Biomedical subjects

G Scarlato

Publications and source records attributed to G Scarlato.

At least 127 records · Page 7Linked to original sources

PubMed1994

NGF-response of EGF-dependent progenitor cells obtained from human sympathetic ganglia.

Signalling molecules are thought to play a significant role in determining the fate of neural crest progenitor cells. The human sympathetic chain was identified at 6.5, 7.5, 8.2, 10.2 and 11.4 postconception (PC) weeks demonstrating low affinity nerve growth factor (NGF) receptors, and was processed for tissue culture. In the presence of epidermal growth factor (EGF), floating spheres of proliferating progenitor cells were developed in vitro. In the absence of EGF progenitor cells differentiated into tyrosine hydroxylase (TH)-immunoreactive neuronal and TH-negative flat cells. NGF treatment significantly increased neurite outgrowth and survival of TH-immunoreactive cells. The multipotent cells we isolated differ from previously reported sympathoadrenal progenitors in that they give rise to TH immunoreactive neurones precociously sensitive to NGF.

Cell Survival↗

PubMed1994

Rapid direct diagnosis of deletions carriers of Duchenne and Becker muscular dystrophies.

13 families and 34 women at risk of being carriers of Duchenne and Becker muscular dystrophies were analysed blind by restriction fragment length polymorphisms (RFLPs) and a new, non-radioactive direct test based on competitive polymerase-chain-reaction (PCR) amplification. All obligate carriers were correctly diagnosed by RFLPs and competitive PCR, and 7 women with a 99% risk of being carriers were also diagnosed carriers by competitive PCR. There were no false positives or false negatives. Only 1 PCR result was non-informative. Our test allows rapid, precise, and direct carrier diagnosis of women whose relatives have a known deletion of the dystrophin gene.

False Negative Reactions↗

PubMed1994

Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-alpha and interleukin-8 mRNA in human neutrophils. Evidence for a role of L-selectin as a signaling molecule.

Sulfatides have been established recently as ligands for L-selectin, and we investigated whether they trigger transmembrane signals through ligation of L-selectin. We found that sulfatides trigger the increase of cytosolic free calcium in neutrophils and that this effect was strictly dependent on sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory. Chymotrypsin and phorbol 12-myristate 13-acetate treatment of neutrophils caused shedding of L-selectin, but not of class I major histocompatibility complex antigens or beta 2 integrins, and blunted the capability of neutrophils to respond to sulfatides with an increase of cytosolic free calcium. Four different anti-L-selectin antibodies (DREG-200, LAM1/3, LAM1/6, and LAM1/10), but not four control antibodies directed against different surface molecules of neutrophils, also triggered an increase of cytosolic free calcium. The anti-L-selectin antibodies were stimulatory both if used in a soluble form, after cross-linking with anti-mouse F(ab')2 fragments, and immobilized to protein A of Staphylococcus aureus through the Fc fragment. With immobilized antibodies, an increase of cytosolic free calcium was found also by plating neutrophils on antibodies bound to protein A-coated coverslips and monitoring the increase of cytosolic free calcium by fluorescence microscopy. Both sulfatides and anti-L-selectin antibody effects were not inhibited by pertussis toxin, thus indicating that a pertussis toxin-sensitive GTP-binding protein was not involved in signal transduction. Sulfatides also triggered an increase of tumor necrosis factor-alpha and interleukin-8 mRNAs in neutrophils. Also to act as stimuli of cytokine mRNA expression, sulfatides required sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory, and depended on expression of L-selectin, as shedding of this molecules induced by chymotrypsin blunted their effects. These findings suggest that L-selectin can transduce signals activating selective cell function.

Calcium↗

PubMed1994

Expression of the low-affinity NGF receptor during human muscle development, regeneration, and in tissue culture.

The expression of the low-affinity NGF receptor (LNGFR) during human muscle development, regeneration, and in tissue culture was analyzed using a murine monoclonal antibody to human LNGFR (MAb ME 20.4). Muscle cells from 12-22-week fetuses stained strongly for LNGFR. In adult normal muscle, only intramuscular nerve endings showed immunoreactivity with MAb ME 20.4, but no staining was detected in muscle fibers. In Duchenne muscular dystrophy, immunohistologically demonstrable LNGFR was present in regenerating muscle fibers. In these fibers, LNGFR gene expression was also demonstrated at the transcriptional level using in situ hybridization with riboprobes coding with human LNGFR. Cultures from human fetal and adult muscle were studied by double label indirect immunofluorescence microscopy with MAb ME 20.4 and antisera against human fetal myosin. Most myosin-positive cells, both at the myoblast and myotube stages, displayed surface LNGFR immunostaining. In cells from fetal muscle, LNGFR was detected during the first 2 weeks in vitro, whereas in cells from adult muscle the expression of LNGFR was observed for up to 7 weeks. These findings suggest a potential involvement of LNGFR in human muscle development and regeneration.

