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Biomedical subjects

E Barbosa

Publications and source records attributed to E Barbosa.

At least 19 recordsLinked to original sources
PubMed2005

Early-onset parkinsonism associated with PINK1 mutations: frequency, genotypes, and phenotypes.

OBJECTIVE: To assess the prevalence, nature, and associated phenotypes of PINK1 gene mutations in a large series of patients with early-onset (<50 years) parkinsonism. METHODS: The authors studied 134 patients (116 sporadic and 18 familial; 77% Italian) and 90 Italian controls. The whole PINK1 coding region was sequenced from genomic DNA; cDNA was analyzed in selected cases. RESULTS: Homozygous pathogenic mutations were identified in 4 of 90 Italian sporadic cases, including the novel Gln456Stop mutation; single heterozygous truncating or missense mutations were found in another 4 Italian sporadic cases, including two novel mutations, Pro196Leu and Gln456Stop. Pathogenic mutations were not identified in the familial cases. Novel (Gln115Leu) and known polymorphisms were identified with similar frequency in cases and controls. In cases carrying single heterozygous mutation, cDNA analysis detected no additional mutations, and revealed a major pathogenic effect at mRNA level for the mutant C1366T/Gln456Stop allele. All patients with homozygous mutations had very early disease onset, slow progression, and excellent response to l-dopa, including, in some, symmetric onset, dystonia at onset, and sleep benefit, resembling parkin-related disease. Phenotype in patients with single heterozygous mutation was similar, but onset was later. CONCLUSIONS: PINK1 homozygous mutations are a relevant cause of disease among Italian sporadic patients with early-onset parkinsonism. The role of mutations found in single heterozygous state is difficult to interpret. Our study suggests that, at least in some patients, these mutations are disease causing, in combination with additional, still unknown factors.

Adolescent↗
PubMed2005

The G6055A (G2019S) mutation in LRRK2 is frequent in both early and late onset Parkinson's disease and originates from a common ancestor.

BACKGROUND: Mutations in the gene Leucine-Rich Repeat Kinase 2 (LRRK2) were recently identified as the cause of PARK8 linked autosomal dominant Parkinson's disease. OBJECTIVE: To study recurrent LRRK2 mutations in a large sample of patients from Italy, including early (<50 years) and late onset familial and sporadic Parkinson's disease. RESULTS: Among 629 probands, 13 (2.1%) were heterozygous carriers of the G2019S mutation. The mutation frequency was higher among familial (5.1%, 9/177) than among sporadic probands (0.9%, 4/452) (p<0.002), and highest among probands with one affected parent (8.7%, 6/69) (p<0.001). There was no difference in the frequency of the G2019S mutation in probands with early v late onset disease. Among 600 probands, one heterozygous R1441C but no R1441G or Y1699C mutations were detected. None of the four mutations was found in Italian controls. Haplotype analysis in families from five countries suggested that the G2019S mutation originated from a single ancient founder. The G2019S mutation was associated with the classical Parkinson's disease phenotype and a broad range of onset age (34 to 73 years). CONCLUSIONS: G2019S is the most common genetic determinant of Parkinson's disease identified so far. It is especially frequent among cases with familial Parkinson's disease of both early and late onset, but less common among sporadic cases. These findings have important implications for diagnosis and genetic counselling in Parkinson's disease.

Adult↗
PubMed2000

Lovastatin therapy for X-linked adrenoleukodystrophy: clinical and biochemical observations on 12 patients.

