Biomedical subjects

R Barresi

Publications and source records attributed to R Barresi.

At least 19 recordsLinked to original sources

PubMed2000

Expression of gamma -sarcoglycan in smooth muscle and its interaction with the smooth muscle sarcoglycan-sarcospan complex.

The sarcoglycan complex in striated muscle is a heterotetrameric unit integrally associated with sarcospan in the dystrophin-glycoprotein complex. The sarcoglycans, alpha, beta, gamma, and delta, are mutually dependent with regard to their localization at the sarcolemma, and mutations in any of the sarcoglycan genes lead to limb-girdle muscular dystrophies type 2C-2F. In smooth muscle beta- and delta-sarcoglycans are associated with epsilon-sarcoglycan, a glycoprotein homologous to alpha-sarcoglycan. Here, we demonstrate that gamma-sarcoglycan is also a component of the sarcoglycan complex in the smooth muscle. First, we show the presence of gamma-sarcoglycan in a number of smooth muscle-containing organs, and we verify the existence of identical transcripts in skeletal and smooth muscle. The specificity of the expression of gamma-sarcoglycan in smooth muscle was confirmed by analysis of smooth muscle cells in culture. Next, we provide evidence for the association of gamma-sarcoglycan with the sarcoglycan-sarcospan complex by biochemical analysis and comparison among animal models for muscular dystrophy. Moreover, we find disruption of the sarcoglycan complex in the vascular smooth muscle of a patient with gamma-sarcoglycanopathy. Taken together, our results prove that the sarcoglycan complex in vascular and visceral smooth muscle consists of epsilon-, beta-, gamma-, and delta-sarcoglycans and is associated with sarcospan.

Amino Acid Sequence↗

PubMed2000

PCR testing for HCV in anti-HCV negative blood donors involved in the so called HCV +ve post-transfusion hepatitis.

UNLABELLED: Although it is infrequent, post-transfusion HCV infection may occur if the donors blood is collected in the window period between exposure and anti-HCV detectability by ELISA testing. STUDY DESIGN: In these last years, despite of routine application of anti-HCV testing, our blood transfusion center has been involved in 53 cases of alleged post-transfusion HCV hepatitis and look-back programs were set up with the goal of finding out the donors possibly involved in viral transmission. Most of these patients were hematological cases with multiple transfusions given because of aplastic anemia (3 cases), leukemia with or without bone marrow transplantation (5/4 cases) but necessitating long-term platelet support, leukemia and solid cancer patients undergoing autologous PBSC transplantation (3/4 cases) and TTP (2 cases). Only 32 patients were of the simple medical or surgical type, 9 transfused because of cardiac or vascular surgery, 8 because of spine surgery, 5 for different diseases and 5 for different types of cancer surgery. Donor's infectivity was determined by ELISA anti-HCV testing, by recombinant immunoblotting assay, and by nucleic acid testing. RESULTS AND CONCLUSION: No donors out of 267 traced of a total of 359 involved was found with anti-HCV seroconversion, or positive on PCR testing. This suggests that the responsibility for HCV transmission can only hypothetically be related to blood or blood components and that other transmission routes should be found out.

Blood Donors↗

PubMed2000

Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.

Two young males with limb-girdle muscular dystrophy (LGMD) resulting from sarcoglycan deficiency died at 27 (patient 1) and 18 years (patient 2) of severe cardiomyopathy. Genetic analysis showed that they were compound heterozygotes for mutations in the beta sarcoglycan gene. One of these mutations, an 8 bp duplication in exon 3, was common to both patients. The second mutation in patient 2 was a 4 bp deletion at the splice donor site of intron 2, not reported previously. Patient 2 had more severe heart and skeletal muscle defects with faster deterioration; no sarcoglycans were detected in his skeletal muscle. The second mutation in patient 1, inferred because the unaffected father carries the 8 bp duplication, was not found. In patient 1, both heart and skeletal muscle were analysed and showed reduction of all sarcoglycans in both tissues and incorrect localisation of alpha and gamma sarcoglycans in heart. Therefore mutations in one sarcoglycan gene can disrupt the entire sarcoglycan complex in both skeletal and cardiac muscle. Differing expression patterns of sarcoglycan components in heart and skeletal muscle could be the result of alternatively spliced transcripts in these tissues. By sequencing an alternative transcript, highly expressed in the heart and skeletal muscle of patient 1, we found an 87 bp cryptic exon not previously reported. Although cardiomyopathy can result from mutations in alpha and gamma sarcoglycans, we show for the first time that the condition can also be caused by mutations in the beta sarcoglycan gene. This report therefore expands the phenotype of sarcoglycanopathies and suggests that cardiac function in LGMD patients with defective sarcoglycan expression should be monitored.

