Biomedical subjects

D Nosi

Publications and source records attributed to D Nosi.

12 recordsLinked to original sources

PubMed2006

Sphingosine 1-phosphate induces myoblast differentiation through Cx43 protein expression: a role for a gap junction-dependent and -independent function.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43(Delta130-136) reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Animals↗

PubMed2006

Serous cutaneous glands of the Pacific tree-frog Hyla regilla (Anura, Hylidae): patterns of secretory release induced by nor-epinephrine.

The serous (poison) cutaneous glands of the Pacific tree-frog Hyla regilla were induced to release their product by 10(-3)M nor-epinephrine stimulation. After discharge structural and ultrastructural features of the cutaneous glands involved in release were observed. Furthermore, the discharged product, consisting of discrete, secretory granules, was collected and processed for transmission electron microscope analysis. As indicated by patterns found in the myoepithelium encircling the syncytial secretory unit, gland discharge is caused by contraction of the peripheral myocytes. Muscle cell compression dramatically affects the syncytium and results in degenerative changes, including expulsion of the secretory unit nuclei. Therefore, the structural collapse in depleted glands has been ascribed to the mechanical activity performed by the myoepithelium during discharge, rather than cytoplasm involution described in conventional, holocrine glands. TEM investigation revealed that the secretory granules collected after discharge maintain their peculiar traits: they consist of recurrent patterns of thin subunits, acquired during serous maturation and provided with remarkable structural stability.

Amphibian Venoms↗

PubMed2005

Damage of cutaneous peripheral nervous system evolves differently according to the disease phase and subset of systemic sclerosis.

OBJECTIVE: Evidence shows that peripheral nervous system (PNS) is involved in systemic sclerosis (SSc), but few morphological studies have assessed the ultrastructural pathological modifications. The aim was to study ultrastructural modifications of skin PNS fibres in SSc according to subsets [limited SSc (lSSc) and diffuse SSc (dSSc)] and phases (early and advanced) of the disease. METHODS: Skin biopsies were taken from the forearms of 23 SSc patients (11 lSSc and 12 dSSc) and 10 controls. Each biopsy was processed for transmission electron microscopy (TEM). RESULTS: At TEM, observation in skin from early lSSc, signs of inflammation were evident, while PNS fibres were not damaged. The microvascular wall showed hypertrophic endothelial cells bulging into the lumen. In advanced lSSc, fibrosis prevailed on inflammation and slight ultrastructural alterations of PNS fibres were evident in the papillary derma. In early dSSc, ultrastructural alterations of PNS fibres, similar to those observed in the advanced phase of lSSc, were found together with signs of inflammation and fibrosis. In advanced dSSc, in the papillary and reticular dermis PNS fibres were reduced and showed relevant ultrastructural alterations. CONCLUSIONS: In SSc, PNS ultrastructure damage is linked to the progression and severity of skin involvement. The alterations evolve from the early to the advanced phase mainly in the diffuse subset. In particular, the severe PNS lesions found in advanced lSSc are already present and widely diffuse in early dSSc and the microvascular involvement in early lSSc seems to precede the modification of the PNS in the skin. Thus, an early therapeutic approach can be useful to reduce the progression of PNS and skin damage in SSc patients.

Adult↗

PubMed2005

Differential expression of tissue kallikrein in the skin of systemic sclerosis.

