Showing papers by "Francesco Salvatore published in 2004"
TL;DR: It is concluded that AKT participates in the settings of endothelial dysfunction in SHR rats by impaired membrane localization and is involved in endothelium dysfunction in hypertension.
Abstract: Background— In hypertension, reduced nitric oxide production and blunted endothelial vasorelaxation are observed. It was recently reported that AKT phosphorylates and activates endothelial nitric oxide synthase and that impaired kinase activity may be involved in endothelial dysfunction. Methods and Results— To identify the physiological role of the kinase in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), we used adenoviral vectors to transfer the human AKT1 gene selectively to the common carotid endothelium. In vitro, endothelial vasorelaxations to acetylcholine, isoproterenol, and insulin were blunted in control carotids from SHR compared with WKY rats, and human AKT1 overexpression corrected these responses. Similarly, blood flow assessed in vivo by Doppler ultrasound was reduced in SHR compared with WKY carotids and normalized after AKT1 gene transfer. In primary cultured endothelial cells, we evaluated AKT phosphorylation, activity, and compartmentalization and observ...
90 citations
TL;DR: Butyrate could be effective in treating congenital chloride diarrhea and is easily administered, useful in preventing severe dehydration episodes, and may be a promising therapeutic approach for a long-term treatment in this rare and severe condition.
89 citations
TL;DR: The cytogenetic and molecular response (CgR, MR) to imatinib in 191 patients with late chronic-phase Philadelphia-positive CML, previously treated with interferon alpha is determined, concluding that in CCgRs the degree of MR may vary from 2 to more than 4 logs, and that there is a progressive decrease of transcript level by time.
84 citations
TL;DR: Six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene were insoluble; molecular graphics data suggest this is due to impaired folding.
Abstract: Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired functioning of human liver aldolase (B isoform; ALDOB). To-date, 29 enzyme-impairing mutations have been identified in the aldolase B gene. Here we report six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene. Three of these are missense mutations (g.6846T>C, g.10236G>T, g.10258T>C), one is a nonsense mutation (g.8187C>T) and two affect splicing sites (g.8180G>C and g.10196A>G). We have expressed in bacterial cells the recombinant proteins corresponding to the g.6846T>C (p.I74T), g.10236G>T (p.V222F), and g.10258T>C (p.L229P) natural mutants to study their effect on aldolase B function and structure. All the new variants were insoluble; molecular graphics data suggest this is due to impaired folding. © 2004 Wiley-Liss, Inc.
29 citations
TL;DR: An extensive molecular graphic analysis of the mutated enzymes suggests that the Glu206-->Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region.
Abstract: We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly346-->Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly346-->Ser mutant, and the Glu206-->Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly346Ser mutant had the same Km as the wild-type enzyme, but a 4-fold lower kcat. The Glu206-->Lys mutant had a Km approx. 2-fold higher than that of both the Gly346-->Ser mutant and the wild-type enzyme, and a kcat value 40% less than the wild-type. The Gly346-->Ser and wild-type enzymes had the same Tm (melting temperature), which was approx. 6-7 degrees C higher than that of the Glu206-->Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu206-->Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly346 is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site.
26 citations
TL;DR: Most laboratories from the present study already have experience in previous European external quality assessments for at least one genetic test; this can explain the higher analytical accuracy in the Italian external quality assessment with respect to quality control programmes in other countries.
Abstract: The first Italian national trial of external quality assessment in genetic testing was organised within the framework of the "Italian National Project for Standardisation and Quality Assurance of Genetic Tests". Sixty-eight Public Health Service laboratories volunteered for the trial, which involved molecular genetic tests (cystic fibrosis, beta-thalassaemia, familial adenomatous polyposis coli and fragile-X syndrome) and cytogenetic tests (prenatal and postnatal, the latter included cancer cytogenetics). The response rate was high (88.2%). The level of analytical accuracy was good, i.e., the percentage of laboratories that correctly genotyped all samples was 89.3% for cystic fibrosis, 90.9% for beta-thalassaemia, 100% for familial adenomatous polyposis coli (despite two laboratories did not complete the analysis because the amount of DNA was considered insufficient), and 90.5% for fragile-X syndrome. Written reports differed widely and were judged "inadequate" in over 50% of cases. Most laboratories from the present study already have experience in previous European external quality assessments for at least one genetic test; this can explain the higher analytical accuracy in the Italian external quality assessment with respect to quality control programmes in other countries. Collaborative networks are strongly suggested to improve the quality of the reports.
26 citations
TL;DR: A rapid, easy method based on amplified fragment length polymorphism to characterize the genetic patterns of C. rubrum was set up and was able to plot a “genetic identity card” of this commercially harvested species, which is also a marker of pollution.
