Publications by authors named "Dorianna Sandonà"

22 Publications

  • Page 1 of 1

An Automated Low-Cost Swim Tunnel for Measuring Swimming Performance in Fish.

Zebrafish 2021 Apr 20. Epub 2021 Apr 20.

Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.

The study of swimming behavior is an important part of fish biology research and the swim tunnel is used to study swimming performance as well as metabolism of fish. In this investigation, we have developed a user-friendly, automated, modular, and low-cost swim tunnel that permits to study the performance of one or more fish separately, as well as a small group of individuals. To validate our swim tunnel, we assessed swimming activity of four different species (zebrafish, medaka, guppy, and cavefish) recording reliable data of swimming behavior and performance. Because swimming behavior has been recently used in different fields from physiology to ecotoxicology, our setup could help researchers with a low-cost solution.
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http://dx.doi.org/10.1089/zeb.2020.1975DOI Listing
April 2021

Non-genomic mechanisms in the estrogen regulation of glycolytic protein levels in endothelial cells.

FASEB J 2020 09 5;34(9):12768-12784. Epub 2020 Aug 5.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.

Few studies have explored the mechanisms coupling estrogen signals to metabolic demand in endothelial cells. We recently showed that 17β-estradiol (E2) triggers angiogenesis via the membrane G-protein coupled estrogen receptor (GPER) and the key glycolytic protein PFKFB3 as a downstream effector. We herein investigated whether estrogenic agents regulate the stability and/or degradation of glycolytic proteins in human umbilical vein endothelial cells (HUVECs). Similarly to E2, the GPER selective agonist G1 rapidly increased PFKFB3 protein amounts, without affecting mRNA levels. In the presence of cycloheximide, E2 and G1 treatment counteracted PFKFB3 degradation over time, whereas E2-induced PFKFB3 stabilization was abolished by the GPER antagonist G15. Inhibitors of selective SCF E3 ubiquitin ligase (SMER-3) and proteasome (MG132) rapidly increased PFKFB3 protein levels. Accordingly, ubiquitin-bound PFKFB3 was lower in E2- or G1-treated HUVECs. Both agents increased deubiquitinase USP19 levels through GPER signaling. Notably, USP 19 siRNA decreased PFKFB3 levels and abolished E2- and G1-mediated HUVEC tubularization. Finally, E2 and G1 treatments rapidly enhanced glucose transporter GLUT1 levels via GPER independent of transcriptional activation. These findings provide new evidence on mechanisms coupling estrogen signals with the glycolytic program in endothelium and unravel the role of USP19 as a target of the pro-angiogenic effect of estrogenic agents.
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http://dx.doi.org/10.1096/fj.202001130RDOI Listing
September 2020

Combined Use of CFTR Correctors in LGMD2D Myotubes Improves Sarcoglycan Complex Recovery.

Int J Mol Sci 2020 Mar 6;21(5). Epub 2020 Mar 6.

Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/b 35131 Padova, Italy.

Sarcoglycanopathies are rare limb girdle muscular dystrophies, still incurable, even though symptomatic treatments may slow down the disease progression. Most of the disease-causing defects are missense mutations leading to a folding defective protein, promptly removed by the cell's quality control, even if possibly functional. Recently, we repurposed small molecules screened for cystic fibrosis as potential therapeutics in sarcoglycanopathy. Indeed, cystic fibrosis transmembrane regulator (CFTR) correctors successfully recovered the defective sarcoglycan-complex in vitro. Our aim was to test the combined administration of some CFTR correctors with C17, the most effective on sarcoglycans identified so far, and evaluate the stability of the rescued sarcoglycan-complex. We treated differentiated myogenic cells from both sarcoglycanopathy and healthy donors, evaluating the global rescue and the sarcolemma localization of the mutated protein, by biotinylation assays and western blot analyses. We observed the additive/synergistic action of some compounds, gathering the first ideas on possible mechanism/s of action. Our data also suggest that a defective α-sarcoglycan is competent for assembly into the complex that, if helped in cell traffic, can successfully reach the sarcolemma. In conclusion, our results strengthen the idea that CFTR correctors, acting probably as proteostasis modulators, have the potential to progress as therapeutics for sarcoglycanopathies caused by missense mutations.
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http://dx.doi.org/10.3390/ijms21051813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084537PMC
March 2020

Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D.