Systemic sclerosis (SSc) is characterised by ischemic damage, impaired angiogenesis and skin fibrosis. Tissue kallikrein (t-kallikrein) is involved through kinins in inflammation, vasorelaxation and angiogenesis. T-kallikrein is synthetised by endothelial, smooth muscle, and inflammatory cells and, in skin, also by dark cells of the sweat glands, where it is involved in sweat formation. Our aim was to analyse, by immunohistochemistry and RT-PCR, the expression of t-kallikrein in the skin of patients with different SSc subsets, limited (lSSc) and diffuse (dSSc), and phases, early and advanced. Skin biopsies were taken from 18 SSc patients and 10 controls. Immunohistochemistry was performed on paraffin sections with an antibody against human urinary t-kallikrein. For RT-PCR, cDNA from skin biopsies was amplified using primers specific for human t-kallikrein. In the control skin, dark cells of the secretory units of sweat glands showed immunopositivity for t-kallikrein as well as blood vessels. In the lSSc skin, immunoreactivity was observed only in some glands, with weak staining in the advanced phase. In early lSSc skin, immunoreactivity was observed in microvessel walls and in the inflammatory infiltrate. In dSSc skin, dark cells of the glandular fundus units, and the few remaining vessels showed scarcity (early phase) or lack (advanced phase) of immunoreactivity for t-kallikrein. RT-PCR confirmed a decrease of t-kallikrein mRNA levels from early to advanced phase in SSc subsets, reaching its lowest level in advanced dSSc. In conclusion, immunohistochemical and biomolecular results indicate that t-kallikrein is decreased in the skin of SSc patients and decreases progressively from the early to advanced phase of lSSc and dSSc. The decreased expression of t-kallikrein may be involved in the impairment of the sweating process, vessel functionality and angiogenesis.

Adult↗

PubMed2004

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

Actins↗

PubMed2004

Effects of S1P on myoblastic cell contraction: possible involvement of Ca-independent mechanisms.

Sphingosine-1-phosphate (S1P) is a lipid mediator, which affects many essential processes such as cell proliferation, differentiation and contraction in many cell types. We have previously demonstrated that the lipid mediator elicits Ca(2+) transients in a myoblastic cell line (C2C12) by interacting with its specific receptors (S1PR(s)). In the present study, we wanted to correlate the Ca(2+) response with activation of myoblastic cell contractility. C2C12 cells were first investigated for the expression and cellular organization of cytoskeletal proteins by immunoconfocal microscopy. We found that myoblasts exhibited a quite immature cytoskeleton, with filamentous actin dispersed as a web-like structure within the cytoplasm. To evaluate intracellular Ca(2+) mobilization, the cells were loaded with a fluorescent Ca(2+) indicator (Fluo-3), stimulated with S1P and simultaneously observed with differential interference contrast and fluorescence optics. Exogenous S1P-induced myoblastic cell contraction was temporally unrelated to S1P-induced intracellular Ca(2+) increase; cell contraction occurred within 5-8 s from stimulation, whereas intracellular Ca(2+) increase was evident only after 15-25 s. To support the Ca(2+) independence of myoblastic cell contraction, the cells were pretreated with a Ca(2+) chelator, BAPTA/AM, prior to stimulation with S1P. In these experimental conditions, the myoblasts were still able to contract, whereas the S1P-induced Ca(2+) transients were completely abolished. On the contrary, when C2C12 cells were induced to differentiate into skeletal myotubes, they responded to S1P with a rapid cell contraction concurrent with an increase in the intracellular Ca(2+). These data suggest that Ca(2+)-independent mechanism of cell contraction may be replaced by Ca(2+)-dependent ones during skeletal muscle differentiation.

Actins↗

PubMed2003

Possible role of acylphosphatase, Bcl-2 and Fas/Fas-L system in the early changes of cardiac remodeling induced by volume overload.

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca(2+) homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca(2+) pump activity was reduced until 48 h (-30%), but a recovery of control values was found at 96 h. The decrease in SR Ca(2+)-ATPase (SERCA2a) expression at 48 h, was more marked (-60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.

Acid Anhydride Hydrolases↗

PubMed2003

Development of serous cutaneous glands in Scinax nasica (Anura, Hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families.

We examined the development of serous (poison) cutaneous glands in larval and juvenile Scinax nasica (Hylidae) at the ultrastructural level. We describe the biosynthesis and maturation of the cutaneous poison in comparison with the corresponding processes in representatives of Discoglossidae, Leptodactylidae, Pelobatidae and Pipidae. Serous biosynthesis in S. nasica starts in discrete adenoblasts and continues in the syncytial secretory unit. Biosynthetic processes involve rough endoplasmic reticulum and the Golgi apparatus, that releases membrane-bounded material, varying from fine grained to flocculent. During the post-Golgian secretory phase, this material undergoes initial maturation, and two products are formed: dense granules and larger vesicles holding a thin substance that will later be structured into a three-dimensional, honeycomb-like net. Both the secretory granules and vesicles change into glomerular-like aggregates of bowed, rod-shaped subunits (modules). In adult frogs, formation of dense granules is bypassed. The modular granule substructure seems to be related to the merocrine release of small amounts of poison, involved in regulating skin homeostasis. Comparison with maturational changes in larval glands of species representing four anuran families discloses similar patterns in the Leptodactylidae, but production of opaque homogeneous granules occurs in the Discoglossidae, clear vesicles in the Pelobatidae and aggregates of dense bars in the Pipidae.

