Sarcoglycan subcomplex during embryonic life of rats: an immunohistochemical study

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other member of dystrophin-glycoprotein complex (DGC) in order to provide a mechano-signaling connection from the cytoskeleton to the extracellular matrix in myocytes. Previous investigations have demonstrated that in skeletal and cardiac muscle, the SGC is a heterotetrameric unit constituted by the a-, b-, g-, and d-sarcoglycans. Other authors demonstrated that the expression of a-sarcoglycan is restricted to striated muscle cells, whereas e-sarcoglycan, is also expressed in several other tissues. Moreover, further analysis showed the presence, in vascular and visceral smooth muscle, of other sarcoglycan subcomplex, consisting of e-, b-, g-, and d-sarcoglycan, associated with sarcospan. Previous our studies have demonstrated presence of sarcoglycans also in non-muscle tissue as prostatic and breast glandular epithelial tissues in normal and pathological conditions, hypothesizing a key role for these glycoproteins in mediating the signalling between cell and extracellular matrix (1,2). Furthermore, some Authors have studied the presence of sarcoglycans also in fetal tissues demonstrating that the signals of these proteins in fetal liver are weak or absent (3), or that their immunostaining is clearly detectable (4). Although these discordant assumptions, insufficient data are present on sarcoglycans during fetal period. On this basis, in order to verify composition of sarcoglycan subcomplex during this period, we analysed fetuses of rat at 8 and 14 days using immunohistochemical techniques, observing several organs at fetal stage, as well liver, kidney and brain. Our results have shown that all sarcoglycans are constantly present in all tested embryonic organs and that their staining pattern was more detectable that that the adult life. In our opinion, these data demonstrated that, also in fetal period, sarcoglycans are present confirming a key function for these proteins in regulating of transduction signalling. In this way, sarcoglycan subcomplex could play a crucial role in cytodifferentiation processes in order to check the development of several organs during embryonic life

Diffusion tensor imaging (DT I) and Transcranial magnetic stimulation (TMS): an integrated approach

In present study we adopted an innovative approach, integrating the “Diffusion Tensor Imaging tractography (DTI)” technique with navigated transcranial magnetic stimulation (nTMS). In this way it is possible to improve accuracy of motor fiber reconstruction by positioning the fiducial seeding from DTI reconstruction, over the motor areas localized by nTMS. This will allow a direct comparison between the density of the motor map obtained with nTMS with the related axonal density obtained with DTI. DTI uses anisotropic water diffusion like test to study in vivo white matter anatomy in order to reconstruct tri-dimensional fiber bundles improving the practical 1 mm. MRI scanning resolution. Therefore we used the appropriate acquisition and reconstruction DTI techniques in order to study local cerebral pathways. Related acquisition must be done following resonance protocol and varying the magnetic gradient, in order to have the following steps: DWI acquisition; tensor calculation; scalar maps; 3d visualization; fiducial seeding. DTI technique can exploit two different classes of fiber tracking algorithms: “deterministic” or “probabilistic” tracking higherorder integration schemes. The deterministic one, allows to calculate the directions of streamline propagation, and reveals only, presence or absence of a connection. On the other hand a probabilistic tracking approach is achieved: starting from a basic point, fibers are propagated multiple times through the tensor field while varying, in a stochastic way, the estimated fiber orientation along the traversed voxels. In addition nTMS is a newly evolving technique for in vivo investigating human motor system combining spatial information from high-resolution MRI with the functionality of non-invasive cortical stimulation. In this way it is possible to target motor areas more precisely obtaining a discrete motor maps of facial, hand and leg muscles. This innovative approach will indeed provide a new tool in neuroscience for an in vivo direct correlation between anatomical circuits and electrophysiological data

Muscle-specific integrins in masseter muscle fibers of chimpanzees: an immunohistochemical study.