Hum Mol Genet 2018 03;27(6):969-984

Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, β-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.
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http://dx.doi.org/10.1093/hmg/ddy013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886177PMC
March 2018

Inhibition of ubiquitin proteasome system rescues the defective sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein causing Chianina cattle pseudomyotonia.

J Biol Chem 2014 Nov 6;289(48):33073-82. Epub 2014 Oct 6.

Comparative Biomedicine and Food Science, University of Padova,35020 Legnaro (Padova), Italy,

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca(2+)-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca(2+) homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca(2+) concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.
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http://dx.doi.org/10.1074/jbc.M114.576157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246067PMC
November 2014

Unveiling the degradative route of the V247M α-sarcoglycan mutant responsible for LGMD-2D.

Hum Mol Genet 2014 Jul 23;23(14):3746-58. Epub 2014 Feb 23.

Department of Biomedical Sciences and

Many membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated into the cytosol and degraded by the proteasome. In applying rigid rules, however, quality control sometimes discharges proteins that, even though defective, retain their function. The unnecessary removal of such proteins represents the pathogenetic hallmark of diverse genetic diseases, in the case of ΔF508 mutant of cystic fibrosis transmembrane conductance regulator being probably the best known example. Recently, the inappropriate proteasomal degradation of skeletal muscle sarcoglycans (α, β, γ and δ) with missense mutation has been proposed to be at the bases of mild-to-severe forms of limb girdle muscular dystrophy (LGMD) known as type 2D, 2E, 2C and 2F, respectively. The quality control pathway responsible for sarcoglycan mutant disposal, however, is so far unexplored. Here we reveal key components of the degradative route of V247M α-sarcoglycan mutant, the second most frequently reported mutation in LGMD-2D. The disclosure of the pathway, which is led by the E3 ligases HRD1 and RFP2, permits to identify new potential druggable targets of a disease for which no effective therapy is at present available. Notably, we show that the pharmacological inhibition of HRD1 activity rescues the expression of V247-α-sarcoglycan both in a heterologous cell model and in myotubes derived from a LGMD-2D patient carrying the L31P/V247M mutations. This represents the first evidence that the activity of E3 ligases, the enzymes in charge of mutant fate, can be eligible for drug interventions to treat sarcoglycanopathy.
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http://dx.doi.org/10.1093/hmg/ddu088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065151PMC
July 2014

Effects of pleiotrophin overexpression on mouse skeletal muscles in normal loading and in actual and simulated microgravity.

PLoS One 2013 28;8(8):e72028. Epub 2013 Aug 28.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, Bari, Italy.

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756024PMC
May 2014

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.

PLoS One 2012 28;7(3):e33232. Epub 2012 Mar 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314659PMC
August 2012

TAT-mediated aequorin transduction: an alternative approach for effective calcium measurements in plant cells.

Plant Cell Physiol 2011 Dec 24;52(12):2225-35. Epub 2011 Oct 24.

Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B, 35131 Padova, Italy.

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.
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http://dx.doi.org/10.1093/pcp/pcr145DOI Listing
December 2011

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation.

Mol Cell Biochem 2011 May 10;351(1-2):183-96. Epub 2011 Feb 10.

Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.
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http://dx.doi.org/10.1007/s11010-011-0726-4DOI Listing
May 2011

Sphingosine 1-phosphate signaling is involved in skeletal muscle regeneration.

Am J Physiol Cell Physiol 2010 Mar 30;298(3):C550-8. Epub 2009 Dec 30.

Dept. of Human Anatomy and Physiology, Univ. of Padova, Via Marzolo 3, 35131 Padua, Italy.

