Debora Di Mauro

IMMUNOLOCALIZATION OF THE COSTAMERES IN HUMAN SKELETAL MUSCLE FIBERS : CONFOCAL SCANNING LASER MICROSCOPE INVESTIGATIONS

The costameres in skeletal muscle fibers were first described by Pardo et al. (1983a) and have been defined as transverse circumferential elements of the cytoskeleton associated to the sarcolemma. Specific immunostaining for vinculin shows that the costameres overlie I bands. However, an exact correlation between the costameres and the Z line is uncertain, although ∼10 proteins so far have been localized in the costameres. To define the exact localization of costameres in human skeletal muscle fibers, we carried out an immunofluorescence study using confocal scanning laser microscopy on the fascia lata muscle of adult males.

Modulation of nicotinic acetylcholine receptor turnover by tyrosine phosphorylation in rat myotubes

The muscle nicotinic acetylcholine receptor (AChR) turns over at different rates depending on stage of synaptogenesis and innervation. Tyrosine phosphorylation modulates desensitization, interaction with cytoskeleton and lateral mobility in the membrane of AChR. To determine whether tyrosine phosphorylation also modulates the turnover of AChR, myotubes in vitro were exposed to the tyrosine phosphatase inhibitor pervanadate. Our data indicate that a transient increase of phosphotyrosine levels stabilized a fraction of AChRs. The effects were limited to the non-epsilon subunit-containing AChRs already present in the membrane. Tyrosine phosphorylation of the receptor occurred on the beta subunit, was transient and stable molecules were not selectively tyrosine phosphorylated. The data indicate that modulation of phosphotyrosine levels in muscle cells provides signals to control AChR metabolic stability.

Altered Cytoskeletal Structure of Smooth Muscle Cells in Ureteropelvic Junction Obstruction

PURPOSE Ureteropelvic junction obstruction is one of the most common causes of hydronephrosis in children. A malfunction of smooth muscle cells is believed to be the underlying mechanism causing obstruction. We investigated the expression of some integrins, talin and β-dystroglycan, considered the main compound of smooth muscle cell cytoskeleton, and active caspase 3 at the level of the ureteropelvic junction obstruction. MATERIALS AND METHODS Specimens were obtained at pyeloplasty in 12 children with ureteropelvic junction obstruction. Six control specimens were obtained during organ explantation. Specimens were divided into renal pelvis, ureteropelvic junction and ureter below the obstruction. Western blot analysis of active caspase 3, and immunofluorescence and polymerase chain reaction analysis were performed for α7A, β1A, α7B and β1D integrins, talin and β-dystroglycan. RESULTS Talin and β-dystroglycan were slightly impaired in ureteropelvic junction obstruction, while α7B and β1D integrins were severely reduced, and α7A, β1A and active caspase 3 were significantly enhanced compared to controls. CONCLUSIONS We demonstrated activation of apoptosis and a critical alteration of cytoskeleton that might explain the altered function and the increased apoptosis in smooth muscle cells in ureteropelvic junction obstruction. The delayed rearrangement of the cytoskeleton of smooth muscle cells in ureteropelvic junction obstruction might be linked to a postnatal splicing from α7A and β1A to α7B and β1D integrins, respectively. This relationship could explain the common clinical scenario of spontaneous improvement of hydronephrosis in children with suspected ureteropelvic junction obstruction.