Most notably, recent comparative genomic analyses strongly indicate that the marked differences between modern human and chimpanzees are likely due more to changes in gene regulation than to modifications of the genes. The most peculiar aspect of hominoid karyotypes is that human have 46 chromosomes whereas gorillas and chimpanzees have 48. Interestingly, human and chimpanzees do share identical inversions on chromosome 7 and 9 that are not evident in the gorilla karyotype. Thus, the general phylogeny suggests that humans and chimpanzees are sister taxa; based on this, it seems that human-chimpanzee sequence similarity is an astonishing 99%. At this purpose, of particular interest is the inactivation of the myosin heavy chain 16 (MYH16) gene, most prominently expressed in the masticatory muscle of mammals. It has been showed that the loss of this gene in humans may have resulted in smaller masticatory muscle and consequential changes to cranio-facial morphology and expansion of the human brain case. Powerful masticatory muscles are found in most primates; contrarily, in both modern and fossil member Homo, these muscles are considerably smaller. The evolving hominid masticatory apparatus shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. To better comprehend the real role of the MYH16 gene, we studied the primary proteins present in the muscle fibers of humans and non-humans, in order to understand if they really can be influenced by MYH16 gene. At this aim we examined the muscle-specific integrins, alpha 7B and beta 1D-integrins, and their relative fetal isoforms, alpha 7A and beta 1A-integrins, analyzing, by immunohistochemistry, muscle biopsies of two components of a chimpanzee's group in captivity, an alpha male and a non-alpha male subjects; all these integrins participate in vital biological processes such as maintenance of tissue integrity, embryonic development, cell differentiation, and cell-extracellular matrix interactions. Our results demonstrated a different quantitative composition of integrins, in alpha male in respect to human and non-alpha male, hypothesizing that the MYH16 gene could modify the expression of integrins, influencing, in turn, the phenotype of muscle. In this way, alpha 7A-and beta 1A-integrin could determine the presence of type II fibers and then they could play a key role in the determination of contraction force. Then, MYH16 gene could be a common interactor of signalling between sarcoglycans and integrins in chimpanzee muscles

Sarcoglycan subcomplex on normal and pathological prostatic tissue: an immunohistochemical and molecular study

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other member of dystrophin-glycoprotein complex (DGC) in order to provide a mechano-signaling connection from the cytoskeleton to the extracellular matrix in myocytes. Previous investigations have demonstrated that in skeletal and cardiac muscle, the SGC is a heterotetrameric unit constituted by the alfa, beta, gamma and delta-sarcoglycans. Other authors demonstrated that the expression of alfa-sarcoglycan is restricted to striated muscle cells, whereas epsilon-sarcoglycan, is also expressed in several other tissues. Moreover, further analysis showed the presence, in vascular and visceral smooth muscle, of other sarcoglycan subcomplex, consisting of epsilon, beta, gamma and delta-sarcoglycan, associated with sarcospan. In order to verify composition of sarcoglycan subcomplex in other tissues, we analyzed glandular epithelium of prostate, testing it both in normal and in pathological conditions. In particular we performed immunofluorescence reactions using all sarcoglycans alfa, beta, gamma, delta and epsilon) on biopsies of ten normal subjects, and ten subjects with prostatic hyperplasia and prostatic cancer. Our results showed that in normal prostate all tested sarcoglycan are detectable both in epithelial and in myoepithelial cells; whereas in biopsies of prostatic hyperplasia all sarcoglycans were less detectable, whereas in prostatic carcinoma they were almost absent in both cell types. These data demonstrated that also in epithelium of prostate, as well as in all epithelia previously tested by us, the sarcoglycan subcomplex play a key role in mediating the signalling between cell and extracellular matrix; moreover, the absence of all sarcoglycans in pathological glandular tissue, and in particular in mioepithelial cells, demonstrated that this glycoprotein can play an important role in signalling for contraction of these cells

Sarcoglycans and GABAA Rε receptors in cerebral cortex, thalamus and hippocampus: an immunohistochemical study

