Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes.

Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function.

BACKGROUND: Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease. METHODS: We studied whether sarcomeric structure and function can be improved by introducing nebulin's Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied. RESULTS: Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression. CONCLUSIONS: This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle.

Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in nebulin cause muscle weakness in nemaline myopathy patients, suggesting that nebulin plays important roles in force generation, yet little is known about nebulin's influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in nebulin (using a conditional nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 Å) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 Å) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.

Microvessel Density Is Associated with VEGF and α-SMA Expression in Different Regions of Human Gastrointestinal Carcinomas.

Tumor angiogenesis is known to be regulated by growth factors secreted by host and tumor cells. Despite the importance of tumor vasculature and angiogenic heterogeneity in solid tumors, few studies have compared the vasculature in different regions of human cancer. Blood vessels from different regions of carcinomas might have morphofunctional implications in tumor angiogenesis. In the present study, therefore, we have examined the relationship between microvascular density (MVD) and vascular endothelial growth factor (VEGF) expression and alpha smooth muscle actin (α-SMA) expression in the center of the tumor (CT), periphery (P) and metastasis (M) regions from gastrointestinal carcinomas (GITC), as well as the association of MVD with clinicopathological factors. Surgically resected specimens corresponding to the CT, P and M from 27 patients were examined for FVIII, VEGF and α-SMA by immunohistochemistry. The MVD was not significantly different in the CT, P and M regions from GITC. The MVD in the VEGF positive group was significantly higher than in the VEGF negative group (CT, p = 0.034; P, p = 0.030; M, p = 0.032). The MVD as a function of α-SMA expression was also significantly higher in the CT and P region compared to the M region (p = 0.0008). In conclusion, the MVD association with VEGF and α-SMA expression, might indicate an increase of the number of neoformed and preexisting blood vessels uniformly or partially covered by pericytes in different regions of GITC, suggesting that not only MVD and VEGF are important parameters to the tumor vasculature, but also blood vessels maturation is a crucial factor for gastrointestinal tumor angiogenesis regulation and possible target of vascular therapy.

Relationship between VEGF and p53 expression and tumor cell proliferation in human gastrointestinal carcinomas.

PURPOSE: The vascular endothelial growth factor (VEGF) and p53 play important roles in the growth of tumor. However, the relationship between the expression of VEGF and p53 and tumor cell proliferation in human gastrointestinal cancer remains unknown. In the present study, therefore, we have examined the relationship between VEGF and p53 expression and tumor cell proliferation in gastrointestinal carcinoma (GITC), as well as the association between these biomarkers and clinicopathological factors. METHODS: Surgical specimens from 30 patients with GITC were examined for VEGF, p53, and proliferating cell nuclear antigen (PCNA) expression by immunohistochemical staining. RESULTS: We found a predominant VEGF expression of moderate intensity in 16(54.84%) of 30 GITC cases, while p53 expression was mainly high in 13(45.16%) of 30 GITC cases. PCNA expression was high in 20(64.52%) of 30 GITC cases. Tumor size, infiltration, vascular invasion, and gastritis were significantly correlated with VEGF, p53, and PCNA expression. There was a significant correlation between VEGF and p53 expression (P = 0.0001), VEGF and PCNA expression (P = 0.00004), and between p53 expression and PCNA expression (P = 0.0016). When the VEGF and p53 expression, and PCNA expression were considered together, both VEGF and p53 expression were not significantly associated with PCNA. A significant correlation between the PCNA expression and the mitotic index (P = 0.0016) was also found. CONCLUSION: These results demonstrate that VEGF and p53 expression are significantly correlated as independent prognostic factors with tumor cell proliferation, and might be associated with relevant events involved in gastrointestinal tumor biology.

Schistosoma mansoni: respuesta celular hepática de ratones Balb/c infectados unisexualmente / Schistosoma mansoni: hepatic cellular response of unisexually-infected Balb/c mice

La respuesta granolomatosa en animales infectados con Schistosoma mansoni es atribuida a la presencia del huevo y aunque los gusanos adultos co-existentes en el sistema porta hepático, el papel en la inmunopatología no está claro. Se evaluó la respuesta celular hepática en ratones Balb/c infectados unisexualmente (UNI) con cercarias de S. mansoni, mediante histología convencional, inmunofluorescencia para CD4+ y CD8+, inmunohistoquímica e inmunomicroscopía electrónica para macrófagos activados. Los resultados muestran que la infección UNI produjo un infiltrado hepático con un área de 33,9 ± 18,86 µm² (media ± ME), constituido por macrófagos, linfocitos, plasmocitos y escasos eosinófilos. En la infección bisexual, la composición celular fue similar pero más intensa con un área de 195,6 ± 74,4 µm². Las células CD4+ y CD8+ en el hígado se ubicaron entre 2 y 4 cel/ µm² cerca del gusano y distante en forma difusa o en grupos, en ambas condiciones de infección. Los macrófagos activados se ubicaron alrededor de los gusanos y en donde se deposita el pigmento esquistosiomal. Los resultados sugieren que el gusano adulto es capaz de inducir una respuesta inflamatoria hepática en ausencia del huevo, la cual debe jugar un papel importante en la respuesta producida contra el huevo y tienen implicaciones en la patogénesis de la esquistosomiasis murina