Expression of Sarcoglycans in the Human Cerebral Cortex: An Immunohistochemical and Molecular Study

The sarcoglycan (SG) complex (SGC) is a subcomplex within the dystrophin-glycoprotein complex (DGC) and is composed of several transmembrane proteins (α, β, δ, γ, ε and ζ). The DGC supplies a transmembranous connection between the subsarcolemmal cytoskeleton networks and the basal lamina in order to protect the lipid bilayer and to provide a scaffold for signaling molecules in all muscle cells. In addition to its role in muscle tissue, dystrophin and some DGC components are expressed in neurons and glia. Very little is known about the SG subunits in the central nervous system (CNS) and some data suggested the presence of ε and ζ subunits only. In fact, mutations in the ε-SG gene cause myoclonus-dystonia, indicating its importance for brain function. To determine the presence and localization of SGC in the human cerebral cortex, we performed an investigation using immunofluorescence, immunoblotting and reverse transcriptase polymerase chain reaction. The results showed that all SG subunits are expressed in the human cerebral cortex, particularly in large neurons but also in astrocytes. These data suggest that the SG subcomplex may be involved in the organization of CNS synapses.

Assessment of glutathione peroxidase-1 polymorphisms, oxidative stress and DNA damage in sensitivity-related illnesses.

AIMS Oxidative stress increase is a key event for development of sensitivity-related illnesses (SRIs). The aim of this work was to evaluate the influence of a genetic variant in the antioxidant enzyme glutathione peroxidase (GPx1) on oxidative stress development in SRIs. MAIN METHODS GPx1 rs1800668 genotype, as well as glutathione, ubiquinone, and DNA damage were assessed in 34 SRI patients and 36 healthy subjects. KEY FINDINGS Total glutathione, reduced/oxidized glutathione, and ubiquinone were significantly decreased in cases compared with controls, while DNA fragmentation was significantly increased in patients. However, these differences were not associated to GPx1 genetic background. SIGNIFICANCE GPx1 rs1800668 polymorphism does not play a major role in SRI-related oxidative stress development.

Expression pattern of transglutaminases in the early differentiation stage of erupting rat incisor

Several studies demonstrated that transglutaminases play a key role in extracellular matrix stabilization needed for cell differentiation. We evaluated transglutaminase expression and activity in the pre-secretory stage of differentiation of the continuously erupting rat incisor. We observed that transglutaminase-mediated incorporation of monodansylcadaverine into protein substrates was specifically located in the apical loop, and along the basement membrane joining mesenchyme and inner dental epithelium in the odontogenic organ. Enzyme activity was associated with mRNAs for transglutaminase 1 and 2. Notably, labelling cells for these isoenzymes were observed in both mesenchymal and epithelial compartments, but not in the basement membrane, in the ameloblast facing pulp anterior region, where ameloblast and odontoblast differentiation begins. These findings demonstrate that transglutaminase 1 and transglutaminase 2 are expressed at a major extent in the pre-secretory stage of regenerating rat incisor, where they probably play complementary roles in cell signalling between mesenchyme and epithelium and extracellular matrix.

Role of subunit composition in determining acetylcholine receptor degradation rates in rat myotubes

During neuromuscular junction maturation, the rapidly degrading receptors (Rr; t1/2 approximately equal to 1 day) are replaced by metabolically stable molecules (Rs; t1/2 approximately equal to 10 days). Rr and Rs do not interconvert, are differently regulated after denervation in adult muscle and are endowed of unique responses to stabilizing agents. In cultured rat myotubes all the epsilon subunit-containing acetylcholine receptors (epsilon-AchRs) are of the Rs type. In the present study we show that Rs exist also in absence of epsilon-AChR and that nonepsilon-(presumably gamma-)AChRs can be included in the Rs pool when epsilon-AChR expression is low. The data indicate that Rs metabolic properties are independent of AChR subunit composition and that epsilon subunit is a signal to efficiently sort AChR molecules to the Rs pool.