The Sarcoglycans sub-complex is a protein system which plays a role in sarcolemma stabilization, protecting the fibers by any injury provoked by muscle activity. This complex is composed by six transmembrane glycoproteins, α-,β-,γ-,δ-,ε- and ξ-sarcoglycans and, although numerous studies have been conducted on this system, there are no many data about its localization in non-muscular tissues. In our previous study we have analyzed the sarcoglycans expression and localization in rat’s cerebral cortex and our results showed that all sarcoglycans are present with a staining pattern in relation to the cerebral cortex area observed. In particular we think that they could be associated with synapse sites such as inhibitory GABAA Rε receptors. In order to verify any association between sarcoglycans and GABAA Rε receptors we performed double immunolabeling to detect α-,β-,γ-,δ-,ε- and ξ-sarcoglycans and GABAA Rε receptor. Our results have shown that in cerebral cortex each sarcoglycans is equally associated with GABAA Rε receptor, showing some point of colocalization around the cellular soma. Moreover, we observed the reaction in thalamus and hippocampus where we found that all sarcoglycans are expressed with the same “spot-like” staining pattern that we observed in cerebral cortex. Instead, in the extension of the neurons the proteins present a linear staining pattern. We have also found that in these districts the fluorescence pattern of GABAA Rε receptor increase together with the sarcoglycans fluorescence pattern, supporting our previous idea about a tight correlation between sarcoglycans and GABAA Rε receptors. These results suggest again a role of sarcoglycans in cellular signalling, regulating the post-synaptic receptor assembly. On this basis it could be hypothesized that sarcoglycans could be involved in some pathologies of the brain becoming, in these districts, as important as in muscle

Sarcoglycans and integrins in masseter muscle of baboons: an immunohistochemical and molecular study

The sarcoglycans subcomplex consists of six transmenbrane proteins (α,β,δ,γ,ε,ζ), functionally connected by a bidirectional signalling with integrins, transmembrane receptors that play a key role in cell adhesion and differentiation. b1D-integrin is detected only in skeletal and cardiac muscle, while low amounts of b1A were detected in striated muscles. b1D was associated with α7A and α7B in adult skeletal. Although numerous studies have been carried out on these proteins in many muscle types, insufficient data exist on their behaviour in masseter muscle, an highly unusual muscle for the presence, in addition to slow and fast fibers, of hybrid fibers types important for the specific functional demands of masseter. Our studies on normal human masseter muscle and on masseter of subjects with right crossbite, showed that integrins play a role in the functional activity of muscle and in the optimization of contractile forces. Also, we studied these proteins in masseter of chimpanzees, alpha and non-alpha male subjects. These results have shown a different quantitative composition of integrins in alpha male in respect to non-alpha male hypothesizing a key role for integrins and sarcoglycans in the determination of contraction force. Here, we analyzed masseter muscle obtained from baboons, animals similar in phylogeny with humans and chimpanzees, individuating subjects with high and low dominance. Our immunohistochemical results, confirmed also by Western Blotting analysis, show that, in high dominance subjects, stainings for sarcoglycans and integrins were normal; interestingly, in low dominance subjects stainings for these proteins were normal, lower or absent in different fibers of the same microscopic field. Thus, preliminary analysis on cell cultures of myoblasts and myotubes, at different days of differentiation, immunolabelled with antibodies against sarcoglycans and integrins, have demonstrated a similar behaviour, showing cells with an higher or lower staining for these proteins. In our opinion, these results provide the first suggestion that integrins and sarcoglycans in masseter muscle play a key role regulating muscular functional activity and allowing the optimization of contractile forces of this muscle

Sarcoglycans and mucin in epithelial tissues of digestive and respiratory tracts: an immunofluorescence study

Sarcoglycans are transmembrane glycoproteins which play a key role in maintaining sarcolemma stabilization during muscle contraction. Several studies have demonstrated that this complex is not muscle specific and that it is also expressed in epithelial tissues as gingival, breast and prostatic epithelia. In the present study we investigated sarcoglycans expression in the epithelia of digestive and respiratory tracts. We performed immunofluorescence reactions using antibody against a-, b-, g-, d-, e- and z-sarcoglycans and against mucin 4 and 16. Mucins are a superfamily of proteins which serve to protect the underlying epithelia against a wide range of injuries (bacteria, virus, parasites, toxins, pH). This protection leads to coordinate cell proliferation, differentiation and apoptosis among other cellular responses; in fact, mucins are promising biomarkers and therapeutic targets in cancer and inflammatory diseases. Our results show the expression of sarcoglycans in the basal, lateral, and apical epithelial cell’s surface; moreover, sarcoglycans show to colocalize with mucins in the cell’s apical surface of bronchi and bronchioles, stomach and intestine but no apical localization has been detected in the esophageal epithelium. These results support the role of sarcoglycans in cell-cell and cell-matrix interaction. Moreover, the colocalization between sarcoglycans and mucins at apical level of epithelia which have high mucosecretory activity suggest that sarcoglycans could interact with mucus, maybe involving in maintainig omeostasis of gastro enteric epithelia. It will be necessary to demonstrate the hypothetical correlation between sarcoglycans and the maintaining epithelial homeostasis