X-linked adrenoleukodystrophy (X-ALD) is a progressive demyelinating disorder whose neurological signs and symptoms can manifest in childhood as cerebral ALD or in adulthood in the form of a progressive myelopathy (AMN). The consistent metabolic abnormality in all forms of X-ALD is an inherited defect in the peroxisomal beta-oxidation of very long chain (VLC) fatty acids (>C(22:0)) which may in turn lead to a neuroinflammatory process associated with demyelination of the cerebral white matter. The current treatment for X-ALD with Lorenzo's oil aims to lower the excessive quantities of VLC fatty acids that accumulate in the patients' plasma and tissues, but does not directly address the inflammatory process in X-ALD. We have previously demonstrated that lovastatin and other 3-HMG-CoA reductase inhibitors are capable of normalizing VLC fatty acid levels in primary skin fibroblasts derived from X-ALD patients. Lovastatin can block the induction of inducible nitric oxide synthase and proinflammatory cytokines in astrocytes, microglia, and macrophages in vitro. In a preliminary report, we demonstrated that lovastatin therapy can normalize VLC fatty acids in the plasma of patients with X-ALD. Here we report our clinical and biochemical observations on 12 patients with X-ALD who were treated with lovastatin for up to 12 months. Our results show that the high plasma levels of hexacosanoic acid (C(26:0)) showed a decline from pretreatment values within 1 to 3 months of starting therapy with 40 mg of lovastatin per day and stabilized at various levels during a period of observation up to 12 months. The percentage decline from pretreatment values varied and did not correlate with the type of ALD gene mutation (point mutation versus gene deletion). In 6 patients, in whom red cell membrane fatty acid composition was studied, a mean correction of 50% of the excess C(26:0) was observed after 6 months of therapy suggesting sustained benefit. In a few patients who discontinued lovastatin therapy plasma C(26:0) levels reverted to pretreatment values suggesting a cause and effect relationship between these events. Two patients dropped out of the study claiming no clinical benefit, 1 was withdrawn due to adverse effects, and an adult patient with cerebral involvement died during the study. A 10-year-old boy with severe cerebral involvement showed worsening of his neurological status. All patients with AMN remained neurologically stable or showed modest subjective improvement. All patients who did not have Addison's disease at the time of enrollment maintained normal adrenal function throughout the study. The implications of our findings for developing an effective therapy for X-ALD are discussed.

Adrenoleukodystrophy↗
PubMed2000

Characterization of fibroblast cytoplasmic proteins that bind to the 3' UTR of human catalase mRNA.

The excessive expression of catalase protein and its activity in cultured skin fibroblast from Zellweger Syndrome (ZS), a disorder of peroxisomal biogenesis, was found to be regulated at the translational level (J. Neurochem. 67: 2373-2378, 1996). Overall there is a considerable increase in the association of catalase mRNA with polysomes in ZS cell lines as compared to control indicating translational upregulation. To investigate the possibility that RNA-protein interactions are involved in the mediation of this increase in translation, the interaction between 3' untranslated region of human catalase mRNA and human fibroblast cytoplasmic proteins were investigated by RNA gel shift assay technique. Competition experiments demonstrated that all the 600 bases of 3' UTR (of human catalase gene) was required for efficient binding. Catalase RNA- protein interaction was sensitive to the altered redox state in these in vitro assays and this RNA-protein interaction could be enhanced by the addition of beta-mercaptoethanol in cytoplasm from control fibroblast but not in cytoplasm from ZS fibroblast. UV cross linked RNA-protein complexes on SDS polyacrylamide gel electrophoresis revealed the presence of at least four protein bands with approximate molecular masses of 38 kDa, 50 kDa, 66 kDa and 80 kDa. The potential role of these mRNA binding proteins in the regulation of catalase gene expression is discussed.

3' Untranslated Regions↗
PubMed1999

Use of the pediatric symptom checklist in the pediatric neurology population.

The purpose of this study was to evaluate the effectiveness of the Pediatric Symptom Checklist (PSC) as a mental health screening instrument in a busy pediatric neurology population in comparison with more lengthy, time-consuming assessment methods. One hundred two children were screened using the PSC. PSC results were compared with scores on the Child Behavior Checklist (CBCL), results from structured interviews, and ratings of adaptive functioning using the Children's Global Assessment Scale (CGAS). Thirty-nine of the patients (38%) scored 63 or above on the CBCL, indicating psychosocial impairment. Using a cutoff score of 22, the PSC correctly identified 35 of these 39 positive cases (sensitivity 89.7) and 48 of the 63 children with CBCL scores below 63 (specificity 76.2). CGAS scores were significantly negatively correlated with PSC scores (r = -0.60, P < 0.05). The PSC correctly identified 85.9% of children who scored 70 or below on the CGAS. Among the 53 children with psychiatric diagnoses on the basis of the interview, 41 scored above the cutoff of 22 on the PSC. Results suggest that the PSC is an efficient and accurate screen for identification of mental health problems in the pediatric neurology population.

