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Biomedical subjects

M Ferrari

Publications and source records attributed to M Ferrari.

At least 145 records · Page 8Linked to original sources
PubMed1997

Rapid detection of 21-hydroxylase deficiency mutations by allele-specific in vitro amplification and capillary zone electrophoresis.

A quick diagnosis of the classic form of 21-hydroxylase deficiency (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treatment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractory mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amplified. In the nested reaction, ARMS-detected wild-type and mutated alleles are separately pooled and resolved by CZE. CZE is performed in coated capillaries in the presence of 30 g/L hydroxyethyl cellulose in the background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophoresis buffer contains the fluorescent dye SYBR Green I. Laser-induced fluorescence detection is obtained by excitation at 488 nm and signal collection at 520 nm. Specificity and reproducibility of the protocols were established by using samples from 75 Italian families with 21-hydroxylase deficiency already genotyped by allele-specific oligonucleotide hybridization or direct sequencing. Whereas dot-blot is time consuming because of the high number of hybridizations with radioactive probes, this present protocol is more rapid, giving sufficient separation on CZE after PCR reactions without preconcentration or desalting of samples.

Adrenal Hyperplasia, Congenital↗
PubMed1997

Bonding mechanism of three "one-bottle" systems to conditioned and unconditioned enamel and dentin.

PURPOSE: This in vivo study investigated the formation of hybrid layer, resin tags and adhesive lateral branches, by use of the latest generation of enamel-dentin bonding systems ("one-bottle" systems) on conditioned and unconditioned enamel and dentin. MATERIALS AND METHODS: The dentin adhesives were tested on 24 flat dentin preparations made on facial surfaces of vital, periodontally compromised teeth. The experimental teeth were randomly divided in six groups of four samples each. Group 1: Prime & Bond 2.1 (conditioned dentin, CD); Group 2: Single Bond (CD); Group 3: Syntac Sprint (CD); Group 4: Prime & Bond 2.1 (unconditioned dentin, UD); Group 5: Single Bond (UD); Group 6: Syntac Sprint (UD). In the first three groups, the "one-bottle" systems were applied following the manufacturers' instructions, while in the other three groups the dentin and the enamel were not treated with phosphoric acid and the primer was applied directly on the prepared surfaces. On top of the primer-adhesive, a thin layer of resin was applied and light-cured for 20 seconds. The sample teeth were extracted immediately after the adhesive material was light-cured. All the samples were split-fractured along their long axis. Half of the samples were deproteinized and decalcified at the interface in order to visualize the hybrid layer and the other halves were completely dissolved in order to observe the morphology of resin tags by use of scanning electron microscopy. RESULTS: Groups 1-3: All the tested products showed the same micromechanical bonding mechanism to conditioned dentin with phosphoric acid: they formed hybrid layer, resin tags and adhesive lateral branches. In the samples of the first three groups, characteristic reverse cone-shaped tags with their corresponding adhesive lateral branches were evident. At the enamel site, traditional pattern of etch enamel was always observed. Groups 4-6: When the bonding systems were applied on unconditioned dentin, hybrid layer was not formed, resin tags were rarely noted and their shape was narrow and they did not completely seal the tubular orifices. The tubules remained mainly closed by smear plugs. At the enamel site, the unetched surface showed absence of characteristic etch pattern and of resin tags.

Acid Etching, Dental↗
PubMed1997

Effect of two etching times on the sealing ability of Clearfil Liner Bond 2 in Class V restorations.

PURPOSE: To investigate the sealing ability of the Clearfil Liner Bond 2 self-etching primer in Class V restorations placed under clinical and laboratory conditions. MATERIALS AND METHODS: Firstly, the influence of two etching times of the self-etching primer were tested in vitro. In 10 Class V cavities (Group 1), the self-etching primer was applied for 30 seconds on the enamel and dentin, while in another 10 Class V cavities (Group 2) the etching time was 60 seconds. Since the Group 2 samples showed the best in vitro sealing ability at both the enamel and cementum-dentin sites, the teeth of Group 3 (10 restorations) were restored as in Group 2 under clinical conditions, applying the self-etching primer for 60 seconds. The sample teeth were extracted after 65-90 days, and then processed for leakage. Two other groups of 10 samples each were prepared under laboratory conditions to investigate hybrid layer, resin tags and adhesive lateral branch formation. A flat buccal surface on each extracted tooth was prepared. Group 4 samples were conditioned for 30 seconds, and those of Group 5 for 60 seconds. RESULTS: In Group 1, 40% of the restorations showed leakage at the cementum-dentin site and 40% at the enamel site. Groups 2 and 3 showed statistically significant difference (less leakage) than Group 1. In Group 2 and 3 no leakage was found at the enamel site, and only a moderate leakage (10% and 20% of restorations, respectively) was found at the cementum-dentin site. The scanning electron microscopy observations of Group 4 samples showed a thin hybrid layer, with well-fitting and smooth resin tags and adhesive lateral branches only sporadically. Group 5 samples presented a thicker hybrid layer, rough and deep resin tags with many adhesive lateral branches. The etch enamel pattern was more uniform and rougher in Group 5 than in Group 4. The 60-second application time of the self-etching primer seems to be more reliable than a shorter conditioning time in day-to-day practice.