Sphingosine 1-phosphate (S1P) is a bioactive lipid known to control cell growth that was recently shown to act as a trophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1P(1) and S1P(3) are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1P(1) receptor, while S1P(3) expression progressively decreased to adult levels. S1P(2) receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of S1P (50 microM) at the moment of myotoxic injury caused a significant increase of the mean cross-sectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating S1P by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1P(1) receptor negatively and S1P(3) receptor positively modulate the early phases of regeneration, whereas S1P(2) receptor appears to be less important. The present results show that S1P signaling participates in the regenerative processes of skeletal muscle.
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http://dx.doi.org/10.1152/ajpcell.00072.2009DOI Listing
March 2010

Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects.

Expert Rev Mol Med 2009 Sep 28;11:e28. Epub 2009 Sep 28.

Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy.

Sarcoglycanopathies are a group of autosomal recessive muscle-wasting disorders caused by genetic defects in one of four cell membrane glycoproteins, alpha-, beta-, gamma- or delta-sarcoglycan. These four sarcoglycans form a subcomplex that is closely linked to the major dystrophin-associated protein complex, which is essential for membrane integrity during muscle contraction and provides a scaffold for important signalling molecules. Proper assembly, trafficking and targeting of the sarcoglycan complex is of vital importance, and mutations that severely perturb tetramer formation and localisation result in sarcoglycanopathy. Gene defects in one sarcoglycan cause the absence or reduced concentration of the other subunits. Most genetic defects generate mutated proteins that are degraded through the cell's quality control system; however, in many cases, conformational modifications do not affect the function of the protein, yet it is recognised as misfolded and prematurely degraded. Recent evidence shows that misfolded sarcoglycans could be rescued to the cell membrane by assisting their maturation along the ER secretory pathway. This review summarises the etiopathogenesis of sarcoglycanopathies and highlights the quality control machinery as a potential pharmacological target for therapy of these genetic disorders.
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http://dx.doi.org/10.1017/S1462399409001203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279956PMC
September 2009

Inhibition of proteasome activity promotes the correct localization of disease-causing alpha-sarcoglycan mutants in HEK-293 cells constitutively expressing beta-, gamma-, and delta-sarcoglycan.

Am J Pathol 2008 Jul 5;173(1):170-81. Epub 2008 Jun 5.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, alpha-, beta-, gamma-, and delta-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (beta, gamma, and delta) of the four sarcoglycans. In these betagammadelta-HEK cells, the lack of alpha-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of alpha-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of betagammadelta-HEK cells with disease-causing alpha-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies.
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http://dx.doi.org/10.2353/ajpath.2008.071146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2438295PMC
July 2008

Trophic action of sphingosine 1-phosphate in denervated rat soleus muscle.

Am J Physiol Cell Physiol 2008 Jan 17;294(1):C36-46. Epub 2007 Oct 17.

Department of Human Anatomy and Physiology, University of Padua, Padua, Italy.

Sphingosine 1-phosphate (S1P) mediates a number of cellular responses, including growth and proliferation. Skeletal muscle possesses the full enzymatic machinery to generate S1P and expresses the transcripts of S1P receptors. The aim of this work was to localize S1P receptors in rat skeletal muscle and to investigate whether S1P exerts a trophic action on muscle fibers. RT-PCR and Western blot analyses demonstrated the expression of S1P(1) and S1P(3) receptors by soleus muscle. Immunofluorescence revealed that S1P(1) and S1P(3) receptors are localized at the cell membrane of muscle fibers and in the T-tubule membranes. The receptors also decorate the nuclear membrane. S1P(1) receptors were also present at the neuromuscular junction. The possible trophic action of S1P was investigated by utilizing the denervation atrophy model. Rat soleus muscle was analyzed 7 and 14 days after motor nerve cut. During denervation, S1P was continuously delivered to the muscle through a mini osmotic pump. S1P and its precursor, sphingosine (Sph), significantly attenuated the progress of denervation-induced muscle atrophy. The trophic effect of Sph was prevented by N,N-dimethylsphingosine, an inhibitor of Sph kinase, the enzyme that converts Sph into S1P. Neutralization of circulating S1P by a specific antibody further demonstrated that S1P was responsible for the trophic effects of S1P during denervation atrophy. Denervation produced the down regulation of S1P(1) and S1P(3) receptors, regardless of the presence of the receptor agonist. In conclusion, the results suggest that S1P acts as a trophic factor of skeletal muscle.
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http://dx.doi.org/10.1152/ajpcell.00164.2007DOI Listing
January 2008

Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells.

