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Biomedical subjects

L Mazzanti

Publications and source records attributed to L Mazzanti.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase.

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.

Adenosine Triphosphate↗
PubMed2000

Age-related modifications in human unstimulated whole saliva: a biochemical study.

Human whole saliva contains a number of antimicrobial agents, and lysozyme, lactoferrin, secretory IgA and peroxidase are among the best known. Peroxidase catalyzes a reaction involved in the inhibition of bacterial growth and metabolism, and the prevention of hydrogen peroxide accumulation, thus protecting proteins from the action of oxygen and reactive oxygen species (ROS). To better understand the role played by the oxidative stress in the aging process, we studied the relationship between total protein content, peroxidase activity, malondialdehyde (MDA) and nitric oxide (NO) content of human unstimulated whole saliva in 169 healthy subjects subdivided into groups according to age. Our results show a significant decrease in peroxidase activity with age. Moreover, the increase in saliva lipid peroxide levels indicates an enhanced free radical production that may contribute to tissue damage. On the other hand, findings concerning human unstimulated whole saliva NO content showed a significant increase in elderly subjects, suggesting that an enhanced NO production might depend on a stimulation of leukocyte-inducible NO synthase (i-NOS) activity. Our results suggest that during aging the oral tissues may become more susceptible to environmental factors due to a modification in the balance between different antimicrobial agents.

Adolescent↗
PubMed2000

Nuclear matrix provides linkage sites for translocated NF-kappa B: morphological evidence.

In response to the binding of extracellular ligands to cell surface receptors, multiple transcription factors are activated in the cytoplasm and translocated into the nucleus where they exert positive or negative control over cellular genes. The human transcription factor NF-kappa B family regulates the expression of a large number of genes involved in the host defence mechanism. They are typically present in the cytoplasm bound to the inhibitory I kappa B proteins. The activation of NF-kappa B involves the signal-induced degradation of these proteins, allowing NF-kappa B to translocate to the nucleus. In this study, by multiparametric analysis, we recognise in RPMI-8402 DMSO-activated cells the intracellular movement of transcription factor NF-kappa B providing its definite intranuclear collocation. Intact cells, purified nuclei and nuclear matrix preparations after 4 h of treatment were processed for morphological and biochemical analyses. Light and electron microscope observations show, in untreated cells, the presence of NF-kappa B protein homogeneously retained in the cytoplasm. Treated cells display a massive presence of NF-kappa B at the nuclear level bound to the interchromatin region. Immunoblotting of the same specimens confirms the strong association of NF-kappa B with the nuclear scaffold. Taken together, the data presented in this manuscript support a model where DMSO treatment provokes the cleavage and translocation of NF-kappa B from the cytoplasm to the nucleus and, in particular, in the proteinaceous network of the nuclear matrix sustaining the active role of this subcellular structure on regulation of eukaryotic gene expression.

Biological Transport↗
PubMed1999

N-dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols.

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.

Animals↗
PubMed1999

Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.

AIMS/HYPOTHESIS: The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. METHODS: Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting. RESULTS: In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. CONCLUSION/INTERPRETATION: Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.

Adult↗
PubMed1999

Involvement of the skull base and vault in chronic idiopathic hyperphosphatasia.

Chronic idiopathic hyperphosphatasia (CIH) is a rare generalised skeletal dysplasia in childhood. The clinical, radiographic and cerebral MR findings in a 5-year-old girl with the severe infantile form of CIH are reported. In spite of cranial enlargement, the intracranial space and the skull base were markedly reduced, the whole brain was compressed and a Chiari I malformation was present. Normal flow in the dural venous sinuses was documented. The patient showed no detectable cranial nerve involvement or hydrocephalus. Cranial MR in this patient enabled us to confirm that CIH involves the skull base and vault.

Abnormalities, Multiple↗
PubMed1999

A study on human umbilical cord endothelial cells: functional modifications induced by plasma from insulin-dependent diabetes mellitus patients.

