The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Collagen fiber reorganization in the rat ventral prostate following androgen deprivation: A possible role for smooth muscle cells

BACKGROUND. Stroma plays an essential role in glandular function in different systems. In the prostate, it is responsible for the development and maintenance of the differentiated state of the epithelium. The marked reduction in the epithelial compartment of the prostate gland following castration is followed by a similarly important reorganization of the stroma. In this work, we characterized the reorganization of collagen fibers in the ventral prostate of castrated rats. METHODS. Histochemical tests and immunohistochemistry for type I and III collagens plus confocal microscopy of triple-labeled (collagen III, actin, and DNA) tissue sections were employed. RESULTS. We showed that collagen fibers are composed of type I and type III collagens and that they are progressively concentrated around the epithelial structures (ducts and acini) and become increasingly undulated and folded. Double-labeling of collagen fibers and F-actin demonstrated that smooth muscle cells (SMC) are intimately associated with collagen fibers. CONCLUSIONS. The results demonstrated a marked reorganization of the collagen fibers, and suggest an active role of the SMC in the reorganization of the fibrillar components of the stroma. (C) 2000 Wiley-Liss, Inc.

Immunohistochemical aspects of basal cell adenoma and canalicular adenoma of salivary glands

Basal cell adenoma is a benign epithelial neoplasm with a uniform histologic appearance dominated by basaloid cells. Those cells may be distributed in various arrangements as solid, trabecular, tubular and membranous. Canalicular adenoma is also a benign neoplasm composed by columnar cells arranged in branching and interconnecting cords of single or double cell thick rows. There is some disagreement among investigators about whether canalicular adenoma should be included within the basal cell adenoma histologic spectrum. In the present study we compared the expression of cytokeratins (CK), vimentin and muscle-specific actin, utilizing immunohistochemical technique, in three cases diagnosed as basal cell adenomas predominantly of the solid type, and three cases of canalicular adenomas. The results obtained showed a distinct immunoprofile for both neoplasms. Solid areas of basal cell adenomas did not stain for any of the tested antibodies; only when there was tubular differentiation, those structures expressed CKs 7, 8, 14, and 19 in luminal cells and vimentin in non-luminal cells. On the other hand, canalicular adenomas strongly expressed CKs 7 and 13. The panel of antibodies utilized supports the separation of the two entities. © 2001 Elsevier Science Ltd.

Morphological aspects of muscle regeneration in the Nile tilapia (Oreochromis niloticus)

The aim of our study was to analyze the morphological events in the skeletal muscle of the Nile tilapia (Oreochromis niloticus) after a traumatic lesion. Thirty-two fish were used, on which a small longitudinal incision was made in the muscle. The fish were sacrificed after 7, 14, 21, and 42 days and muscle samples were collected from the lesion and processed for morphological analysis. Muscle regeneration in the tilapia occurred gradually through the analyzed period, possibly due to the proliferation and differentiation of myosatellite cells, which were more morphologically evident 7 and 14 days after lesion.

γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. © 2008 Elsevier B.V. All rights reserved.

Hypertrophied myocardium is more dependent on extracellular calcium than the normal cardiac muscle

Background: The aim of this study was to analyze stable hypertrophied myocardial function and its response to inotropic maneuvers in rats submitted to renovascular hypertension for a 10-week period (RHT group, n=10). Material/Methods: Myocardial performance was studied in isolated left ventricle papillary muscles in isometric contraction under the following conditions: at postrest contraction of 30 seconds (PRC), at extracellular calcium (ECa 2+) chloride concentration of 1.25 and 5.20 mM, and after beta-adrenergic stimulation with 10 -6 M isoproterenol (ISOP). Results: The results were compared with normotensive Wistar controls rats (C group, n=10). In basal condition, resting tension, and contraction time (TPT) were greater, while relaxation time (RT 50) tended to be longer in RHT than C group. PRC and ISOP promoted a similar change in muscle function response intensity (Δ) in both groups. ECa 2+ shift did not change TPT in the C group and decreased TPT in the RHT animals; Δ was different between these groups. RT 50 increased in C and decreased in RHT, both without statistical significance; however, Δ was different. Conclusions: These results suggest that hypertrophied myocardial dysfunction may be attibuted to changes in intracellular calcium cycling. © Med Sci Monit, 2010.

Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1α

Pós-graduação em Ciências Fisiológicas - FOA

Grafts of porcine small intestinal submucosa seeded with cultured homologous smooth muscle cells for bladder repair in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of maternal diabetes on male offspring: high cell proliferation and increased activity of MMP-2 in the ventral prostate

This study presents a comprehensive view of the histological and functional status of the prostate of adult rat offspring of mothers subjected to gestational diabetes induced by alloxan. The ventral prostate of male adult offspring of diabetic (DP) or normal (CP) mothers was evaluated for collagen fibres, cell death, fibroblasts, smooth muscle cells, cell proliferation, matrix metalloproteinases (MMPs), androgen receptors (AR), transforming growth factor beta 1 (TGF beta-1), catalase and total antioxidant activity. The prostates of DP animals were lower in weight than those of the CP group. The DP group also exhibited hyperglycaemia and hypotestosteronemia, higher cell proliferation and AR expression, a reduction in alpha-actin (possibly interfering with the reproductive function of the prostate), and enhanced activity of MMP-2, although the absolute content of MMP-2 was lower in this group. These findings were associated with increased TGF beta-1 and decreased collagen distribution. The prostates of DP rats additionally exhibited reductions in catalase and total antioxidant activity. Thus, rats developing in a diabetic intrauterine environment have glycaemic and hormonal changes that impact on the structure and physiology of the prostate in adulthood. The increased AR expression possibly leads to elevated cell proliferation. Stromal remodelling was characterized by enhanced activity of MMP-2 and collagen degradation, even with increased TGF beta-1 activation. These changes associated with increased oxidative stress might interfere with tissue architecture and glandular homeostasis.

Stromal myofibroblasts in squamous cell carcinoma of the tongue in young patients - a multicenter collaborative study

Objective. The purpose of this study was to evaluate the presence of myofibroblasts, frequently associated with a more aggressive neoplastic behavior, in oral tongue squamous cell carcinoma (TSCC) of young patients and to compare with the distribution observed in older patients.Study Design. Tumor samples from 29 patients younger than 40 years old affected by TSCC were retrieved and investigated for the presence of stromal myofibroblasts by immunohistochemical reactions against a smooth muscle actin, and the results obtained were compared to TSCC cases affecting older patients.Results. No positive reaction could be found in the stromal areas devoid of neoplastic tissue, whereas myofibroblasts were present in 58.6% of the lesions in young patients and in 75.9% of the older ones. No significant difference was found when comparing the invasive front and the overall stroma of both groups, and no correlation could be obtained with stromal a smooth muscle actin expression, higher tumor grades or clinical stage (P > .05).Conclusion. There was no significant difference between the presence of stromal myofibroblasts of TSCC affecting young and old individuals.

Benefits of kinesiotherapy and aquatic rehabilitation on sickle cell anemia. A case report

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as delta lesions were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. © 1991.

Ultrastructural features of the myotendinous junction of the sternomastoid muscle in Wistar rats: from newborn to aging

The myotendinous junction (MTJ) is a major area for transmitting force from the skeletal muscle system and acts in joint position and stabilization. This study aimed to use transmission electron microscopy to describe the ultrastructural features of the MTJ of the sternomastoid muscle in Wistar rats from newborn to formation during adulthood and possible changes with aging. Ultrastructural features of the MTJ from the newborn group revealed pattern during development with interactions between muscle cells and extracellular matrix elements with thin folds in the sarcolemma and high cellular activity evidenced through numerous oval mitochondria groupings. The adult group had classical morphological features of the MTJ, with folds in the sarcolemma forming long projections called finger-like processes and sarcoplasmic invaginations. Sarcomeres were aligned in series, showing mitochondria near the Z line in groupings between collagen fiber bundles. The old group had altered finger-like processes, thickened in both levels of sarcoplasmic invaginations and in central connections with the lateral junctions. We conclude that the MTJ undergoes intense activity from newborn to its formation during adulthood. With increasing age, changes to the MTJ were observed in the shapes of the invaginations and finger-like processes due to hypoactivity, potentially compromising force transmission and joint stability. Microsc. Res. Tech. 75:12921296, 2012. (C) 2012 Wiley Periodicals, Inc.

Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle

The effect of short-term creatine (Cr) supplementation upon content of skeletal muscle-derived-reactive oxygen species (ROS) was investigated. Wistar rats were supplemented with Cr (5 g/kg BW) or vehicle, by gavage, for 6 days. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated for evaluation of ROS content using Amplex-UltraRed reagent. The analysis of expression and activity of antioxidant enzymes (superoxide dismutase 1 and 2, catalase and glutathione peroxidase) were performed. Direct scavenger action of Cr on superoxide radical and hydrogen peroxide was also investigated. Short-term Cr supplementation attenuated ROS content in both soleus and EDL muscles (by 41 and 33.7%, respectively). Cr supplementation did not change expression and activity of antioxidant enzymes. Basal TBARS content was not altered by Cr supplementation. In cell-free experiments, Cr showed a scavenger effect on superoxide radical in concentrations of 20 and 40 mM, but not on hydrogen peroxide. These results indicate that Cr supplementation decreases ROS content in skeletal muscle possibly due to a direct action of Cr molecule on superoxide radical.

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background: The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods: A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results: Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion: We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.