Animals↗

PubMed2003

Altered Cx43 expression during myocardial adaptation to acute and chronic volume overloading.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

Adaptation, Physiological↗

PubMed2002

Ca+2 homeostasis and cytoskeletal rearrangement operated by sphingosine 1-phosphate in C2C12 myoblastic cells.

In hypogravity conditions unloading of skeletal muscle fibres causes alterations in skeletal muscle structure and functions including growth, gene expression, cell differentiation, cytoskeletal organization, contractility and plasticity. Recent studies have identified sphingosine I -phosphate (SPP) as a lipid mediator capable of eliciting intracellular Ca2+ transients, cell proliferation, differentiation, suppression of apoptosis, as well as cell injury repair. The aim of this research is to evaluate a possible involvement of SPP in skeletal muscle cells differentiation and repair from space-flight damage. Particularly, we investigated the Ca2+ sources and the changes on the cytoskeletal rearrangement induced by SPP in a mouse skeletal (C2C12) myoblastic cell line. Confocal fluorescence imaging revealed that SPP elicited Ca2+ transients which propagated throughout the cytosol and nucleus. This response required extracellular and intracellular Ca2+ mobilization. SPP also induced cell contraction through a Ca2(+)- independent/Rho-dependent pathway. The nuclear Ca2+ transients are suggestive for an action of SPP in the differentiation program and damage repair.

Journal Article↗

PubMed2001

Secretory granule-cytoplasm relationships in serous glands of anurans: ultrastructural evidence and possible functional role.

A survey covering the serous (granular) cutaneous glands in several anuran families from the Old and New Worlds (Bombinatoridae, Discoglossidae, Ranidae, Hylidae, Pseudidae and Leptodactylidae) has revealed consistent patterns of complex interactions between the syncytial secretory unit and serous deposits (granules). These relationships involve outgrowths from the syncytial cytoplasm encircling the granules and complex invaginations of the perigranular compartment (halo) into the syncytium. The outgrowths are branched, cytoplasm processes resembling ramified microvilli, or can be larger, dome-like to cylindrical structures. Despite their different features and origins, all these structures are efficient devices for amplifying the cytoplasmic surfaces round the granules, so improving exchange between the secretory syncytium and serous product. These complex secretory granule-cytoplasm interactions affect the product released from the Golgi apparatus and are consistent with the hypothesis of prolonged serous maturation following the initial phase of biosynthesis. Post-Golgian maturation modifies the secretory material on a centripetal gradient, causing condensation and, possibly, the transfer of component molecules from and/or to the cytoplasm.

Animals↗

PubMedDate not supplied

[Peripheral nervous system in limited systemic sclerosis].

OBJECTIVES: PNS is involved in Systemic Sclerosis (SSc) since the earliest phases. Our aim is to perform an ultrastructural study on skin PNS fibers in SSc. METHODS: Skin biopsies were taken from forearms of 8 patients affected by limited SSc (lSSc) and 3 controls and processed for transmission electron microscopy (TEM). The semithin sections (2 mm) were observed at light microscope and optical fields were chosen for ultrathin sections (1 mm) preparation and TEM examination. RESULTS: In lSSc skin, in the semithin sections, damaged areas are close to apparently spared areas. At TEM, in early lSSc patients, signs of inflammation and damaged microvessels are visible in derma. PNS fibers are no damaged. In advanced lSSc, fibrosis prevails on inflammation, and slight ultrastructural alterations of PNS fibers are evident in papillar derma. CONCLUSIONS: PNS lesions are different in severity in lSSc according to the disease duration, resulting more severe in advanced than in early phase.

Biopsy↗