Adolescent↗

PubMed1999

Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.

To investigate mechanisms in the pathogenesis of cardiomyopathy associated with mutations of the dystrophin-glycoprotein complex, we analyzed genetically engineered mice deficient for either alpha-sarcoglycan (Sgca) or delta-sarcoglycan (Sgcd). We found that only Sgcd null mice developed cardiomyopathy with focal areas of necrosis as the histological hallmark in cardiac and skeletal muscle. Absence of the sarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiac membranes was observed in both animal models. Loss of vascular smooth muscle SG-SSPN complex was only detected in Sgcd null mice and associated with irregularities of the coronary vasculature. Administration of a vascular smooth muscle relaxant prevented onset of myocardial necrosis. Our data indicate that disruption of the SG-SSPN complex in vascular smooth muscle perturbs vascular function, which initiates cardiomyopathy and exacerbates muscular dystrophy.

Animals↗

PubMed1997

X-linked Emery-Dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

Adolescent↗

PubMed1997

Concomitant deficiency of beta- and gamma-sarcoglycans in 20 alpha-sarcoglycan (adhalin)-deficient patients: immunohistochemical analysis and clinical aspects.

We have investigated the expression, using immunohistochemistry, of beta- and gamma-sarcoglycans in the muscles of 20 patients in whom previous screening had revealed a deficiency of alpha-sarcoglycan. alpha-, beta- and gamma-sarcoglycans were absent in 7 patients and variably reduced in 8 patients, in 2 of whom beta-sarcoglycan was more reduced than the alpha- and gamma-proteins. In 5 other patients with variably reduced alpha- and beta-sarcoglycans, gamma-sarcoglycan was completely absent. In all patients the distribution of hyposthenia at disease onset was similar, and predominantly involved pelvic girdle muscles; however, the age at onset and rate of disease progression were highly variable. In severely compromised patients, the onset of disease was before 10 years of age and gamma-sarcoglycan or all three sarcoglycans were absent from muscles. Immunohistochemical analysis of sarcoglycans should be part of routine screening for muscle dystrophies to identify patients with sarcoglycanopathy. Gene analysis is necessary to identify the primary defect; however, sarcoglycan immunohistochemistry may be useful for indicating which gene to investigate. Further biochemical characterization of the interactions between these proteins is required to fully elucidate their roles in causing severe, moderate or mild muscular dystrophy.

Adolescent↗

PubMed1997

Heart-specific localization of emerin: new insights into Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked inherited disease characterized by early contracture of the elbows, Achilles tendons and post-cervical muscles, slow progressive muscle wasting and weakness and cardiomyopathy presenting with arrhythmia and atrial paralysis: heart block can eventually lead to sudden death. The EDMD geneencodes a novel ubiquitous protein, emerin, which decorates the nuclear rim of many cell types. Amino acid sequence homology and cellular localization suggested that emerin is a member of the nuclear lamina-associated protein family. These findings did not explain the role of emerin nor account for the skeletal muscle- and heart-specific clinical manifestations associated with the disorder. Now we report that emerin localizes to the inner nuclear membrane, via its hydrophobic C-terminal domain, but that in heart and cultured cardiomyocytes it is also associated with the intercalated discs. We propose a general role for emerin in membrane anchorage to the cytoskeleton. In the nuclear envelope emerin plays a ubiquitous and dispensable role in association of the nuclear membrane with the lamina. In heart its specific localization to desmosomes and fasciae adherentes could account for the characteristic conduction defects described in patients.