Morpho-structural alterations of sub-chondral bone tissue in patients with osteoarthritis: a scanning electron microscopy study

Osteoarthritis focuses principally on the degeneration of articular cartilage as a primary cause of the disease. The pathophysiological process of osteoarthritis is characterized by alteration of chondrocytes and the increased bone formation by sub-chondral osteoblasts. Infiltration of macrophages and perivascular T and B lymphocytes is observed, and these infiltrates have been demonstrated in both early and advanced disease. The morphological and phenotypic characteristics of osteocytic cells attached to the normal and the osteoarthritic matrix differ from each other, suggesting that specific signalling pathways arise or are altered between matrix and cells. On this basis, we have examined biopsies of bone obtained by normal femur and by femur of subjects affected by osteoarthritis using techniques of scanning electron microscopy in order to identify the morphostructural alterations that occur in the sub-chondral bone. Our results have shown that the bone tissue of subjects not affected by any disease of bone presents a well-organized structure, while the bone tissue obtained by patients affected by osteoarthritis shows a derangement of tissue itself possibly correlated with altered function of the osteoblasts, that during the pathological process produce a less mineralized extracellular matrix with consequent loss of the normal bone structure. In our opinion, during the osteoarthritic process there would be a defective signalling between bone cells leading to the production of an irregular, amorphous extracellular matrix by osteoblasts, characteristic of the pathological condition

muscle specific integrins in masseter muscle fibers of chimpanzees an immunohistochemical study

Most notably, recent comparative genomic analyses strongly indicate that the marked differences between modern human and chimpanzees are likely due more to changes in gene regulation than to modifications of the genes. The most peculiar aspect of hominoid karyotypes is that human have 46 chromosomes whereas gorillas and chimpanzees have 48. Interestingly, human and chimpanzees do share identical inversions on chromosome 7 and 9 that are not evident in the gorilla karyotype. Thus, the general phylogeny suggests that humans and chimpanzees are sister taxa; based on this, it seems that human-chimpanzee sequence similarity is an astonishing 99%. At this purpose, of particular interest is the inactivation of the myosin heavy chain 16 (MYH16) gene, most prominently expressed in the masticatory muscle of mammals. It has been showed that the loss of this gene in humans may have resulted in smaller masticatory muscle and consequential changes to cranio-facial morphology and expansion of the human brain case. Powerful masticatory muscles are found in most primates; contrarily, in both modern and fossil member Homo, these muscles are considerably smaller. The evolving hominid masticatory apparatus shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. To better comprehend the real role of the MYH16 gene, we studied the primary proteins present in the muscle fibers of humans and non-humans, in order to understand if they really can be influenced by MYH16 gene. At this aim we examined the muscle-specific integrins, alpha 7B and beta 1D-integrins, and their relative fetal isoforms, alpha 7A and beta 1A-integrins, analyzing, by immunohistochemistry, muscle biopsies of two components of a chimpanzee's group in captivity, an alpha male and a non-alpha male subjects; all these integrins participate in vital biological processes such as maintenance of tissue integrity, embryonic development, cell differentiation, and cell-extracellular matrix interactions. Our results demonstrated a different quantitative composition of integrins, in alpha male in respect to human and non-alpha male, hypothesizing that the MYH16 gene could modify the expression of integrins, influencing, in turn, the phenotype of muscle. In this way, alpha 7A-and beta 1A-integrin could determine the presence of type II fibers and then they could play a key role in the determination of contraction force. Then, MYH16 gene could be a common interactor of signalling between sarcoglycans and integrins in chimpanzee muscles