Acid Etching, Dental↗
PubMed1997

Interdiffusion of a traditional glass ionomer cement into conditioned dentin.

PURPOSE: To investigate whether a new auto-curing glass ionomer cement (Fuji IX) in combination with polyacrylic acid (Cavity Conditioner) forms an interdiffusion zone both under laboratory and clinical conditions. MATERIALS AND METHODS: Seven vital human posterior teeth were selected. A flat dentin surface was created on the buccal surface in order to apply the glass ionomer material. Seven other extracted human molars were prepared following the same procedure as with the in vivo experimental teeth. After extraction of the in vivo restored teeth, the samples were cut and prepared for hybrid layer observation under scanning electron microscope. RESULTS: An interdiffusion zone, of approximately 6 microns thick, was observed both under in vivo and in vitro conditions. At the interface between the two substrates, the interdiffusion zone was acid resistant and uniform.

Acrylic Resins↗
PubMed1996

Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4.

Genes for familial hemiplegic migraine (FHM) and episodic ataxia type-2 (EA-2) have been mapped to chromosome 19p13. We characterized a brain-specific P/Q-type Ca2+ channel alpha1-subunit gene, CACNL1A4, covering 300 kb with 47 exons. Sequencing of all exons and their surroundings revealed polymorphic variations, including a (CA)n-repeat (D19S1150), a (CAG)n-repeat in the 3'-UTR, and different types of deleterious mutations in FHM and EA-2. In FHM, we found four different missense mutations in conserved functional domains. One mutation has occurred on two different haplotypes in unrelated FHM families. In EA-2, we found two mutations disrupting the reading frame. Thus, FHM and EA-2 can be considered as allelic channelopathies. A similar etiology may be involved in common types of migraine.

Base Sequence↗
PubMed1996

Autoantibodies in insulin-dependent diabetes recognize distinct cytoplasmic domains of the protein tyrosine phosphatase-like IA-2 autoantigen.

Protein tyrosine phosphatase-like IA-2 is a major autoantigen in insulin-dependent diabetes. It has been identified as both a specificity of cytoplasmic islet cell Abs and one of the precursors of the 40- and 37-kDa tryptic fragment islet autoantigens. To characterize autoantibody binding to IA-2 and determine whether humoral autoimmunity extends to other tyrosine phosphatases, we analyzed serum reactivity in 100 patients with insulin-dependent diabetes against different in vitro translated portions of the IA-2 protein as well as the tyrosine phosphatase domains of HPTPbeta and HPTPdelta. All autoantibody reactivity was confined to the cytoplasmic portion of IA-2 (amino acids 601-979). At least four epitopes were found. These were contained within amino acids 605 to 620 and 605 to 682 of the juxtamembrane region and within amino acids 777 to 937 and 687 to 979 in the IA-2 tyrosine phosphatase-like domain. Footprinting studies confirmed the presence of multiple epitopes. Fifty-six percent of sera with IA-2 Abs bound epitopes within the juxtamembrane region, and 83% bound epitopes in the tyrosine phosphatase-like domain; 39% had Abs to both regions. No reactivity against the IA-2 ectodomain or the tyrosine phosphatase domains of HPTPbeta and HPTPdelta was found. These data suggest that the cytoplasmic region, in particular the tyrosine phosphatase-like domain, is the major target of IA-2 Abs in insulin-dependent diabetes, and that autoantibody reactivity is specific for IA-2 or IA-2-like molecules.

Adolescent↗
PubMed1996

In vivo detection of mouse liver nitric oxide generation by spin trapping electron paramagnetic resonance spectroscopy.

Nitric oxide, a paramagnetic molecule synthesized in biological systems, plays an important role in many pathophysiological processes. In vivo electron paramagnetic resonance spectroscopy/imaging could be a useful tool to study, in situ and in real time, nitric oxide generation. In this study the intracellular production of nitric oxide in tissues of living septic-shock mouse was detected by the spin trapping technique in combination with electron paramagnetic resonance spectroscopy. A lipophilic spin trap agent was used and nitric oxide formation was determined by the intensity of its iron-mononitrosyl complex. Among all examined tissues at 20 degrees C, the highest signal intensity of the trapped nitric oxide was found in the liver homogenates (n = 5). The amount of complex found in the kidneys was about 40% of that found in the liver. In the brain and lung, around 10% was found. This study reports, for the first time, the in vivo detection of nitric oxide generation in the upper abdomen of septic-shock mice (n = 3). Within 1 h after the trap injection, the signal was stable, indicating that the formation had reached a steady state.

