The synaptic vesicle proteins synapsin I and synaptophysin (protein P38) are concentrated both in efferent and afferent nerve endings of the skeletal muscle

Synapsin I and synaptophysin (protein p38) are 2 major protein components of the membranes of small synaptic vesicles of virtually all presynaptic nerve endings. Synapsin I, a phosphoprotein regulated by both Ca2+ and cAMP, is a peripheral protein of the cytoplasmic surface of the vesicle membrane. It is thought to anchor the vesicle surface to the cytoskeleton of the terminal and to play a regulatory role in neurotransmitter release. Synaptophysin is an intrinsic transmembrane glycoprotein. We report here that both proteins are present and concentrated also in afferent nerve endings, which provide the sensory innervation of the skeletal muscle and of the tendon. The distribution of both antigens in sensory nerve endings is consistent with their localization on the microvesicles that have been described in such endings. Thus, our results suggest the existence of important biochemical, and possibly functional, similarities between small synaptic vesicles of presynaptic nerve endings and microvesicles of sensory endings. Such findings provide new clues to the understanding of the physiology of sensory endings

Optimization of Naked DNA Delivery for Interferon Subtype Immunotherapy in Cytomegalovirus Infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200mg IFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMV gB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases

cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis

Reduced amount of the glucose-regulated protein GRP94 in skeletal myoblasts results in loss of fusion competence

We previously showed that skeletal myocytes of the adult rabbit do not accumulate the endoplasmic reticulum glucose-regulated protein GRP94, neither constitutively nor inducibly, at variance with skeletal myocytes during perinatal development (5), Here we show that C2C12 cells upregulate GRP94 during differentiation and, similarly to primary cultures of murine skeletal myocytes, specifically display GRP94 immunoreactivity on the cell surface. Stable transfection of C2C12 cells with grp94 antisense cDNA shows lack of myotube formation in clones displaying >40% reduction in GRP94 amount. The same result is obtained after irt vivo injection of grp94-antisense myoblasts, Conversely, GRP94 overexpression is accompanied by accelerated myotube formation. Analyses of BrdU incorporation, p21 nuclear translocation, and muscle-gene expression show that muscle differentiation is not apparently affected in grp94-antisense clones. In contrast, cell-surface GRP94 is greatly reduced in grp94-antisense clones, as shown by immunocytochemistry and precipitation of cell-surface biotinylated proteins. Thus, cell-surface expression of GRP94 is necessary for maintenance of fusion competence. Furthermore, differentiating C2C12 cells grown in the presence of anti-GRP94 antibody show decreased myotube number suggesting that cell-surface GRP94 is directly involved in myoblast fusion process

Grp94 (HSP90B1)

nessun abstract disponibil