Culture Techniques↗

PubMed1994

Anionic phospholipids calcium binding sites in Duchenne and murine X-linked muscular dystrophy.

Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx) are genetically homologous and both characterized by absence of dystrophin. The function of this protein is not defined nor is the pathogenesis of the severe muscle necrosis and progressive weakness found in DMD but not in mdx. Recently we found that anionic phospholipid (AP) calcium binding sites are lacking at the muscle cell surface in DMD and we correlated these data with dystrophin deficiency and muscle necrosis. In order to verify the role of AP lack in the pathogenesis of muscle necrosis in DMD we studied the ultrastructural localization of these Ca++ receptors in mdx muscle membrane showing that they are normally represented as they are in control mouse and normal human muscle. The absence of AP in DMD compared with a normal distribution in mdx suggests that these calcium binding site alterations play an important and specific role in muscle fiber necrosis.

Animals↗

PubMed1994

Development of dopaminergic neurons in the human mesencephalon and in vitro effects of basic fibroblast growth factor treatment.

The origin and development of the mesencephalic dopaminergic (mesDA) neurons within the substantia nigra were characterized in human embryos from Postconception (PC) Week 5.0 to 12.0. Tyrosine hydroxylase (TH) immunoreactive cells were first demonstrated in the ventral mesencephalon at PC Week 5.5 next to the ventricular zone. Cell migration and neurite outgrowth of TH-positive neurons were timed. Early expression of ganglioside GM1 was demonstrated in developing neurons. Glial fibrillary acidic protein (GFAP) was first observed at PC Week 10.0 instead, after the dopaminergic neurotransmitter phenotype expression. In vitro complementary information was obtained: TH-positive cells represented about 3% of the total cell population after a week in culture, based upon accurate anatomical dissection. They tended to form microaggregates and to grow in close contact with glial cells. MesDA neuronal expression of TH activity was measured by a biochemical microassay. TH-positive cells responded to basic fibroblast growth factor (bFGF) both with increased TH activity and neuronal survival. bFGF effects were mediated by the proliferative action on glial cells. Astroglial GFAP-positive cells express nerve growth factor-low-affinity receptor in culture. Information on in vitro and in vivo sequences of mesDA neuronal development and their response to identified neurotrophic molecules may be invaluable for selection of the most appropriate tissue donor age for brain grafting and development of alternative treatment strategies for Parkinson's disease.

Cell Movement↗

PubMed1994

Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level.

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxigenin-labeled, polymerase chain reaction (PCR)-amplified DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.

Adolescent↗

PubMed1994

Multiple sclerosis and mitochondrial myopathy: an unusual combination of diseases.

A woman with definite multiple sclerosis (MS) and mitochondrial myopathy is described. There were widespread white matter lesions on magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) abnormalities and evoked response changes. Muscle biopsy showed ragged red fibres (RRFs) and cytochrome c oxidase (CoX) deficiency. Southern blot analysis revealed a large deletion of mitochondrial DNA (mtDNA). The patient may be affected by two unrelated diseases, MS and mitochondrial myopathy, but this combination has never previously been reported.

Biopsy↗

PubMed1994

General paresis: neuropsychological and SPECT study of five cases.

Although much less common than before the advent of the antibiotics, cognitive impairment due to general paresis can still be found in clinical practice. Five patients with general paresis underwent longitudinal neuropsychological assessment; on the occasion of the final evaluation, Single Photon Emission Tomography (SPECT) was also performed in four cases. The most frequent cognitive deficit at admission was long term spatial memory impairment, which was present in four patients. Only the two patients who received the highest dosage of intravenous penicillin therapy showed significant improvement at neuropsychological tests after treatment. SPECT findings included areas of cortical hypoperfusion and/or perfusion asymmetry in three out of four patients.

Brain↗

PubMed1994

The Italian Longitudinal Study on Aging (ILSA): design and methods.

