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M Scarpa

Publications and source records attributed to M Scarpa.

At least 37 records · Page 2Linked to original sources
PubMed1999

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin.

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.

Amylopectin↗
PubMed1999

Identification of a D579G homozygote cystic fibrosis patient with pancreatic sufficiency and minor lung involvement. Mutations in brief no. 221. Online.

Here we describe the identification of an italian patient homozygote for the D579G mutation affected by a mild form of Cystic Fibrosis with pancreatic sufficiency, minor lung involvement and marked viscosity of the cervical mucous. The D579G mutation causes an A1868G transition, a substitution of an aspartic acid to a glycine residue, generating an important amino acid change (charged to hydrophobic) in the nucleotide-binding domain (NBD). The mutation was first described by Brancolini et al. (1995) on two pancreatic sufficient CF patients, compound heterozygotes for delta508F. Patients were from Southern Italy (Puglia) as the D579G homozygote one, who is a 30 years old woman from Taranto (Puglia), daughter of second cousins born in Bari (Puglia). The identification of a homozygote D579G patient might confirm that this mutation does correlate with pancreatic sufficiency and a mild pulmonary phenotype.

Adult↗
PubMed1999

Enzyme mimics complexing Cu(II) ion: structure-function relationships.

Five peptides containing (His-X2)-His or (His-X3)-His motifs have been designed and synthesized to coordinate Cu(II). Structural information was obtained by various spectroscopic techniques and was used as constraint to search for local conformational energy minima by molecular mechanics. Thermodynamic stability constants of the Cu(II) chelates was obtained by 19F-NMR. The synthesized Cu(II)-peptide chelates were tested as catalysts of some important red-ox processes occuring in biological systems, in particular oxidation of ascorbate and dismutation of superoxide ion. The catalytic efficiency of the five chelates was much lower than that of ascorbate oxidase. On the contrary, two of them showed kinetic constants for superoxide dismutation about one order of magnitude lower than that of the enzyme Cu,Zn superoxide dismutase. In both cases, the catalytic properties were dependent on the peptide sequence. The relationships between structure and activity are discussed to find the structural parameters crucial for catalytic activity that can be modulated by appropriate design and synthesis of the peptides.

Amino Acid Sequence↗
PubMed1999

Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.

BACKGROUND: The search for the eight most frequent mutations (i.e., DeltaF508, G542X, W1282X, N1303K, 1717-1G-->A, R553X, 2183AA-->G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles. METHODS: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations. RESULTS: The screening revealed five mutations, R1158X, 711+1G-->T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3-1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype. CONCLUSIONS: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.

Adult↗
PubMed1998

Continuous flow immunosensor for atrazine detection.

The hapten atrazine was detected under continuous flow conditions using a micro-column which contained immobilized monoclonal antibodies (Ab) against atrazine and atrazine labeled with alkaline phosphatase (An*). The equilibrium of the antibody-hapten system, was achieved by a continuous flow of the tracer An* through the micro-column containing the immobilized antibodies. The activity of the tracer was monitored continuously, after the micro-column, by an amperometric detector using p-hydroquinone phosphate as substrate. When pulses of unlabeled atrazine (An) were added to the An* flowing continuously through the micro-column, An* bound to the antibody was displaced, with a consequent change of the detector signal. By this method atrazine concentrations in the range 9-180 micrograms/l were monitored under conditions of continuous operation. Since the equilibrium condition for the system Ab-An* was continuously restored by the flow of An* through the micro-column the regeneration of the antibody was not required.

Animals↗
PubMed1998

In vitro and in vivo antitumor effects of retrovirus-mediated herpes simplex thymidine kinase gene-transfer in human medulloblastoma.

Herpes simplex virus thymidine kinase gene (HSVtk) transfer together with treatment with the prodrug ganciclovir (GCV) represents the most commonly used suicide gene approach for the gene therapy of human central nervous system malignancies. Despite encouraging results reported in clinical trials conducted in adults, very little is known about the feasibility of this approach for the treatment of CNS tumors of childhood. We studied the effects of the HSVtk/GCV system on human medulloblastoma cells in vitro and in vivo. The transfer of tk gene to medulloblastoma cells was capable of mediating cell suicide in vitro and in vivo upon treatment with GCV, but the overall effect in vivo appeared to be suboptimal. The relatively low sensitivity of the medulloblastoma cells to viral infection and a limited bystander effect, coupled with a low expression of connexin-43 protein, might partially explain these results. Whether this is a peculiarity of the cell line studied or a general characteristic of medulloblastoma remains to be determined. These findings should be taken into account for the future planning of gene therapy trials for human medulloblastoma.