Arrhythmias, Cardiac↗

PubMed1996

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle.

Dystrophin, utrophin and the dystrophin-associated glycoproteins, beta-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins beta-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than beta-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12-16 weeks. From 8 weeks all fetal myotubes expressed beta-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.

Blotting, Western↗

PubMed1996

Clinical heterogeneity of adhalin deficiency.

We report adhalin deficiency in 8 patients with clinically diagnosed muscular dystrophy, dystrophic histopathological features, high plasma creatine kinase levels, normal expression of dystrophin, and marked variability of symptoms. Although the distribution of hyposthenia was similar in all 8 patients and predominantly involved muscles in the pelvic girdle, age at onset and rate of disease progression were highly variable: In 2 patients onset, at ages 24 and 25, was later than has been previously observed. We found no apparent relation between disease severity and the quantity of adhalin expressed. Two kinds of myopathy with adhalin deficiency have been reported: one caused by a mutation in the adhalin gene on chromosome 17 (primary adhalinopathy) and the other linked to chromosome 13. The product of the gene on chromosome 13 is probably associated with adhalin and its deficiency results in secondary adhalinopathy. The severity of clinical phenotypes in these adhalinopathies seems to relate more to the kind and site of the mutations than to the residual amount of the protein. We also detected a variable reduction in the laminin beta 1 subunit by immunohistochemistry in most patients, confirming that this is commonly associated with adhalin deficiency.

Adolescent↗

PubMed1996

Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers.

The absence of dystrophin in muscle fibers is associated with a major reduction in dystrophin-associated proteins (DAPs) and disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. We investigated the expression of the DAPs beta-dystroglycan, alpha-sarcoglycan, gamma-sarcoglycan and syntrophin as well as utrophin in the muscles of 13 Duchenne muscular dystrophy (DMD) carriers (with variable percentages of dystrophin-deficient fibers and with a range of clinical symptoms), 2 Becker muscular dystrophy (BMD) carriers (expressing a highly truncated protein in some fibers), 2 girls with a DMD-like phenotype, and 11 BMD carriers with almost normal dystrophin expression (reduced or patchy distribution in a few fibers only and rare dystrophin-deficient fibers). DAPs were highly reduced in all fibers lacking dystrophin in the DMD carriers, but were almost normal in the dystrophin-deficient fibers of the 2 BMD carriers with highly truncated dystrophin. In the 11 BMD carriers with nearly normal dystrophin, the few fibers with reduced or patchy dystrophin immunostaining also showed reduced DAP expression in correlation with dystrophin expression. Immunoblot for beta-dystroglycan and alpha-sarcoglycan confirmed the immunohistochemical findings. Utrophin expression was slightly increased in a proportion of fibers in the DMD and BMD carriers with dystrophin mosaicism. We found no correlation between utrophin expression and DAP expression. We conclude that absence or reduction of dystrophin in muscle fibers of DMD and BMD carriers causes a reduction of DAPs in the same fibers, as observed in DMD and BMD patients, while utrophin does not seem to play a role in DAP expression in adult muscle.

Adolescent↗

PubMed1996

Major histocompatibility complex class II molecule expression on muscle cells is regulated by differentiation: implications for the immunopathogenesis of muscle autoimmune diseases.

Major histocompatibility complex (MHC) class II molecules are expressed on myoblasts after interferon-gamma (IFN-gamma) treatment, suggesting a muscle cell involvement in antigen presentation in inflammatory myopathies. However, they were not observed on normal or pathological myofibers. This discrepancy might be related to different responsiveness of developmentally differentiated muscle cells to IFN-gamma. Myoblasts expressed class II transcripts and proteins after IFN-gamma, while myotubes and innervated contracting muscle cells did not show staining for class II molecules. At all cell stages no loss of IFN-gamma receptor was detected indicating that myofiber maturation blocks their capacity to express MHC class II molecules. This suggests that completely differentiated myofibers cannot participate in class II restricted immunological reactions.