The Italian Longitudinal Study on Aging (ILSA) is a population-based, longitudinal study of the health status of Italians aged 65-84 years. The main objectives of ILSA are the study of the prevalence and incidence rates of common chronic conditions in the older population, and the identification of their risk and protective factors. ILSA is also designed to assess age-associated physical and mental functional changes. A random sample of 5632 individuals, stratified by age and gender using the equal allocation strategy, was identified on the demographic lists of the registry office of eight municipalities: Genova, Segrate (Milano), Selvazzano-Rubano (Padova), Impruneta (Firenze), Fermo (Ascoli Piceno), Napoli, Casamassima (Bari), and Catania. An extensive investigation, including interviews, physical exams, and laboratory tests, was conducted at baseline to identify the presence of cardiovascular disease (ischemic heart disease, hypertension, congestive heart failure, arrhythmia, intermittent claudication), diabetes, impaired glucose tolerance, thyroid dysfunction, dementia, parkinsonism, stroke, and peripheral neuropathy, as well as assess physical and mental functional status. The baseline examination was carried out between March 1992 and June 1993; a second comprehensive examination will begin in March 1995. An interim hospital discharge data survey and a mortality survey are currently ongoing to assess the hospitalization rate and the cause-specific mortality rate in this study cohort.

Aged↗

PubMed1994

Multiple deletions of mitochondrial DNA in sporadic and atypical cases of encephalomyopathy.

Multiple deletions of mitochondrial DNA (mtDNA) were first identified in patients with mitochondrial encephalomyopathy with a clear mendelian inheritance. We found this genetic alteration in four atypical and sporadic cases of mitochondrial encephalomyopathy, characterized by RRF and partial COX deficiency. One patient was affected by essential hyperCPKemia, 1 by subacute onset flaccid tetraplegia and 2 by parkinsonism. Southern blot and PCR revealed mtDNA multiple deletions in muscle tissue of these patients. These findings indicate that these alterations are not confined to the families with mendelian transmission, but can be present in sporadic cases with heterogeneous phenotypic features.

Aged↗

PubMed1994

Chronic progressive external ophthalmoplegia: a correlative study of quantitative molecular data and histochemical and biochemical profile.

We studied muscle biopsies of 5 patients with Kearns-Sayre syndrome and 3 patients with chronic progressive external ophthalmoplegia all with the common deletion. Steady state levels of normal and deleted mitochondrial DNA (mtDNA) measured in each patient by quantitative PCR were correlated with histochemical and biochemical features. We found that (1) normal mtDNA levels were higher in many patients than in controls; (2) as levels of deleted mtDNA increased, so did levels of normal mtDNA; (3) cytochrome c oxidase (COX) activity and the percentage of COX negative fibers were both related to the levels of deleted mtDNA; and (4) as percentage of ragged red fibers increased, so did levels of total, deleted and normal mtDNA. The quantity of deleted mtDNA plays a key role in determining the severity of COX deficiency, which is responsible for the overaccumulation of mitochondria in muscle.

Adolescent↗

PubMed1994

Serum IgG antibodies to a 35-kDa P0-related glycoprotein in motor neuron disease.

Using an immunoblot technique we found a significantly higher frequency of serum IgG antibodies to a 35-kDa peripheral nerve myelin glycoprotein in patients with motor neuron disease (MND) (39% of 70) than in patients with neuropathy (13% of 61), other neurological disease (9% of 32) and normal subjects (5% of 20) (P < 0.005 in all cases), but not with multiple sclerosis (MS) (20% of 30) or non-neural immune diseases (25% of 32). Most positive patients had antibody titers of 1:200 or 1:2000 while higher titers were only found in seven patients with MND, one with chronic inflammatory demyelinating neuropathy, two with MS, two with non-neural immune diseases and one with stroke. The reacting protein had a higher molecular mass than P0 and was only faintly bound by an anti-P0 antiserum, but had the same N-terminal amino acid sequence of P0. The difference in molecular mass between P0 and the 35-kDa protein and the IgG reactivity of one patient's IgG with the 35-kDa protein persisted after its deglycosylation and dephosphorylation. Although there is no evidence that these antibodies are pathogenic, their frequent occurrence in MND and other immune-mediated conditions supports the hypothesis of an activation of the immune system in MND.

Antibodies↗

PubMed1994

Frontal dysfunction in early Parkinson's disease.

Recent studies have suggested that patients with Parkinson's disease (PD) share many of the behavioral deficits found following lesions to the pre-frontal cortex. We assessed the performance of a group of 22 mildly impaired, not-demented parkisonians (I or II Hoehn & Yahr stage) in a test of classification and recall of pictures of familiar objects, which has been demonstrated to be sensitive to frontal damage in patients with unilateral cerebral excision. Parkinsonians utilized fewer categories than normal controls for object classification, while no significant difference was found in the immediate and delayed recall scores. These results support the contention that a subclinical dysfunction of frontal type may be present even in the early stages of PD. A subanalysis of the data suggests that this dysfunction could possibly be aggravated by anticholinergic drugs.

Adult↗

PubMed1994

Gangliosides. Their role in clinical neurology.