Blood 2007 May 27;109(9):3856-64. Epub 2006 Dec 27.

Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, I-44100 Ferrara, Italy.

Dendritic cells (DCs) are professional antigen-presenting cells that initiate the immune response by activating T lymphocytes. DCs express plasma membrane receptors for extracellular nucleotides named P2 receptors (P2Rs). Stimulation of P2Rs in these cells is known to cause chemotaxis, cytokine release, and cell death and to modulate LPS-dependent differentiation. Here we show that stimulation of the P2X(7) receptor subtype (P2X(7)R) causes fast microvesicle shedding from DC plasma membrane. Vesicle release occurs from both immature and mature DCs; however, only vesicles from mature DCs, due to their previous exposure to LPS, contain IL-1beta. Microvesicles, whether from immature or mature DCs, also contain caspase-1 and -3 and cathepsin D. They also express the P2X(7)R in addition to other P2Rs and known markers of immune cells such as major histocompatibility complex II (MHC II) and CD39. Activation of the P2X(7)R by extracellular ATP causes IL-1beta release from the vesicle lumen. Previous studies demonstrated that high extracellular K(+) inhibits IL-1beta processing and release; here we show that high ionic strength reduces microvesicle shedding when compared with a low ionic strength medium but strongly increases microvesicle IL-1beta loading.
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http://dx.doi.org/10.1182/blood-2005-06-031377DOI Listing
May 2007

Transition of Homer isoforms during skeletal muscle regeneration.

Am J Physiol Cell Physiol 2006 Mar 19;290(3):C711-8. Epub 2005 Oct 19.

Dipartimento di Scienze Biomediche Sperimentali, Università degli Studi di Padova, viale G. Colombo 3, 35121 Padova, Italy.

Homer represents a new and diversified family of proteins that includes several isoforms, Homer 1, 2, and 3; some of these isoforms have been reported to be present in striated muscles. In this study, the presence of Homer isoforms 1a, 1b/c/d, 2b, and 3 was thoroughly investigated in rat skeletal muscles under resting conditions. Transition in Homer isoforms compositon was studied under experimental conditions of short-term and long-term adaptation, e.g., fatigue and regeneration, respectively. First, we show that Homer 1a was constitutively expressed and was transiently upregulated during regeneration. In C(2)C(12) cell cultures, Homer 1a was also upregulated during formation of myotubes. No change of Homer 1a was observed in fatigue. Second, Homer 1b/c/d and Homer 2b were positively and linearly related to muscle mass change during regeneration, and third, Homer 3 was not detectable under resting conditions but was transiently expressed during regeneration although with a temporal pattern distinct from that of Homer 1a. Thus a switch in Homer isoforms is associated to muscle differentiation and regeneration. Homers may play a role not only in signal transduction of skeletal muscle, in particular regulation of Ca(2+) release from sarcoplasmic reticulum, but also in adaptation.
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http://dx.doi.org/10.1152/ajpcell.00217.2005DOI Listing
March 2006

Deficiency of alpha-sarcoglycan differently affects fast- and slow-twitch skeletal muscles.

Am J Physiol Regul Integr Comp Physiol 2005 Nov 7;289(5):R1328-37. Epub 2005 Jul 7.

Department of Human Anatomy and Physiology, University of Padova, Italy.

