Search for genetic variants in the p66Shc longevity gene by PCR-single strand conformational polymorphism in patients with early-onset cardiovascular disease.

Background: Among the possible candidate genes for atherosclerosis experimental data point towards the longevity gene p66(Shc). The p66(Shc) gene determines an increase of intracellular reactive oxygen species (ROS), affecting the rate of oxidative damage to nucleic acids. Knock-out p66(Shc-/-) mice show reduction of systemic oxidative stress, as well as of plasma LDL oxidation, and reduced atherogenic lesions. Thus, p66(Shc) may play a pivotal role in controlling oxidative stress and vascular dysfunction in vivo. Methods: We searched for sequence variations in the p66(Shc) specific region of the Shc gene and its upstream promoter by PCR-SSCP in a selected group of early onset coronary artery disease ( CAD) subjects (n. 78, mean age 48.5 +/- 6 years) and in 93 long-living control subjects ( mean age 89 +/- 6 years). Results: The analysis revealed two variant bands. Sequencing of these variants showed two SNPs: -354T > C in the regulatory region of p66(Shc) locus and 92C > T in the p66 specific region (CH2). Both these variants have never been described before. The first substitution partially modifies the binding consensus sequence of the SpI transcription factor, and was detected only in two heterozygous carriers (1 CAD subjects and 1 control subject). The 92C > T substitution in the CH2 region consists in an amino acid substitution at codon 31 (proline to leucine, P31L), and was detected in heterozygous status only in one CAD subject. No subjects homozygous for the two newly described SNPs were found. Conclusion: Only two sequence variations in the p66(Shc) gene were observed in a total of 171 subjects, and only in heterozygotes. Our observations, in accordance to other studies, suggest that important variations in the p66(Shc) gene may be extremely rare and probably this gene is not involved in the genetic susceptibility to CAD

Biochemical assessment of red blood cells during storage by 1H nuclear magnetic resonance spectroscopy. Identification of a biomarker of their level of protection against oxidative stress

Background. Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions. Materials and methods. Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of 1H-NMR spectra. Results. The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile. Discussion. We proved the analytical power of 1H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy

Search for genetic variants in the p66^Shclongevity gene by PCR-single strand conformational polymorphism in patients with early-onset cardiovascular disease

Abstract Background Among the possible candidate genes for atherosclerosis experimental data point towards the longevity gene p66Shc. The p66Shc gene determines an increase of intracellular reactive oxygen species (ROS), affecting the rate of oxidative damage to nucleic acids. Knock-out p66Shc-/- mice show reduction of systemic oxidative stress, as well as of plasma LDL oxidation, and reduced atherogenic lesions. Thus, p66Shc may play a pivotal role in controlling oxidative stress and vascular dysfunction in vivo. Methods We searched for sequence variations in the p66Shc specific region of the Shc gene and its upstream promoter by PCR-SSCP in a selected group of early onset coronary artery disease (CAD) subjects (n. 78, mean age 48.5 ± 6 years) and in 93 long-living control subjects (mean age 89 ± 6 years). Results The analysis revealed two variant bands. Sequencing of these variants showed two SNPs: -354T>C in the regulatory region of p66Shc locus and 92C>T in the p66 specific region (CH2). Both these variants have never been described before. The first substitution partially modifies the binding consensus sequence of the Sp1 transcription factor, and was detected only in two heterozygous carriers (1 CAD subjects and 1 control subject). The 92C>T substitution in the CH2 region consists in an amino acid substitution at codon 31 (proline to leucine, P31L), and was detected in heterozygous status only in one CAD subject. No subjects homozygous for the two newly described SNPs were found. Conclusion Only two sequence variations in the p66Shc gene were observed in a total of 171 subjects, and only in heterozygotes. Our observations, in accordance to other studies, suggest that important variations in the p66Shc gene may be extremely rare and probably this gene is not involved in the genetic susceptibility to CAD.</p

Characterizing macular edema in retinitis pigmentosa through a combined structural and microvascular optical coherence tomography investigation

Abstract The aim of the study was to characterize macular edema (ME) in retinitis pigmentosa (RP) by means of quantitative optical coherence tomography (OCT)-based imaging. The study was designed as observational, prospective case series, with 1-year follow-up. All RP patients underwent complete ophthalmologic assessment, including structural OCT, OCT angiography, and microperimetry (MP). The primary outcome was the characterization through quantitative OCT-based imaging of RP eyes complicated by ME. A total of 68 RP patients’ eyes (68 patients) and 68 eyes of 68 healthy controls were recruited. Mean BCVA was 0.14 ± 0.17 LogMAR at baseline and 0.18 ± 0.23 LogMAR at 1-year follow-up (p > 0.05). Thirty-four eyes (17 patients; 25%) showed ME, with a mean ME duration of 8 ± 2 months. Most of the eyes were characterized by recurrent ME. The ME was mainly localized in the inner nuclear layer in all eyes. LogMAR BCVA was similar in all RP eyes, whether with or without ME, although those with ME were associated with higher vessel density values, as well as thicker choroidal layers, than those without ME. In conclusion, the inner retina is closely involved in the pathogenesis of ME. The impairment of retinal-choroidal exchanges and Müller cell disruption might be a major pathogenic factor leading to the onset of ME in RP