Animals↗
PubMed1996

Phosphorylation of the DNA polymerase alpha-primase B subunit is dependent on its association with the p180 polypeptide.

The B subunit of the DNA polymerase (pol) alpha-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner. In this report, we show that the four subunits of the yeast DNA polymerase alpha-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol alpha (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180.p86 interaction. Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180.p86 subcomplex is required for B subunit phosphorylation.

Cell Cycle↗
PubMed1996

PTCA with the use of cardiac assist devices: risk stratification, short- and long-term results.

Percutaneous cardiopulmonary assist devices (PCPS) have become available in interventional cardiology within recent years. These tools offer the opportunity of performing percutaneous transluminal coronary angioplasty (PTCA) in high-risk patients characterized by significant stenoses of several coronary arteries and a poor left ventricular function. It is unclear for which patients PCPS are necessary and which patients will profit by PTCA as compared to coronary artery bypass grafting (CABG). Therefore, the anticipated risk of CABG and of PTCA without assist devices was calculated according to risk scores and compared with our results of assisted PTCA. In addition the long-term survival rate was investigated. In 35 patients (mean 65.5 years of age, 12 females, 23 males), we performed PTCA concomitant with the use of cardiac assist devices. The indications for the use of a cardiac assist device were severely impaired LV function (EF 30% +/- 8.9%) in combination with significant coronary artery disease (2.7 +/- 0.3 vessels) and a significant supply area of the vessel to be dilated. In 6 patients, PCPS was started before coronary angioplasty because of hemodynamic instability. In 21 cases, PCPS was on a standby basis without being connected to the patient's circulation. In 8 patients, a left heart assist device, the 14F-Hemopump, was inserted percutaneously. The patients were analyzed using risk scores of angioplasty and of coronary bypass graft surgery. The calculated risk of hemodynamic compromise during PTCA according to the risk scores was more than 50%. The anticipated risk of a fatal outcome following CABG would have been 19.8%. PTCA was performed on an average of 2.0 coronary arteries per patient and was successful in 85%. We observed a decline in angina pectoris classification (CCS) from 3.5 to 1.6. An average reduction of 1.1 NYHA class was achieved. The in-hospital mortality was 8.6% (3 patients: 1 x sepsis, 1 x early reocclusion, 1 x cerebral embolism). At 24 months follow-up, a re-PTCA was necessary in four cases because of restenosis. In the remainder, NYHA and CCS class were stable during the follow-up period. An additional five patients died during the first year and two patients in the second year. We conclude that PTCA with the use of a cardiac assist device shows favorable short-term results in a subset of patients with extended coronary artery disease and severely impaired LV function who are not suitable for nonsupported PTCA or CABG due to their risk profile. However, the long term results are not satisfying and stress the need for complete revascularisation with CABG once the patient's condition is stabilized by means of supported PTCA.

Aged↗
PubMed1996

Genetic contribution of polymorphism of the GLUT1 and GLUT4 genes to the susceptibility to type 2 (non-insulin-dependent) diabetes mellitus in different populations.

Polymorphic variation of genes encoding the glucose transporters glycoproteins (GLUT) may contribute to the genetic susceptibility to type 2 (non-insulin-dependent) diabetes. In this study we evaluated the allele and genotype frequencies of GLUT1 and GLUT4 restriction fragment length polymorphism (RFLP), revealed by digestion with XbaI for GLUT1 and KpnI for GLUT4, in Caucasian, Chinese, Japanese, Asian Indian and American black populations. No differences of the KpnI GLUT 4 RFLP were found between control and diabetic subjects in any ethnic group or when all data are combined. In contrast, positive results were found for the XbaI RFLP: (1) most ethnic groups showed an association of allele 1 with type 2 diabetes, and this association was maintained when all groups were analysed together; (2) after stratifying for sex and obesity, this association was significant only for overweight/obese women. This joint analysis suggests that GLUT1 polymorphism may contribute to susceptibility to type 2 diabetes in some populations, and especially in overweight/obese women.

Adult↗
PubMed1996

Present status of electron paramagnetic resonance (EPR) spectroscopy/imaging for free radical detection.