Gangliosides are normal constituent of mammalian vertebrate cell membranes and are particularly abundant in the central and peripheral nervous systems. The biological effects of exogenously administered gangliosides have been extensively investigated in vitro and in experimental animal models where they have neuronotrophic and neuritogenic properties. Despite these findings there is still little evidence that treatment with parenteral gangliosides in humans can be effective in peripheral neuropathies or other neuromuscular diseases. The initial preliminary reports on the positive effects of GM1 in cerebrovascular diseases and spinal cord injury need to be confirmed in larger controlled trials. At the same time the occasional development of an acute motor neuropathy clinically presenting as the Guillain-Barré syndrome and associated with high titres of anti-ganglioside antibodies highlights the risks of their widespread use before more consistent data on their efficacy become available.

Animals↗

PubMed1994

Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers.

In the present work, we demonstrate the possibility of using human blood platelets as mitochondrial donors for the repopulation of mtDNA-less (rho 0) cells. The noninvasive nature of platelet isolation, combined with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, has substantially increased the applicability of the rho 0 cell transformation approach for mitochondrial genetic analysis and for the study of mtDNA-linked diseases. This approach has been applied to platelets from several normal human individuals and one individual affected by the myoclonic-epilepsy-and-ragged-red-fibers (MERRF) encephalomyopathy. A certain variability in respiratory capacity was observed among the platelet-derived rho 0 cell transformants from a given normal subject, and it was shown to be unrelated to their mtDNA content. The results of sequential transfer of mitochondria from selected transformants into a rho 0 cell line different from the first rho 0 acceptor strongly suggest that this variability reflected, at least in part, differences in nuclear gene content and/or activity among the original recipient cells. A much greater variability in respiratory capacity was observed among the transformants derived from the MERRF patient and was found to be related to the presence and amount of the mitochondrial tRNALys mutation associated with the MERRF syndrome. An analysis of the relationship between proportion of mtDNA carrying the MERRF mutation and degree of respiratory activity in various transformants derived from the MERRF patient revealed an unusual complementation behavior of the tRNALys mutation, possibly reflecting the distribution of mutant mtDNA among the platelet mitochondria.

Adult↗

PubMed1993

Decrease of nerve Na+,K(+)-ATPase activity in the pathogenesis of human diabetic neuropathy.

A decrease in Na+,K(+)-ATPase activity is claimed to play a central role in the pathogenesis of electrophysiological and morphological abnormalities that characterize the neuropathic complications in different animal models of diabetes mellitus. The peripheral nerves from 17 patients with either type I or type II diabetes mellitus were studied to assess the importance of changes in Na+,K(+)-ATPase activity in chronic human diabetic neuropathy. Sixteen nerves from age- and sex-matched normal individuals, and 12 nerves from non-diabetic neuropathic subjects undergoing vascular or orthopedic surgery served as negative and positive controls, respectively. All specimens were processed blind. Ouabain-sensitive ATPase activity was measured by a modified spectrophotometric coupled-enzyme assay. Standard histology, fiber teasing and electron microscopy were used to establish the normal or neuropathological patterns of surgical material. Morphometric analysis permitted calculation of fiber density in each nerve specimen and correlation of this figure with the relevant enzymatic activity. Na+,K(+)-ATPase activity was approximately 59% lower in nerves from diabetic patients than in normal controls (P < 0.01) and approximately 38% lower in nerves from non-diabetic patients with neuropathy (P < 0.01). Although nerves from both neuropathic conditions had significantly fewer fibers than those from normal individuals (diabetic -33%, and non-diabetic -22%), the decreases in Na+,K(+)-ATPase activity and fiber density were not correlated only in specimens from diabetic patients (r2 = 0.096; P = 0.22). Taken together with data from experimental animal models, these results suggest that the reduction in Na+,K(+)-ATPase activity in diabetic nerves is not an epiphenomenon secondary to fiber loss; rather, it may be an important factor in the pathogenesis and self-maintenance of human diabetic neuropathy.

Adult↗

PubMed1993

Myelin protein transcripts increase in experimental diabetic neuropathy.

A Northern blot analysis of P0 and MBP myelin protein transcripts in the sciatic nerve from rats with alloxan-induced diabetes at two different time points is described. After 5 weeks of diabetes induction, only P0 mRNA is significantly increased by 39%, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels, partially counteracts P0 mRNA increase at both stages of diabetes and delays MBP mRNA increase present only in chronic animals. We suggest that increased transcript levels of P0 and MBP in Schwann cells may represent a higher turnover of myelin sheath specific proteins in diabetic syndrome, as attempt to repair the hyperglycemia-induced nerve damage, which is partially prevented by insulin treatment.

Animals↗