Cerebellar Neoplasms↗
PubMed1997

The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio rerio) embryos.

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model.

Animals↗
PubMed1997

Deuterium distribution in lactate as tracer of metabolic pathways.

2H-NMR spectroscopy has been used to study the distribution of 2H atoms in brain lactate, after intraperitoneal injection of 2H2O. Information on brain metabolism was obtained from the ratio [(3-2H) lactate]/[(2-2H)-lactate] which was found very sensitive to the PO2 in the breathing air. The dependence of this ratio on the rates of metabolic pathways is an example of the possibility to use the distribution of 2H atoms in a molecule to probe metabolic flow through different pathways.

Air↗
PubMed1997

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors.

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial beta-galactosidase (betaGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7betaGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7betaGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the 'leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.

Adenoviridae↗
PubMed1997

Learning and forgetting processes in Parkinson's disease: a model-based approach to disentangling storage, retention and retrieval contributions.

Learning and forgetting a prose passage was studied in 20 patients with Parkinson's disease and in 20 normal control subjects by means of stochastic models, with the aim of identifying the learning and retaining abilities that are affected by Parkinson's disease. Results suggested that Parkinson's disease patients are impaired in developing automatic processing both during learning and retaining, while functions that require active attention are spared. The automatic/intentional dissociation, which is the hallmark of motor disturbance in Parkinson's disease, extends to memory abilities, and, on the grounds of neuroanatomical, neurochemical and neurophysiological correlates, suggests that the memory deficit in Parkinson's disease may be contingent on a dysfunction of the medial prefrontal-cingulate cortex.

Aged↗
PubMed1997

In vitro correction of iduronate-2-sulfatase deficiency by adenovirus-mediated gene transfer.

Hunter syndrome is a lethal lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase and characterized by severe skeletal and neurological symptoms. Only symptomatic treatments are available and, although bone marrow transplantation has been suggested, no encouraging results have been obtained so far. Therefore, gene therapy might be a route to be pursued for treatment of the disease. In this respect, one major goal to achieve is the generation of an overexpressing vector able to correct, in particular, central nervous system (CNS) cells. Adenoviruses have been shown to infect CNS cells efficiently with minor or even absent immunological response. We describe the generation of a replication-defective adenoviral vector, AdRSVIDS, which is able to express in vitro high levels of iduronate-2-sulfatase. After infection, accumulation of mucopolysaccharides in treated Hunter cells was normalized. Furthermore, endocytosis of the transduced IDS did occur via the mannose-6-phosphate (M6P) receptor. Since no animal model for the disease is available, we developed a system based on the generation of derma-equivalents which enabled us to verify the expression of high levels of sulfatase up to 30 days after infection.

Adenoviridae↗
PubMed1997

ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo.

Nonviral vectors might represent a safe alternative to adenovirus for gene therapy of lung disorders, in particular cystic fibrosis (CF). Cationic lipids have been shown to correct the CF defect both in vitro and in vivo, but more efficient vectors are needed to improve the low gene transfer efficiency. Here, we show that the cationic polymer ExGen 500, a linear polyethylenimine derivative, is more efficient than cationic lipids in transferring reporter genes to lung epithelial cells in vitro. In vivo ExGen 500 was able to mediate gene transfer into both newborn and adult rabbit lungs with comparable efficiencies. The best levels of transfection were obtained using neutral complexes. Under such conditions, luciferase activities corresponding to about 10(3) RLU/10 s/mg of protein were reproducibly obtained 2 days after transfection throughout the four lung lobes of newborn and adult rabbits. A nlslacZ reporter gene showed transfected cells around the lumen of large and small bronchi. No signs of acute toxicity (inflammation, cellular infiltration etc) were detected by direct histopathological analysis. Within 1 week after instillation, transgene expression decreased by two orders of magnitude.

Animals↗
PubMed1997

An automated method for mapping human tissue permittivities by MRI in hyperthermia treatment planning.