Behavioral Symptoms↗
PubMed1999

The use of hypothermia: a role in the treatment of neonatal asphyxia?

Perinatal asphyxia remains one of the most devastating neurologic processes. Although the understanding of the pathophysiology after perinatal asphyxia is extensive, there are few therapeutic interventions available to prevent or even mitigate the devastating process that unfolds after injury. The search for a safe and efficacious therapy has prompted scientists and clinicians to consider various promising therapies. One such therapy is therapeutic hypothermia. On the basis of adult, pediatric, and animal research, there is increasing evidence to suggest that therapeutic hypothermia may be an effective intervention to lessen the secondary neuronal injury that ensues after a hypoxic-ischemic insult. In this article the historic and modern-day uses of therapeutic hypothermia are first reviewed. The pathophysiology of neonatal asphyxia is examined next, with emphasis on the changes that occur when therapeutic hypothermia is implemented. Potential side-effects of the therapy in the neonate and the debate over systemic vs selective hypothermia are discussed. Lastly, although hypothermia as a potential treatment modality for neonates with hypoxic-ischemic encephalopathy is supported by numerous studies, the need for well-designed multicenter trials with detailed patient entry criteria and therapeutic conditions is emphasized.

Adult↗
PubMed1999

Involvement of sulfated proteoglycans in embryonic brain expansion at earliest stages of development in rat embryos.

The expansive force generated by the positive pressure of the neural tube fluid confined inside the brain vesicles has been shown to be a key factor during the earliest stages of brain morphogenesis and development of chick embryos. In a previous study, we demonstrated the existence of an intracavity extracellular matrix rich in condroitin sulfate in this species, which could be involved in the regulation of the expansive process. In this report, scanning electron microscopy and immunohistochemistry show that, after neurulation, a similar extracellular matrix rich in chondroitin sulfate is present inside the brain vesicles of rat embryos during early enlargement of the brain anlage. In vitro treatment of rat embryos with beta-D-xyloside (a chemical compound which disrupts chondroitin sulfate synthesis) shows that changes in intralumen chondroitin sulfate concentration are accompanied by significant changes in brain anlage growth. These results support the hypothesis that intracerebral chondroitin sulfate plays a relevant role in the regulation of the expansive process of the brain anlage of rat embryos, and could represent a general mechanism in the early brain development of avian and mammalian embryos.

Animals↗
PubMed1998

Restoration of phytanic acid oxidation in Refsum disease fibroblasts from patients with mutations in the phytanoyl-CoA hydroxylase gene.

Refsum disease (RD) is biochemically characterized by the excessive accumulation of phytanic acid in tissues and body fluids due to deficiency of phytanoyl-CoA hydroxylase (PAHX). In this study, we screened three RD patients and identified a novel deletion (88 amino acids), and a missense mutation (Arg275Trp) in the previously reported PAHX cDNA (Jansen et al., 1997; Mihalik et al., 1997). Moreover, transfection of skin fibroblasts from two RD patients with wild-type PAHX gene restored the activity for alpha-oxidation of phytanic acid. Southern analysis on a somatic cell hybrid panel detected the PAHX gene on chromosome 10, corroborating radiation hybrid and homozygosity mapping data (Mihalik et al., 1997; Nadal et al., 1995).

Chromosome Mapping↗
PubMed1998

Abnormality in catalase import into peroxisomes leads to severe neurological disorder.

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H2O2 or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the in situ levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H2O2 toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease.