Ultraestructura de la microvasculatura en lesiones premalignas y malignas de la mucosa bucal / Microvasculature ultrastructure in premalignant and malignant lesions of oral mucosa

El proceso de angiogénesis y remodelado vascular es esencial para el crecimiento, la invasión y la metástasis tumoral. Los vasos sanguíneos tumorales presentan anormalidades estructurales y funcionales. El objetivo de este trabajo fue caracterizar las alteraciones ultraestructurales de la microvasculatura de displasias y carcinomas espinocelulares de la mucosa bucal como un modelo de progresión tumoral, mediante microscopía electrónica de transmisión convencional. Las alteraciones encontradas en los casos de displasia leve y moderada incluyeron la distribución y el número variable de las vesículas pinocíticas y cavéolas, así como la proliferación de algunos organelos. Asimismo, en ambos tipos de displasia se apreció la membrana basal reduplicada y el pericito en algunos casos se observó alterado. En los carcinomas espinocelulares bucales también se encontró el engrosamiento y reduplicación de la membrana basal. La hipertrofia del endotelio fue frecuente, con el edema del mismo y del pericito. Las prolongaciones del endotelio hacia la luz fueron observadas en las displasias leve y moderada y en los carcinomas bucales. Tales resultados podrían sugerir anormalidades en el funcionamiento de los capilares, en relación a cambios en la actividad metabólica y la permeabilidad de las células endoteliales y los pericitos.

O processo do angiogénesis e o remodelado vascular é essencial para o crescimento, a invasão e o metástasis do tumor. A embarcação de sangue presente do tumor estruturais e funcionais anomalias. O objetivo deste trabalho era caracterizar o ultrastructural alterações do microvasculatura dos displasias e dos carcinomas os espinocelulares da mucosa oral gostam de um modelo do tumor progressão, por meio do microscopio eletrônico da transmissão convencional. As alterações encontradas nos exemplos do displasia pesam e moderam incluíram a distribuição e o número variável do spinocíticase vesicles dos cavéolas, o proliferacion de alguns organelos. Também, em ambos os tipos de displasia a membrana basal do reduplicada estava engrossada e o pericito em alguns casos foi observado alterou-se. Em carcinomas orais os espinocelulares eram também engrossamento e reduplicación da membrana basal. Hipertrofia do endotelio era freqüente, com o edema do mesmo e o pericito. Os prolongations do endotelio para a luz foram observados dentro displasias ligeiros e moderados e os carcinomas orais. Tais resultados podiam sugerir anomalias na operação do capilar, com relação às mudanças na atividade metabolica e na permeabilidade das pilhas dos endoteliales e dos pericitos.

The process of angiogenesis and vascular remodelling is essential for tumor growth, invasion and metastasis. Tumor blood vessels show structural and functional abnormalities. The aim this work was characterize the ultrastructural alterations of microvasculature in dysplasias and squamous carcinomas of oral mucosa as a model of tumor progression by conventional transmission electron microscopy. Alterations included a variable distribution and number of pinocytotic vesicles and caveolae, as well as proliferation of some organelles in slight and moderate dysplasias. Likewise, the basement membrane looked reduplicated in both cases and pericytes in some cases was alterations. In oral squamous carcinomas capillary basement membrane was thickened and reduplicated. Endothelial hypertrophy was frequent with edema and also in the pericytes. Endothelial cell cytoplasm infoldings into the lumen were observed in slight and moderate dysplasias and oral squamous carcinoma. The results could suggest an abnormal capillary funtion in relation to metabolic activity and permeability of endothelial cells and pericytes.