Our group and others have been working on the development of EPR imaging to obtain "in vivo" free radical images. Using a 280 MHz apparatus a pyrrolidine nitroxide free radical was localized in the rat abdomen and thorax during its kinetic in whole body. We investigated the role of different experimental and instrumental parameters on the resolution of the images. The conclusion of these studies is that the instrumentation presents the advantage to perform EPR measurements on whole body rats with limited sensitivity (50 microM). The present resolution (8 mm) allows to get images in which it is not possible to resolve all the organs; instead L-band instruments provide images of single organs (up to 25 mm in size) with a higher resolution.

Animals↗
PubMed1996

Clinicopathological and genetic studies of two further Italian families with cerebral autosomal dominant arteriopathy.

We report on two Italian families with an early-adult onset autosomal dominant disorder, characterized by leukoencephalopathy, migraine, psychiatric disturbances, stroke and dementia. These findings fulfill the diagnostic criteria for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome. Moreover, to confirm the CADASIL gene location to 19p12, we performed a linkage analysis with four microsatellite markers. The results of the genetic study gave positive but not significant lod scores, indicating only weak evidence of a linkage with 19p12. In one autopsy case, we found extensive ischemic changes due to the selective involvement of the small muscular arteries of the cerebral white matter. The lesions consisted of a thickening of the media with deposition of granular eosinophilic material. Ultrastructural examination of the arterial walls showed graded damage to smooth muscle cells, mostly of the longitudinal layer, and an abnormal proliferation of basal lamina components. Immunocytochemical analysis showed strong reactivity using antibodies to collagen IV and smooth myosin proteins. The results suggest a primary involvement of the smooth muscle cells of small cerebral arteries, with a secondary alteration of basal lamina components and elastic tissue.

Adult↗
PubMed1996

Short direct repeats at the breakpoints of a novel large deletion in the CFTR gene suggest a likely slipped mispairing mechanism.

In the cystic fibrosis conductance transmembrane regulator (CFTR) gene a few small deletions and only a large, complex, 50-kb deletion have been described so far. We report a second large deletion, which had been hypothesized in a patient affected by cystic fibrosis on the basis of an abnormal pattern of inheritance of the intragenic microsatellites IVS17b/TA and IVS17b/CA. Southern blot analysis revealed the presence of an anomalous band in the patient and her father, in the region encompassing exons 13 - 19, approximately 0.6 kb shorten than the one present in normal controls, in addition to the band of the correct size. Cloning and sequencing the DNA fragments spanning the region of interest demonstrated the presence of a 703-bp deletion causing complete removal of exon 17b in the paternal cystic fibrosis chromosome. This analysis revealed the presence of two short direct repeats flanking the breakpoints. The 3' repeat partially overlapped the IVS17b/CA microsatellite and the number of CA repeated units present in the paternal cystic fibrosis allele was the shortest ever found among chromosomes so far analyzed. These data may suggest that the mechanism for the generation of the deletion may have involved a slipped mispairing during DNA replication, which has not previously been described in the CFTR gene.

Adult↗
PubMed1996

Detection of a de novo R1066H mutation in an Italian patient affected by cystic fibrosis.

Search for mutations in a cystic fibrosis patient, compound heterozygous for 1717-1G-->A and another uncharacterized molecular defect, revealed the presence of a de novo R1066H mutation on the affected chromosome of paternal origin. Three additional rare mutations (R1066C, R1066S and R1066L), occurring at the CpG dinucleotide at position 3328-3329 of the cystic fibrosis transmembrane conductance regulator gene, have so far been reported. The identification of a R1066H de novo mutation further suggests that this dinucleotide may constitute a mutational hotspot.

Adolescent↗
PubMed1996

Point mutations in Italian patients with classic, non-classic, and cryptic forms of steroid 21-hydroxylase deficiency.

Seventy-three Italian patients affected by steroid 21-hydroxylase deficiency were studied by a PCR-allele-specific oligonucleotide protocol in order to evaluate the presence of eight known point mutations. The majority of chromosomes were found to carry point gene conversions normally present in the pseudogene. Within the classic form, the most common mutations were the splicing mutation A/C-655 to G in intron 2 (34.2%), the nonsense mutation C-1993 to T in exon 8 (10.8%), and the missense mutation T-999 to A in exon 4 (10%). Within the non-classic form, the missense mutation G-1683 to T was the most common (57.7%). Other mutations were either absent, such as the three clustered missense mutations T-1380, T-1383, T-1389 to A in exon 6, or very rare, like the 1761 + T in exon 7 and the C-2108 to T in exon 8. Family genotyping revealed the presence of ten asymptomatic parents carrying mutations in both chromosomes, thus identifying the gene defect in cryptic subjects. Interestingly, the same mutations were found in both symptomatic and asymptomatic forms.

Adrenal Hyperplasia, Congenital↗