Alpha-sarcoglycan (Sgca) is a transmembrane glycoprotein of the dystrophin complex located at skeletal and cardiac muscle sarcolemma. Defects in the alpha-sarcoglycan gene (Sgca) cause the severe human-type 2D limb girdle muscular dystrophy. Because Sgca-null mice develop progressive muscular dystrophy similar to human disorder they are a valuable animal model for investigating the physiopathology of the disorder. In this study, biochemical and functional properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of the Sgca-null mice were analyzed. EDL muscle of Sgca-null mice showed twitch and tetanic kinetics comparable with those of wild-type controls. In contrast, soleus muscle showed reduction of twitch half-relaxation time, prolongation of tetanic half-relaxation time, and increase of maximal rate of rise of tetanus. EDL muscle of Sgca-null mice demonstrated a marked reduction of specific twitch and tetanic tensions and a higher resistance to fatigue compared with controls, changes that were not evident in dystrophic soleus. Contrary to EDL fibers, soleus muscle fibers of Sgca-null mice distinctively showed right shift of the pCa-tension (pCa is the negative log of Ca2+ concentration) relationships and reduced sensitivity to caffeine of sarcoplasmic reticulum. Both EDL and soleus muscles showed striking changes in myosin heavy-chain (MHC) isoform composition, whereas EDL showed a larger number of hybrid fibers than soleus. In contrast to the EDL, soleus muscle of Sgca-null mice contained a higher number of regenerating fibers and thus higher levels of embryonic MHC. In conclusion, this study revealed profound distinctive biochemical and physiological modifications in fast- and slow-twitch muscles resulting from alpha-sarcoglycan deficiency.
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http://dx.doi.org/10.1152/ajpregu.00673.2004DOI Listing
November 2005

The T-tubule membrane ATP-operated P2X4 receptor influences contractility of skeletal muscle.

FASEB J 2005 Jul 26;19(9):1184-6. Epub 2005 Apr 26.

Department of Biomedical and Experimental Sciences, University of Padova, Padova, Italy.

Evidence indicates that extracellular ATP may have relevant functions in skeletal muscle, even though the physiological role and distribution of specific signaling pathway elements are not well known. The present work shows that P2X4 receptor, an extracellular ATP-regulated cell membrane channel permeable to Ca2+, is expressed in several tissues of the rat, including skeletal muscle. A specific antibody detected a protein band of approximately 60 kDa. Immunofluorescence demonstrated that P2X4 has an intracellular localization, and confocal analysis revealed that the receptor colocalizes with the T-tubule membrane DHP receptor. Considering that the natural agonist of P2X4 is ATP, we explored if changes of extracellular ATP levels could occur in contracting skeletal muscle to regulate the channel. In vitro experiments showed that substantial ATP is released and rapidly hydrolyzed after electrical stimulation of rat muscle fibers. Results show that the presence of ATP-degrading enzymes (hexokinase/apyrase), inhibitors of P2X receptors or Ca2+-free conditions, all abolished the progressive twitch tension potentiation produced in soleus muscle by low-frequency (0.05 Hz) stimulation. These data reveal that ATP-mediated Ca2+ entry, most likely through P2X4 receptor, may play an important role in modulating the contractility of skeletal muscle.
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http://dx.doi.org/10.1096/fj.04-3333fjeDOI Listing
July 2005

Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix.

Cell Calcium 2005 Feb;37(2):129-36

Department of Biomedical Sciences, CNR Institute of Neurosciences, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.

The role of mitochondria in Ca2+ handling has acquired renewed interest in recent years in the field of cell signaling. Detailed studies of Ca2+ dynamics in this organelle at the single cell level have been hampered by technical problems in the available Ca2+ probes. Some of the latest generation GFP-based Ca2+ probes (Camgaroos, Cameleons and Pericams) show great potential to address this issue. Our data show that the choice of targeting sequence influences not only the overall efficiency of subcellular localization of the probes, but also their functional characteristics within the matrix. In particular, we here show that the use of a tandemly duplicated mitochondrial targeting sequence is capable of improving the delivery efficacy of all tested probes into the organelle's matrix, in particular that of Cameleon, a GFP-based Ca2+ probe that is otherwise largely mistargeted to the cytosol. The devised strategy should be generally applicable to other proteins that are characterized by poor targeting. Last, but not least, we also demonstrate that if the targeting sequence is not removed from the imported protein, the fluorescent properties and the Ca2+ affinity of the probe can be grossly affected.
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http://dx.doi.org/10.1016/j.ceca.2004.08.002DOI Listing
February 2005

Characterization of the ATP-hydrolysing activity of alpha-sarcoglycan.