Actins↗

PubMed1996

Mild clinical phenotype in a 12-year-old boy with partial merosin deficiency and central and peripheral nervous system abnormalities.

We found partial merosin deficiency in a boy presenting at 12 yr with marked limb weakness and a waddling gait. Magnetic resonance imaging (MRI) showed the characteristic white matter abnormalities of merosin-negative congenital muscular dystrophy. There were also peripheral demyelinating polyneuropathy and evoked potential abnormalities. Unlike classic merosin-negative congenital muscular dystrophy, however, our patient was less hypotonic and weak and was able to achieve independent walking. Both by immunohistochemistry and Western blot merosin was shown to be moderately reduced. By immunostaining the alpha 1 laminin chain was overexpressed and beta 1 laminin chain was reduced. A spectrum of clinical phenotypes is likely to become evident in merosin-deficient patients in relation to the discovery of a range of molecular defects in, and variable expression of, this protein.

Central Nervous System Diseases↗

PubMed1995

Dystrophin characterization in BMD patients: correlation of abnormal protein with clinical phenotype.

We have investigated protein expression and genotype in 59 Becker muscular dystrophy (BMD) patients. The aim was to identify possible causes of the marked variability in phenotype in patients with similar deletions/mutations. The patients were examined neurologically and functionally and underwent Manual Muscle Testing. Dystrophin expression was analysed by immunohistochemistry and western blot using antibodies against six different segments of the protein. DNA mutations were investigated by PCR amplification of 30 exons. Based on dystrophin expression at the sarcolemma, two groups of patients were identified: group A (29 patients) with the classic patchy distribution of dystrophin and group B (30 patients) with absence or reduction of one or more dystrophin portions and variable, although mostly normal, expression of the other portions of the protein. Dystrophin molecular weight was normal or slightly reduced in group A and was variably reduced, generally conspicuously so, in group B. The quantity of dystrophin expressed varied markedly in both groups. The pattern of immunohistochemical staining in group B patients correlated with milder clinical phenotype, suggesting that small dystrophin molecules lacking a portion in the N-terminus or in the rod domain, are more functional than proteins with normal or slightly reduced molecular weight that display the BMD-typical patchy distribution at the sarcolemma.

Activities of Daily Living↗

PubMed1995

Expression of transforming growth factor-beta 1 in dystrophic patient muscles correlates with fibrosis. Pathogenetic role of a fibrogenic cytokine.

Duchenne muscular dystrophy is a fatal disorder characterized by progressive muscular weakness, wasting, and severe muscle contractures in later disease stages. Muscle biopsy reveals conspicuous myofiber degeneration and fibrosis substituting muscle tissue. We quantitatively determined mRNA of the potent fibrogenic cytokine transforming growth factor-beta 1 by quantitative PCR in 15 Duchenne muscular dystrophy, 13 Becker muscular dystrophy, 11 spinal muscular atrophy patients, and 16 controls. Higher transforming growth factor-beta 1 expression was greater in Duchenne muscular dystrophy patients than controls (P = 0.012) and Becker patients (P = 0.03). Fibrosis was significantly more prominent in Duchenne muscular dystrophy than Becker muscular dystrophy, spinal muscular atrophy, and controls. The proportion of connective tissue in muscle biopsies increased progressively with age in Duchenne muscular dystrophy patients, while transforming growth factor-beta 1 levels peaked at 2 and 6 yr of age. Transforming growth factor-beta 1 protein was also detected by immunocytochemistry and immunoblotting. Our findings suggest that transforming growth factor-beta 1 stimulates fibrosis in Duchenne muscular dystrophy. Expression of transforming growth factor-beta 1 in the early stages of Duchenne muscular dystrophy may be critical in initiating muscle fibrosis and antifibrosis treatment could slow progression of the disease, increasing the utility of gene therapy.

Age Factors↗

PubMed1994

A negative cis-acting G-fer element participates in the regulation of expression of the human H-ferritin-encoding gene (FERH).