Sarcoglycan Complex in Human Normal and Pathological Prostatic Tissue: An Immunohistochemical and RT‐PCR Study

The sarcoglycan complex is a trans‐membrane system playing a key role in mechano‐signaling the connection from the cytoskeleton to the extracellular matrix. While β‐, δ‐, and ε‐sarcoglycans are widely distributed, γ‐ and α‐sarcoglycans are expressed exclusively in skeletal and cardiac muscle. Insufficient data are available on the distribution of sarcoglycans in nonmuscular tissue. In the present study, we used immunohistochemical and RT‐PCR techniques to study the sarcoglycans also in normal human glandular tissue, a type of tissue never studied in relation to the sarcoglycan complex, with the aim of verifying the real wider distribution of this complex. To understand the role of sarcoglycans, we tested specimens collected from patients affected by benign prostatic hyperplasia and adenocarcinoma. For the first time, our results showed that all sarcoglycans are detectable in normal samples both in epithelial and in myoepithelial cells; in pathological prostate, sarcoglycans appeared severely reduced in number or were absent. These data demonstrated that all sarcoglycans have a wider distribution suggesting a new unknown role for these proteins. The decreased number of sarcoglycans, containing cadherin domain homologs in samples of prostate affected by hyperplasia, and the absence of proteins in prostate biopsies, in cases affected by adenocarcinoma, could be responsible for the loss of adhesion between epithelial cells, which in turn facilitates the progression of benign tumors and the invasive potential of malignant tumors. Anat Rec, 297:327–336, 2014.

The Limbic and Sensorimotor Pathways of the Human Amygdala: A Structural Connectivity Study

The amygdala plays a key role in gathering social cues to context-appropriate responses that require refined motor behavior, involving either direct or indirect connections with sensorimotor-related areas. Although, several studies investigated the structural and functional limbic connectivity of the amygdala both in animals and in humans, less is known about the limbic modulation on sensorimotor-related areas. However, recent evidences suggest the amygdala as a possible cornerstone in the limbic-motor interface. Herein, we used high-resolution diffusion data of the Massachusetts General Hospital-University of Southern California (MGH-USC) Adult Diffusion Dataset, constrained spherical deconvolution-based signal modeling and probabilistic tractography aimed at identifying and reconstructing the connectivity patterns linking the amygdala to the limbic- and sensorimotor-related areas. As regards the limbic network, our results showed that the amygdala has high probability to be connected with the fusiform gyrus and the lateral orbitofrontal cortex. On the other hand, our connectomic analysis revealed a close interplay between the amygdala and the inferior parietal lobule, followed by the postcentral gyrus, the precentral gyrus and the paracentral lobule. The findings of the present study are in line with previous literature and reinforce the idea of the existence of a limbic-motor interface, which is likely to be involved in the emotional modulation of complex functions such as spatial perception and movement computation. Considering that these pathways may play an important role, not on in physiological conditions, but also in pathological context, further studies should be fostered in order to confirm the existence of a limbic-motor interface and its precise functional meaning.

Analysis on sarcoglycans expression as markers of septic cardiomyopathy in sepsis-related death

The post-mortem assessment of sepsis-related death can be carry out by many methods recently suggested as microbiological and biochemical investigations. In these cases, the cause of death is a multiple organ dysfunction due to a dysregulated inflammatory response occurring after the failure of infection control process. It was highlighted also that the heart can be a target organ in sepsis which determines the so-called septic cardiomyopathy characterized by myocardial depression. Several mechanisms to explain the pathophysiology of septic cardiomyopathy were suggested, but very few studies about the structural alterations of cardiac cells responsible for myocardial depression were carried out. The aim of this study was to evaluate whether sarcoglycans (SG) were involved in septic cardiac damage analyzing their expression in sepsis-related deaths and, particularly, if these proteins can be used as markers of septic myocardial dysfunction. Cases of septic-related death confirmed by clinical and autopsy records were investigated and compared to a control group of traumatic deaths. Indirect immunofluorescence analysis was performed to analyze α-SG, β-SG, δ-SG, ζ-SG, ε-SG, and γ-SG. Decrease of fluorescence staining pattern for all tested sarcoglycans was observed in the septic-related deaths compared to normal fluorescence staining pattern of control group. These results provide new findings about the myocytes structural alterations due to sepsis and suggest that these proteins could be used in forensic assessment of septic cardiomyopathy.