Biochem J 2004 Jul;381(Pt 1):105-12

Department of Biomedical Sciences, University of Padova, Viale G. Colombo, 3, 35121 Padova, Italy.

Alpha-Sarcoglycan is a glycoprotein associated with the dystrophin complex at sarcolemma of skeletal and cardiac muscles. Gene defects in alpha-sarcoglycan lead to a severe muscular dystrophy whose molecular mechanisms are not yet clear. A first insight into the function of alpha-sarcoglycan was obtained by finding that it is an ATP-binding protein and that it probably confers ability to hydrolyse ATP to the purified dystrophin complex [Betto, Senter, Ceoldo, Tarricone, Biral and Salviati (1999) J. Biol. Chem. 274, 7907-7912]. In the present study, we present definitive evidence showing that alpha-sarcoglycan is an ATP-hydrolysing enzyme. The appearance of alpha-sarcoglycan protein expression was correlated with the increase in ecto-nucleotidase activity during differentiation of C2C12 cells. Approx. 25% of ecto-nucleotidase activity displayed by the C2C12 myotubes was inhibited by preincubating cells with an antibody specific for the ATP-binding motif of alpha-sarcoglycan. This demonstrates that alpha-sarcoglycan substantially contributes to total ecto-nucleotidase activity of C2C12 myotubes. To characterize further this activity, human embryonic kidney 293 cells were transfected with expression plasmids containing alpha-sarcoglycan cDNA. Transfected cells exhibited a significant increase in the ATP-hydrolysing activity that was abolished by the anti-alpha-sarcoglycan antibody. The enzyme had a substrate specificity for ATP and ADP, did not hydrolyse other triphosphonucleosides, and the affinity for ATP was in the low mM range. The ATPase activity strictly required the presence of both Mg2+ and Ca2+ and was completely inhibited by suramin and reactive blue-2. These results show that alpha-sarcoglycan is a Ca2+, Mg2+-ecto-ATPDase. The possible consequences of the absence of alpha-sarcoglycan activity in the pathogenesis of muscular dystrophy are discussed.
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http://dx.doi.org/10.1042/BJ20031644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133767PMC
July 2004

Subcellular distribution of Homer 1b/c in relation to endoplasmic reticulum and plasma membrane proteins in Purkinje neurons.

Neurochem Res 2003 Aug;28(8):1151-8

Dipartimento di Scienze Biomediche Sperimentali dell'Università degli Studi di Padova, viale G. Colombo 3, 35121 Padova, Italy.

The subcellular distribution of endoplasmic reticulum proteins (IP3R1 and RYR), plasma membrane (PM) proteins (mGluR1 and PMCA Ca(2+)-pump), and scaffolding proteins, such as Homer 1b/c, was assessed by laser scanning confocal microscopy of rat cerebellum parasagittal sections. There appeared to be two classes of Ca2+ stores, nonjunctional Ca2+ stores and junctional Ca2+ stores, possibly referable to central cisternae/tubules and sub-PM cisternae, respectively, in soma, dendrites, and dendritic spines. Only some IP3R1s appeared to be part of multimeric, junctional Ca2+ signaling networks, whose composition is shown to include PMCA, mGluR1, Homer 1b/c and, not always, RYR1.
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http://dx.doi.org/10.1023/a:1024264025401DOI Listing
August 2003

A structural investigation of the central chlorophyll a binding sites in the minor photosystem II antenna protein, Lhcb4.

Biochemistry 2002 Feb;41(7):2305-10

Università di Verona, Facoltà di Scienze MM.FF.NN., Biotechnologie Vegetali, Strada Le Grazie, I-37134 Verona, Italy.

Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10056-10061]. The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations. By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides.
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http://dx.doi.org/10.1021/bi015639+DOI Listing
February 2002