Ferritin (Fer) is the major iron storage protein in man. Its synthesis is regulated both at the translational and transcriptional levels. In previous studies on transcriptional regulation of the human H-ferritin-encoding gene (FERH), a 160-bp promoter segment was analyzed [Bevilacqua et al., Gene 111 (1992) 255-260]. In order to obtain a more complete view of the elements involved in the transcriptional regulation of FERH, we have studied, in a further upstream region of the human FERH promoter (pFERH), a sequence between -272 and -291, named G-fer, because it contains a stretch of ten G, which binds a nuclear factor present in different cell types. DNA-binding assays and competition experiments suggest that the factor binding to G-fer has binding properties very similar to inhibitory factor-1 (IF-1), an ubiquitous factor that interacts with G-rich elements in the promoters of the mouse type-I collagen genes. DNA transfection experiments in HeLa cells, using either a wild-type or mutated pFERH fused to a reporter gene, showed that a 3-bp substitution mutation, that abolished the binding of the specific factor to G-fer, increased the promoter activity, thus suggesting an inhibitory role for the G-fer element and its cognate trans-acting factor.

Binding Sites↗

PubMed1994

Fetus-like dystrophin expression and other cytoskeletal protein abnormalities in centronuclear myopathies.

We have investigated supposed maturational arrest of muscle in centronuclear myopathies (CNMs) by characterizing the expression of dystrophin, other cytoskeletal proteins, and fetal myosin in the muscle fibers of 9 CNM patients (4 sporadic, 3 familial, 2 adult sporadic). Dystrophin and beta-spectrin localized intracytoplasmically in centrally nucleated fibers. Talin and vinculin were normally expressed. Desmin was radially organized in several fibers in all patients. Scattered vimentinpositive fibers were found in 3 cases. Six myotonic dystrophy cases and 4 inflammatory myopathy cases with regenerating fibers were also studied: dystrophin and the membrane cytoskeletal proteins were normally expressed in the former; and dystrophin, spectrin, and vinculin were reduced in the latter. Intracytoplasmic dystrophin is further evidence of maturational arrest in CNMs. Spectrin and dystrophin codistribute in these pathological conditions as in normal muscle. We conclude that the altered cytoskeletal network found in CNMs likely plays a pathogenetic role in these conditions.

Adult↗

PubMed1988

Characterization, localization, and biosynthesis of acetylcholinesterase in K 562 cells.

K 562 cell acetylcholinesterase (AChE), identifiable by active site labeling with radioactive diisopropylfluorophosphate (DFP), showed a Mr around 55,000 in both a crude lysate and a purified sample. The K 562 AChE was reactive with one polyclonal and two monoclonal antibodies produced against human erythrocyte AChE. Subcellular localization, investigated by assay on cell fractions, showed that AChE is membrane bound and that it is located on the cell surface as well as on microsomal and Golgi membranes. Biosynthesis of new enzyme molecules, after inactivation of the constitutive AChE with the irreversible inhibitor DFP, allowed us to follow the kinetics of reappearance in the intracellular compartment and at the cell surface (4 and 8 h, respectively).

Acetylcholinesterase↗

PubMed1988

Electrophoretic pattern of NADPH-dependent oxidoreductive activities in K 562 and HL 60 leukemic cell lines.

Morphological and functional differentiation of hemopoietic cells is accompanied by the expression of lineage-specific protein markers. NADPH-oxidoreductive enzymatic activities in HL 60 and K 562 leukemic cell lines, compared with granulocytes and erythrocytes, show a NADPH-oxidizing and a NADPH-diaphorase activity. The oxidizing activity, absent in erythrocytes, has the same electrophoretic migration in HL 60 cells and granulocytes while it is different in K 562 cells. The diaphorase, absent in HL 60 cells and granulocytes, has the same migration in erythrocytes and K 562 cells, although with a slightly different quantitative expression. K 562 cells induced to differentiation with arabinofuranosylcytosine show the appearance of a band of NADPH-oxidizing activity of granulocytic type, together with the major band found in these cells.

Cell Differentiation↗