Angiogénesis en lesiones premalignas y malignas de la mucosa bucal / Angiogenesis in malignant and premalignant oral mucosal lesions

Se ha establecido que el crecimiento, la progresión y las metástasis de los tumores sólidos, constituyen procesos dependientes de la angiogénesis (formación de nuevos vasos sanguíneos a partir de capilares preexistentes). En la mucosa bucal se producen diversas lesiones premalignas y malignas, las cuales se han empleado como un modelo de progresión tumoral. En el presente estudio se determinó la densidad vascular, como una medida de la respuesta angiogénica en grupos de lesiones con diferentes grados de displasia y carcinomas espinocelulares, mediante la cuantificación de vasos sanguíneos identificados con el Factor VIII de von Willebrand, por técnicas de inmunohistoquímica. Los resultados revelaron un incremento en la densidad vascular con la severidad del diagnóstico. La densidad vascular (media +- desviación estándar) fue menor en el grupo de displasia leve (12.70 +- 3.04), con un progresivo aumento en el de displasia moderada (21.62 +- 1.99) y más significativo aún en el grupo de displasia severa (59.50 +- 8,73) y carcinoma espinocelular (60.62 +- 8.74). Sin embargo, no se encontraron diferencias estadísticamente significativas (p=0.05) en la densidad vascular entre la displasia leve y moderada, ni entre la displasia severa y los carcinomas espinocelulares. Estos hallazgos sugieren que la angiogénesis, posiblemente ocurre tempranamente en el desarrollo de la carcinomgénesis bucal, principalmente en la displasia severa y carcinoma

Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line.

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.

Mass determination of native smooth muscle myosin filaments by scanning transmission electron microscopy.

The thick filaments of vertebrate smooth muscle have a fundamentally different arrangement of myosin molecules from the bipolar, helical organization present in striated muscle filaments. This side-polar, non-helical structure is probably critical to the ability of smooth muscles to shorten by large amounts; however, details of myosin organization beyond this general description are unknown. The non-helical arrangement of myosin precludes the use of helical reconstruction methods for structural determination, and a tomographic approach is required. As a first step towards this goal we have determined the number of myosin molecules present at each 14.5 nm repeat in native smooth muscle myosin filaments by scanning transmission electron microscopy. The mass-per-length of myosin filaments was 159 kDa/nm, corresponding to 4.38(+/-0.11) (mean+/-s.e.m.) myosin molecules at each 14.5 nm level. The mass of thin filaments in the preparation (intrinsic control) was 21 kDa/nm, consistent with current models of smooth muscle thin filament structure, and the mass of tobacco mosaic virus (mass standard) was within 5% of the known value. We conclude that native smooth muscle myosin filaments contain four myosin molecules at each 14.5 nm level, two on each side of the side-polar structure.

Descripción de lisosomas en fibras musculares esqueléticas del capibara: un estudio ultraestructural / Description of lysosomes in skeletal muscle fibres of the capybara: an ultrastructural study

El potencial económico del chigüire (Hydrochaeris hydrochaeris) para producción de carne se puede enfatizar a través de estudios ultraestructurales de sus fibras musculares. Se utilizaron técnicas convencionales de microscopía electrónica de transmisión y se empleó un buffer de 350 mOsm, a pH 7.4 El presente estudio describe los lisosomas encontrados en las fibras musculares de chigüires sin ninguna lesión patológica, formando la base para futuras comparaciones ultraestructurales con animales enfermos, lesiones o de edad avanzada y su relación con la calidad de la carne

Skeletal muscle capillary ultrastructure in Toxoplasma gondii parasitized mice / Ultraestructura de los capilares del músculo esqueléticos en ratones parasitados con Toxoplasma gondii

A light and transmission electron microscopic study was perfomed in skeletal muscles from mice experinmentally infected with Toxoplasma gondii. Parasite cysts were not observed. Capillary endothelial cytoplasm abnormalities included proliferation of organelles, decrease of pynocytic vesicles, degenerative changes and necrosis. In some capillaries the lumen was reduced or absent. Pericytes also were altered. In all animals (n=13), the basement membrane was normal. The cellular infiltrate consisted of macrophages, lymphocytes, mastocytes and eosinophils. The alterations observed in muscle microvasculature in absence of Toxoplasma gondii cysts, could be due to a host-immune response to the parasite

Ultraestructura de los capilares intramusculares en el paciente con carcinoma broncogénico / Capillary ultrastructure of the skeletal muscle in patients with bronchogenic carcinoma

En el presente trabajo se estudiaron los efectos del carcinoma broncogénico no tratado y sin manifestaciones metastásicas sobre la ultraestructura de los capilares del músculo QUADRICEPS FEMORIS. Para ello se emplearon biopsias provenientes de pacientes remitidos al Hospital "José Ignacio Baldo". La microspopía electrónica de transmisión reveló la existencia en las biopsias analizadas (n=7) de una amplia gama de alteraciones que comprendieron : engrosamiento y reduplicación de la membrana basal, proliferación endotelial, oclusión de la luz capilar y en algunos casos necrosis de los mismos. El infiltrado mononuclear consistió de linfocitos y macrófagos. Tales anormalidades en los capilares intramusculares podrían constituir un factor importante en la etiopatogenia de las manifestaciones clínicas musculares del paciente con carcinoma broncogénico en ausencia de metástasis