The aim of the present study was to evaluate the action of plasma from insulin-dependent diabetic (IDDM) pregnant women on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs). We also studied the effect of the plasma on cytosolic calcium and on Na+/K+-adenosine triphosphatase (ATPase) activity. Dynamic fluorescence studies of membrane fluidity were contemporarily performed to detect a direct effect of plasma on the endothelial cell membrane. We observed a significant increase in NOS activity, intracellular calcium, and Na+/K+-ATPase activity in cultured HUVECs exposed to IDDM plasma. Our dynamic fluorescence study showed a different microenvironmental organization of the cellular membrane after incubation with plasma from IDDM pregnant women, with a marked decrease in microheterogeneity as evaluated in terms of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) lifetime distribution width. The present investigation suggests that plasma from IDDM pregnant women can cause a generalized disturbance in the function of endothelial cells cultured from healthy subjects. Such a modification might play a central role in the pathogenesis of the vascular complications of the disease.

Adult↗
PubMed1999

Increased susceptibility to peroxidation of VLDL from non-insulin-dependent diabetic patients: a possible correlation with fatty acid composition.

Recent studies suggested that both oxidized very low density lipoproteins (VLDL) and oxidized high density lipoproteins (HDL) might play a role in the pathogenesis of atherosclerosis. The aim of the present work was to analyse the susceptibility to in vitro peroxidation of VLDL and HDL from apparently normolipidemic subjects affected by insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) in good metabolic control and to examine the possible relations between oxidisability and lipoprotein fatty acid composition. VLDL and HDL were isolated from 13 IDDM patients, 12 NIDDM patients and 18 healthy subjects. The degree of lipoprotein oxidation was determined by the measurement of hydroperoxide levels and thiobarbituric acid-reactive substances (TBARS) before and after in vitro peroxidative stress with CuSO4. Fatty acid analysis was performed by gas chromatography. VLDL and HDL from NIDDM patients showed a decrease in the saturated fatty acid content with a concomitant increase in unsaturated fatty acids and higher basal peroxide levels compared with healthy subjects. Oxidisability of VLDL from NIDDM subjects was higher than in controls and was significantly related with the unsaturated fatty acid content. The present work suggests that alterations in the composition and functions of both VLDL and HDL able to produce more atherogenic lipoproteins are present in NIDDM.

Adult↗
PubMed1999

Physicochemical and functional modifications induced by obesity on human erythrocyte membranes.

BACKGROUND: Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated. DESIGN: Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance. RESULTS: Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels. CONCLUSION: Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.

Adult↗
PubMed1999

Reduced spontaneous growth hormone secretion in patients with Turner's syndrome.

We evaluated growth hormone (GH) secretion in 81 patients with Turner's syndrome (TS) (mean age 10.7+/-3.6 y) with respect to karyotype, auxological characteristics and growth response to GH treatment (1 IU/kg/wk). None of the patients had spontaneous puberty or had started replacement therapy with estrogens. Thirty-nine patients (48%) had monosomia 45X, 29 (36%) structural abnormalities of the X chromosome and 13 (16%) X mosaicism. Before the start of GH therapy, each patient underwent an evaluation of mean nocturnal GH concentration (MGHC) and 75 patients also underwent 2 pharmacological tests. MGHC of the TS patients did not differ from that of 29 prepubertal GH-deficient girls (GH peaks < 8 microg/l after pharmacological tests) and both groups were lower (p < 0.0001 and p < 0.0005, respectively) than MGHCs of 27 short normal girls (GH peak > 8 microg/l). MGHC of the patients with TS was negatively correlated (p < 0.001) with bodyweight excess (BWE) at multiple regression analysis. MGHC of the TS patients with BWE < 20% was significantly higher (p < 0.02) than that of the TS patients with BWE > 20%, but again did not differ from that of the GH-deficient patients and was lower (p < 0.001) than that of the short normal girls. MGHC did not significantly differ between the 3 groups subdivided according to karyotype. Forty-four percent of the TS patients showed GH responses to pharmacological tests < 8 microg/l. Height velocity SDS at first and second year of therapy was not influenced by MGHC levels, chronological or bone age, target height or BWE. In conclusion, spontaneous secretion in our patients with TS was lower than that of the short normal prepubertal girls and did not differ from that of GH-deficient subjects, even if we excluded overweight patients. The level of GH secretion was unable to predict GH response to treatment.