Animals↗
PubMed1998

Disruption of proteoglycans in neural tube fluid by beta-D-xyloside alters brain enlargement in chick embryos.

Following neurulation, the anterior end of the neural tube undergoes a dramatic increase in size due mainly to the enlarging of the brain cavity. This cavity is filled with so-called neural tube fluid (NTF), whose positive pressure has been shown to play a key role in brain morphogenesis. This fluid contains a water-soluble matrix, rich in chondroitin sulfate (CS), which has been proposed as an osmotic regulator of NTF pressure genesis. The purpose of the present study is to observe the influence of CS on NTF osmolality and its relation to NTF hydrostatic pressure and brain expansion. NTF was obtained by means of microaspiration from the mesencephalic cavity of chick embryos. The osmolality of NTF between H.H. stages 20 and 29 was measured on the basis of its cryoscopic point. CS synthesis was disrupted by using beta-D-xyloside and the induced variations in brain volume were measured by means of morphometry. We also measured the variations in NTF osmolality, hydrostatic pressure, and the concentration of CS and sodium induced by means of beta-D-xyloside. Our data reveal that, at the earliest stages of development analyzed, variations in NTF osmolality show a characteristic pattern that coincides with the developmental changes in the previously described fluid pressure. Chick embryos treated with beta-D-xyloside, a chemical that disrupts CS synthesis, displayed a notable increase in brain volume but no other apparent developmental alterations. Morphometric analysis revealed that this increase was due to hyperenlargement of the brain cavity. Beta-D-xyloside brings about specific changes in the biochemical composition of NTF, which entails a large increase in CS concentration, mainly in the form of free chains, and in that of sodium. As a result, the fluid's osmolality and brain intraluminal pressure increased, which could account for the increase in size of the brain anlage. These data support the hypothesis that the intraluminal pressure involved in embryonic brain enlargement is directly dependent on NTF osmolality, and that the concentrations of CS and its associated microions could play a key role in the regulation of this process.

Animals↗
PubMed1998

Local increase level of chondroitin sulfate induces changes in the rhombencephalic neural crest migration.

Numerous studies suggest that chondroitin sulfate proteoglycan (CSPG) inhibits neural crest cells (NCC) migration at the trunk level. However, its action on the cephalic neural crest is not clear. To determine this action, we have microinjected 0.5 nl of different concentrations of chondroitin sulfate (CS) at the anterior rhombencephalon level in 9 stage chick embryos, as well as subgerminally administering beta-D-xyloside to 8 stage chick embryos. Beta-D-xyloside disrupts CSPG synthesis, producing an increase in CS free chains in several embryonal anlages. Chondroitin sulfate microinjected embryos and beta-D xyloside treated embryos were reincubated until attaining 12 stage. Results obtained for both experimental groups were similar. Immunoreactivity with HNK-1 antibody revealed that NCC did not migrate, remaining near the rhombencephalon dorsal wall; in addition, several NCC did not separate from the neural fold, becoming invaginated towards the rhombencephalon cavity. Our findings indicate that an increase in CS free chains in cephalic neural crest migratory routes not only disrupts their migration, but also impedes delamination and detachment of the rhombencephalic neuroepithelium NCC. These data suggest that the inhibitory action upon the neural crest migration attributed to CSPG may rest on its glycosaminoglycan (GAG). We cannot, however, rule out the possibility that increases in other GAGs apart from CS, may produce similar effects on neural crest migration.

Animals↗
PubMed1997

Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils.

