Morphological and biochemical patterns in skeletal muscle apoptosis.

Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.

Proteomics-based investigation in C2C12 myoblast differentiation.

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified.To further highlight this process, we studied cells at undifferentiated, intermediate and highly differentiated stages, and we analyzed, for each condition, morphological and proteomic changes. We also identified the proteins that showed statistical significant changes by a ESI-Q-TOF mass spectrometer. This work provides further evidence of the involvement of particular proteins in skeletal muscle development. Furthermore, the high level of expression of many heat shock proteins, suggests a relationship between differentiation and cellular stress. Intriguingly, the discovery of myogenesis-correlated proteins, known to play a role in apoptosis, suggests a link between differentiation and this type of cell death.

Identification of a novel polymorphism in the fibronectin type II domain of the SEL1L gene and possible relation to the persistent hyperinsulinemic hypoglycemia of infancy.

SEL1L, a human gene located on chromosome 14q24.3-q31, is highly expressed in adult pancreas. It is proximal to D14S67 (IDDM11) a proposed type I diabetes susceptibility locus. Considering the organ specific expression of SEL1L, a fundamental role of SEL1L in pancreatic growth can be hypothesized. While screening for mutations in young diabetic patients, in children affected by persistent hyperinsulinemic hypoglycemia of infancy (PHHI), in patients with non-functional endocrine tumours and in over 100 control subjects, we identified a novel polymorphism (D162G) residing on the fourth exon of the gene. This exon encodes for the fibronectin type II domain and the nucleotide change involves a highly conserved amino acid. The D162G polymorphism induces a major change in the amino acid composition producing a possible disruptive role in collagen binding.

Bacterial bio-mediated calcite precipitation for monumental stones conservation: methods of evaluation.

The weathering of monumental stones is a complex process inserted in the more general 'matter transformation cycle' operated by physical, chemical and biological factors. The consequence of these combined actions is a loss of cohesion with dwindling and scaling of stone material and the induction of a progressive mineral matrix dissolution. In the case of calcareous stones, calcite leaching increases the material porosity and decreases its mechanical features with a general weakening of the superficial structural strength. Attempts to stop, or at least to slow down, deterioration of monumental stones has been made by conservative treatments with both inorganic or organic products. More recent studies show a new approach to hinder these phenomena by inducing a bio-mediated precipitation of calcite directly inside the stone porosity. This can be achieved either through the application of organic matrix macromolecules extracted from sea shells or of living bacteria. The effectiveness of the treatment using calcinogenic bacteria has been evaluated with laboratory tests specifically developed to evaluate the parameters such as : porosity, superficial strength and chromatic changes, influenced by the treatment itself. The results obtained seem to indicate that this type of treatment might not be suitable for monumental stone conservation.

Deletion polymorphism in angiotensin converting enzyme gene associated with carotid wall thickening in a healthy male population.

BACKGROUND: Intimal thickening is an adaptive process of the arterial wall, presumably related to atherogenesis. Sex and interethnic differences in intimal thickening, as demonstrated histologically on autoptic material, would indicate a strong genetic control on this process. An insertion/deletion (ID) polymorphism in the angiotensin-converting enzyme (ACE) gene has been shown to be an independent risk factor for cardiovascular disease, especially in subjects otherwise at low risk for coronary heart disease. The aim of the present study was to evaluate the relationship between intimal plus medial thickness (IMT) and ACE-I/D genotype. METHODS: 132 healthy male subjects from Southern Italy were enrolled. IMT has been evaluated from high resolution B-mode echo-Doppler images. Blood lipids and glucose were measured using standard methods. Cigarette consumption was recorded by questionnaire. ACE genotypes were analysed by polymerase chain reaction. RESULTS: Blood lipids, blood pressure and percentage of smokers were similar in the three groups. IMT was greatest in DD subjects, lowest in II subjects and intermediate in heterozygotes. The association between the presence of the D allele and IMT values was statistically significant. Significance was maintained after the elimination of subjects with carotid atherosclerotic plaques. CONCLUSIONS: The present data suggest that the ACE gene seems to be a candidate gene that strongly influences the IMT of the arterial wall and might therefore be involved in the individual's predisposition to the development of atherosclerosis.