crónico en la Región Andina de Colombia

Describir y analizar la carga financiera del cuidado familiar de los enfermos crónicos en la región andina colombiana. Materiales y métodos: este estudio es parte del Programa para la Reducción de la carga de enfermedades crónicas en Colombia. La muestra incluyó a 92 familias que residen en la región andina colombiana. Los instrumentos "gcpc-un-d" se utilizaron para caracterizar a los sujetos y el costo financiero de la encuesta de atención de enfermedades crónicas de Montoya et al, para identificar el consumo real efectivo en el hogar. La carga financiera atribuible al cuidado familiar se determinó bajo la metodología Caracol. Resultados: los costos que más afligen a las familias de la región andina colombiana están en su orden de salud, transporte, vivienda, alimentación y comunicaciones. El cuidado familiar de una persona con enfermedad crónica afecta su consumo efectivo en el hogar. Discusión: Las familias colombianas que residen en la región andina del país tienen una alta carga financiera atribuible al cuidado de una persona con enfermedad crónica.To describe and analyze the financial burden of family care of the chronically ill in the Colombian Andean region. Materials and methods: This study is part of the Program for the Reduction of the burden of chronic disease in Colombia. The sample included 92 families residing in the Colombian Andean region. The Instruments “gcpc-un-d” were used to characterize the subjects and the Survey Financial cost of chronic disease care of Montoya et al, to identify the real effective household consumption. The financial burden attributable to family care was determined under the Caracol methodology. Results: Costs that most afflict families of the Colombian Andean Region are in their order health, transportation, housing, food and communications. Family caring for a person with chronic illness affects its effective household consumption. Discussion: Colombian families residing in the Andean region of the country have a high financial burden attributable to caring for a person with chronic disease

Carga financiera del cuidado familiar del enfermo crónico en la Región Andina de Colombia

Objective: To describe and analyze the financial burden of family care of the chronically ill inthe Colombian Andean region. Materials and methods: This study is part of the Program forthe Reduction of the burden of chronic disease in Colombia. The sample included 92 familiesresiding in the Colombian Andean region. The Instruments “gcpc-un- d” were used to characterize the subjects and the Survey Financial cost of chronic disease care of Montoya et al, to identify the real effective household consumption. The financial burden attributable to family care was determined under the Caracol methodology. Results: Costs that most afflict families of the Colombian Andean Region are in their order health, transportation, housing, food and communications. Family caring for a person with chronic illness affects its effective household consumption. Discussion: Colombian families residing in the Andean region of the country have a high financial burden attributable to caring for a person with chronic disease.Introdução: Descrever e analisar a carga financeira do cuidado familiar do doente crónico na Região Andina da Colômbia. Materiais e métodos: Estudo que faz parte do Programa para a diminuição da carga da doença crónica na Colômbia. A sua amostra a constituíram 92 famílias que residem na região Andina da Colômbia. Se empregaram os instrumentos “GCPC-UN- D” para caracterizar aos sujeitos e o Questionário “Custo financeiro do cuidado da Doença crónica” para conhecer o consumo real efetivo familiar. A carga financeira atribuível ao cuidado familiar se determinou sob a metodologia caracol. Resultados: os custos que mais afligem às famílias da Região Andina colombiana são em sua ordem, os de saúde, transporte, vivenda, alimentação e comunicações. O consumo real efetivo familiar se modifica ao cuidar a uma pessoa com doença crónica. Discussão: as famílias colombianas que residem na região Andina do País têm uma elevada carga financeira atribuível ao cuidado de uma pessoa com doença crónica na Colômbia.Introducción: Describir y analizar la carga financiera del cuidado familiar del enfermo crónico en la Región Andina de Colombia. Materiales y métodos: Estudio que hace parte del ‘Programa para la disminución de la carga de la enfermedad crónica en Colombia‘. Su muestra la constituyeron 92 familias que residen en la región Andina de Colombia. Se emplearon los instrumentos “GCPC-UN- D”, para caracterizar a los sujetos y la Encuesta “Costo financiero del cuidado de la Enfermedad crónica”, para conocer el consumo real efectivo familiar. La Carga financiera atribuible al cuidado familiar se determinó bajo la metodología Caracol. Resultados: Los costos que más agobian a las familias de la Región Andina colombiana son, en su orden, los de salud, transporte, vivienda, alimentación y comunicaciones. El consumo real efectivo familiar se modifica al cuidar a una persona con enfermedad crónica. Discusión: Las familias colombianas que residen en la región Andina del país tienen una elevada carga financiera atribuible al cuidado de una persona con enfermedad crónica

Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures

Nuclear localization of Dp71s during neuronal differentiation process.

<p>The temporal course of the nuclear expression of both Dp71s was performed, in primary cultures of hippocampal neurons. Hippocampal neurons were cultured for 0 (1h), 2, 4, 10, 15, and 21 days <i>in vitro</i> (DIV), and were double stained for Dp71d (Dys-2 Ab, green color, panel A) or Dp71f (5F3 Ab, green color, panel B) and GAD67 or GLUR1 Ab, respectively (red color, panel A and B). Both Dp71s were expressed in nuclei of neurons cultured for 0, 2, 4, 10, 15, and 21 DIV. Quantification of fluorescence intensity in the nucleus of each Dp71 is shown in panel C. The highest level of nuclear expression of both Dp71 proteins were at 10 DIV. Values are means ± SEM, ٭٭p˂0.01. Scale bar = 10 μm.</p

Colocalization of Dp71s with SC35 or DAP in bipolar and multipolar hippocampal neurons.

<p>Cultured hippocampal neurons at 21 DIV were used for immunostaining assays. Hippocampal neurons were double stained with either Dp71d (Dys-2 Ab, green color, panel A) or Dp71f (5F3 Ab, green color, panel B) and SC35, α-DG (K8), β-DG (JAF), α1-, α2-DB (D124), β-DB (β-Brevin), and α-SYN (C4) Abs, respectively (red color, panel A and B). DAPI was used to stain the nuclei, and the fucsia color indicates the colocalization of Dp71s and each DAP in nuclear speckles. The colocalization rate of each Dp71 with SC35 or DAP is shown in the panel C. Values are means ± SEM, ٭٭p˂0.01. Scale bar = 5 μm.</p