The expression of cell-surface adhesion proteins and the release of extracellular-matrix degradative enzymes constitute crucial processes for the attachment of neutrophils to the endothelium and for the subsequent extravasation of these cells through the endothelial layer. We have analysed in resting human neutrophils the subcellular localization of heparanase, a heparan-sulphate-degrading endoglycosidase that can degrade basement-membrane components, thereby facilitating neutrophil passage into the tissue during an inflammatory reaction. By subcellular fractionation of postnuclear supernatants from resting human neutrophils on continuous sucrose gradients, we have found that heparanase activity was mainly located in gelatinase-containing tertiary granules. Using a specific antibody, the 96-kDa heparanase protein was further located in the gelatinase-rich subcellular fractions. Following immunoblotting and immunoprecipitation analysis in the distinct subcellular fractions, we also found co-localization of heparanase and Mo1 (CD11b/CD18), a leucocyte integrin involved in the attachment of neutrophils to the endothelium, in the fractions enriched in gelatinase-containing tertiary granules. Treatment of human neutrophils with tumour necrosis factor or granulocyte/macrophage colony-stimulating factor induced an increase in the CD11b/CD18 cell-surface expression, as well as the release of both gelatinase (matrix metalloproteinase-9) and heparanase, but not of other granule markers, indicating a major co-localization of gelatinase, heparanase and CD11b/CD18 in the same organelle. Furthermore, confocal laser scanning microscopy using specific antibodies against gelatinase and heparanase revealed a major co-localization of both enzymes in intracellular cytoplasmic granules. The major localization of heparanase and CD11b/CD18 in the gelatinase-containing tertiary granule supports the notion that mobilization of this organelle can regulate extravasation of human neutrophils.

CD18 Antigens↗
PubMed1995

Molecular cloning and sequencing of human palmitoyl-CoA ligase and its tissue specific expression.

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.

Amino Acid Sequence↗
PubMed1995

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Blood↗
PubMed1995

Genetic and biochemical normalization in female carriers of Duchenne muscular dystrophy: evidence for failure of dystrophin production in dystrophin-competent myonuclei.

We studied 19 symptomatic female carriers of the Duchenne muscular dystrophy (DMD) gene. Most of these dystrophinopathy patients had had an erroneous or ambiguous diagnosis prior to dystrophin immunofluorescence testing. We assessed clinical severity by a standardized protocol, measured X-chromosome inactivation patterns in blood and muscle DNA, and quantitated the dystrophin protein content of muscle. We found that patients could be separated into two groups: those showing equal numbers of normal and mutant dystrophin genes in peripheral blood DNA ("random" X-inactivation), and those showing preferential use of the mutant dystrophin gene ("skewed" X-inactivation). In the random X-inactivation carriers, the clinical phenotype ranged from asymptomatic to mild disability, the dystrophin content of muscle was > 60% of normal, and there were only minor histopathologic changes. In the skewed X-inactivation patients, clinical manifestations ranged from mild to severe, but the patients with mild disease were young (5 to 10 years old). The low levels of dystrophin (< 30% on average) and the severe symptoms of the older patients suggested a poor prognosis for those with skewed X-inactivation, and they all showed morphologic changes of dystrophy. The random inactivation patients showed evidence of biochemical "normalization," with higher dystrophin content in muscle than predicted by the number of normal dystrophin genes. Seventy-nine percent of skewed X-inactivation patients (11/14) showed genetic "normalization," with proportionally more dystrophin-positive nuclei in muscle than in blood. In 65% of the skewed X-inactivation patients, dystrophin was not produced by dystrophin-positive nuclei; an average of 20% of myofiber nuclei were genetically dystrophin-positive but did not produce stable dystrophin. Biochemical normalization seems to be the main mechanism for rescue of fibers from dystrophin deficiency in the random X-inactivation patients. In the skewed X-inactivation patients, genetic normalization is active, but production failure of dystrophin by dystrophin-normal nuclei may counteract any effect of biochemical normalization. In the skewed X-inactivation patients, the remodeling of the muscle through cycles of degeneration and regeneration led to threefold increase in the number of dystrophin-competent nuclei in muscle myofibers (3.3 +/- 4.6), while dystrophin content was on the average 1.5-fold less then expected (-1.54 +/- 3.38). Our results permit more accurate prognistic assessment of isolated female dystrophinopathy patients and provide important data with which to estimate the potential effect of gene delivery (gene therapy) in DMD.

Adult↗