Abstract: Corallium rubrum taxonomy is based on morphologic criteria; little is known about its genome. We set up a rapid, easy method based on amplified fragment length polymorphism to characterize the genetic patterns of C. rubrum in an attempt to understand better the evolutionary relations between species from diverse geographic areas and to help define migration patterns. Applying this procedure to C. rubrum specimens from Spain and Italy, we identified 6 AFLP amplification fragments common to the 4 coral populations studied and 4 fragments that differentiated between these populations. Using this characterization we were able to plot a “genetic identity card” of this commercially harvested species, which is also a marker of pollution.
17 citations
TL;DR: A quantitative PCR method is reported, followed by separation by capillary gel electrophoresis of the fluorescently labeled amplified exons of hot spot regions of the dystrophin gene, which allowed it to detect ∼99% patients (affected males and female carriers) with macrodeletions and 89% with macroduplications, and to identify small insertions or deletions in those regions.
Abstract: Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked allelic neuromuscular disorders that have a prevalence of 1 in 3500 live-born males. The genetic defect is the result of mutations of the dystrophin gene, which encodes a 427-kDa rod-shaped cytoskeletal protein (1). The locus is very unstable: one-third of all DMD/BMD cases represent new mutations without a family history of the disease (1). Approximately 50–70% of DMD/BMD cases are the result of macrodeletions, and partial gene duplications have been reported in ∼6% of patients (2). Both macrodeletions and macroduplications are preferentially clustered in two areas, the amino-terminal (exons 3–7) and the central (exons 44–55) regions (1). The remaining cases are presumably attributable to point mutations or small insertions/deletions (2) scattered along the entire gene. PCR detection of macrodeletions is very useful in the analysis of affected males (3)(4), but it provides no information about the carrier status of at-risk women. Carrier status within families is usually assessed by haplotype analysis (5), fluorescence in situ hybridization (6), amplification of ectopic transcripts (7)(8), dosage analysis on Southern blots (9), or separation of quantitative PCR products by gel electrophoresis (10). Semiquantitative methods, based on the separation of fluorescently labeled amplified exons by gel or capillary electrophoresis, are also available (11)(12)(13)(14)(15).
Here we report a quantitative PCR method, followed by separation by capillary gel electrophoresis of the fluorescently labeled amplified exons of hot spot regions of the dystrophin gene, which allowed us to detect ∼99% patients (affected males and female carriers) with macrodeletions and 89% with macroduplications, and to identify small insertions or deletions in those regions. This method is rapid, and unlike other procedures, it includes an internal standard to normalize amplification efficiency and to calculate a diagnostic index. We validated …
16 citations
TL;DR: Analysis of the whole coding regions of the CFTR gene revealed the rare mutation D579G in homozygosity, which is found in 10-month-old boy with hypotonic dehydration and metabolic alkalosis.
14 citations
TL;DR: In asymptomatic middle-aged women, HDL-C levels were independently and negatively associated with preclinical atherosclerotic changes of the carotid artery wall.
Abstract: BACKGROUND: Controversy remains on the relationship between high-density lipoprotein cholesterol (HDL-C) and atherosclerotic cerebrovascular disease. METHODS: Over 5000 women living in the area of Naples, Southern Italy, were recruited for a prospective study on the etiology of cardiovascular disease in the female population (the 'Progetto ATENA' study). A sample of 310 participants underwent high-resolution B-mode ultrasound examination and the intima-media thickness and diameters of common carotid artery were measured. In addition to routine biochemical tests, these women also had oxidation markers determined. RESULTS: Women in the upper HDL-C quartile (HDL-C>1.89 mmol/L) had significantly lower body mass index and waist-to-hip ratio values, and triglycerides concentrations when compared with women in the first three quartiles. A linear negative association was found between HDL-C and carotid intima-media thickness (1.07+/-0.16 mm for the IV quartile versus 1.10+/-0.20 mm for the III quartile, 1.15+/-0.26 mm for the II quartile and 1.19+/-0.23 mm for the I quartile; P<0.01 by ANOVA). No difference was found between groups with regard to carotid diameters and oxidation markers. After adjustment for other cardiovascular risk factors, women in the highest quartile of HDL-C had a decreased risk of carotid intima-media thickening (OR 0.42, 95%CI 0.23-0.94). CONCLUSIONS: In asymptomatic middle-aged women, HDL-C levels were independently and negatively associated with preclinical atherosclerotic changes of the carotid artery wall.
13 citations
TL;DR: The human aldolase C gene sequences studied drive endogenous‐like expression in the brain, and the 200 bp proximal promoter region is sufficient to drive low brain‐specific and stripe‐like β‐gal expression in Purkinje cells.
TL;DR: Imatinib is the first-line treatment for CML: its efficacy is very high and it seems to be well tolerated across age groups, and the effects of age on response and tolerance within the phase II trial of the italian GIMEMA CML Working Party are performed.