This paper presents an automatic method to obtain tissue complex permittivity values to be used as input data in the computer modelling for hyperthermia treatment planning. Magnetic resonance (MR) images were acquired and the tissue water content was calculated from the signal intensity of the image pixels. The tissue water content was converted into complex permittivity values by monotonic functions based on mixture theory. To obtain a water content map by MR imaging a gradient-echo pulse sequence was used and an experimental procedure was set up to correct for relaxation and radiofrequency field inhomogeneity effects on signal intensity. Two approaches were followed to assign the permittivity values to fat-rich tissues: (i) fat-rich tissue localization by a segmentation procedure followed by assignment of tabulated permittivity values; (ii) water content evaluation by chemical shift imaging followed by permittivity calculation. Tests were performed on phantoms of known water content to establish the reliability of the proposed method. MRI data were acquired and processed pixel-by-pixel according to the outlined procedure. The signal intensity in the phantom images correlated well with water content. Experiments were performed on volunteers' healthy tissue. In particular two anatomical structures were chosen to calculate permittivity maps: the head and the thigh. The water content and electric permittivity values were obtained from the MRI data and compared to others in the literature. A good agreement was found for muscle, cerebrospinal fluid (CSF) and white and grey matter. The advantages of the reported method are discussed in the light of possible application in hyperthermia treatment planning.

Brain↗
PubMed1997

A peroxidase-catalyzed sulfoxidation of promethazine.

Lactoperoxidase, when incubated with increasing amounts of promethazine (P) and promethazine sulfoxide (PO) catalyzes the formation of promethazine sulfoxide accompanied by oxygen consumption. An intermediate radical of PO can be detected by electron spin resonance (ESR). Catalase or superoxide dismutase do not inhibit the reaction while dopamine does. The lactoperoxidase-catalyzed formation of dopaminochrome in the presence of hydrogen peroxide is inhibited by P. Both P and PO inhibit acetyl- and butyrylcholinesterase. Purified enzymes were used throughout the study and horseradish peroxidase but not myeloperoxidase had an activity similar to that of lactoperoxidase.

Catalase↗
PubMed1997

[Ureteroscopic lithotripsy using the laser].

The Cagliari urology team is very satisfied with the use of the dye laser for ureteroscopic lithotripsy. This apparatus is recommended in units possessing a well equipped endourology room, otherwise it is simpler to use endoureteric ballistic lithotripsy, which also provides good results.

Aluminum Oxide↗
PubMed1996

In vitro and in vivo gene transfer to pulmonary cells mediated by cationic liposomes.

Cationic liposomes have been proposed as alternative to adenovirus in the treatment of cystic fibrosis lung disease. Therefore, we have investigated the efficiency of two lipid mixtures in mediating gene transfer in in vitro and in vivo models. The cationic lipid DOTMA (N-(1-(2,3(dioleyloxy)propyl)-n,n,n-trimethylammoniumchloride++ +) and DOGS (dioctadecylamidoglycylspermine) were used in combination with the neutral lipid DOPE (dioleoylphosphatidylethanolamine). The relative transfection efficiencies of the two cationic liposomes were tested using the bacterial beta-galactosidase (lacZ) and the firefly luciferase genes. Gene expression was detected in both cell limes and primary culture of rhesus monkey airway epithelium after transfection with plasmid DNA complexed with DOGS/DOPE or DOTMA/DOPE. Transfection efficiency of both types of lipids was higher in the mouse fibroblast 3T3 cell line as compared to human carcinoma A549 cells and primary epithelial cultures. Administration of DNA-liposome complexes via intratracheal instillation resulted in expression of the lacZ and luciferase marker gene in the mouse airways. In vivo transfection mediated by both types of liposomes were proven to be far less efficient than adenovirus treatment.

3T3 Cells↗
PubMed1996

Horseradish peroxidase-catalyzed sulfoxidation of promethazine and properties of promethazine sulfoxide.

Promethazine sulfoxide was obtained with a quantitative yield in a horse radish peroxidase-catalyzed reaction of promethazine and hydrogen peroxide and was also prepared by direct chemical synthesis. The enzymatic sulfoxidation of promethazine was studied in vitro as a function of pH, promethazine, and hydrogen peroxide concentration. Promethazine sulfoxide inhibits with an apparent K(i) of 59.7 microM at pH 5.5 the enzymatic reaction, followed spectrophotometrically, polarographically, potentiometrically, and luminometrically. The reaction was also inhibited by ascorbic acid (K(i) 26.8 microM) and glutathione (K(i) 41.8 microM). The spectrophotometric techniques employed, together with ESR spectrometry, allowed the identification of at least three radical species formed in the course of the reaction. Promethazine sulfoxide is devoid of the antioxidant effect exhibited by promethazine on rat brain synaptosomes. The sulfoxide also lacks photosensitizing action, while retaining the neuroleptic effect of the parent compound.

Animals↗