Adolescent↗
PubMed1999

Helicobacter pylori and type 1 diabetes mellitus in children.

BACKGROUND: Helicobacter pylori is a recognized gastroduodenal pathogen and H. pylori infection is one of the most common bacterial infections, usually acquired during childhood. However, diabetes mellitus is characterized by an increased susceptibility to infections. METHODS: We compared the prevalence of H. pylori infection as well as cytotoxin-associated gene A-CagA-and vacuolating cytotoxin gene A-VacA-positivity in 103 children and adolescents with type 1 diabetes mellitus and in 236 nondiabetic children. We used a novel Recombinant ImmunoBlot Assay-Strip (RIBA SIA) with individual band for whole H. pylori lysate and recombinant CagA and VacA. RESULTS: H. pylori-positive subjects, both diabetics and controls, were significantly older than negative subjects. In the whole group of diabetic patients the prevalence of each of the three reactivities was higher than in control subjects, reaching significance only for lysate. Only diabetic patients over 12 years of age, with a longer disease duration, had a higher prevalence of positive cases, although not significantly so. CONCLUSIONS: In the first few years of disease, diabetic children do not differ from the nondiabetic population. Subsequently they show an H. pylori seroprevalence tendentially higher than that of controls of the same age. Therefore, H. pylori infection acquired in childhood and lasting several years, could be one of the causes of chronic atrophic gastritis, which is more frequent in longstanding diabetes mellitus.

Adolescent↗
PubMed1999

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Adenosine Diphosphate↗
PubMed1999

Final height of patients with Turner's syndrome treated with growth hormone (GH): indications for GH therapy alone at high doses and late estrogen therapy. Italian Study Group for Turner Syndrome.

We report final height data of patients with Turner's syndrome collected by the Italian Study Group for Turner's Syndrome. One hundred and thirty-five patients reached their final height during GH therapy with different therapeutic regimens (dose and combination). They were divided into 3 groups: group A, 74 patients with high doses of GH (1 IU/kg/week) for at least 2 yr; group A1, GH alone and estrogen therapy added not before 14 yr of chronological age (47 patients, of whom 30 were treated for >4 yr and 10 for >6 yr); group A2, GH plus ethinyl estradiol (17 patients) or GH plus oxandrolone (10 patients); group B, 51 patients with low doses of GH (0.5 IU/kg-week) and high doses of GH for less than 2 yr; and group C, 10 patients with high doses of GH with spontaneous menarche. In contrast to the patients of groups B and C, the patients of group A showed a significantly higher final height (mean, 147.5+/-6.5 cm) than their projected height (mean, 142.9+/-6.4 cm). They showed also a significantly higher final height compared to the subjects of groups B (mean, 145.6+/-5.7 cm) and C (mean, 143.0+/-5.3). Among the patients of group A, the best results were obtained in the patients of group A1 treated with GH alone at high doses and for a longer period (4 yr, 149.3+/-6.4 cm; 6 yr, 153.8+/-4.0 cm). Karyotype, GH secretion, and birth weight did not influence the efficacy of GH therapy. A low target height and a high prevalence of a spontaneous ovarian activity or menarche may negatively influence the effect of GH therapy. Estrogens did not improve final height when added to GH therapy. The use of small doses of oxandrolone was not effective in our experience. GH therapy provides a satisfactory auxological result, especially with high doses of GH alone, given for a long period of time. Optimization of the treatment would seem to require the identification of the ideal age for starting therapy, and this is only possible with a specially designed multicenter study.

Adolescent↗
PubMed1999

Modifications induced by LDL from type 1 diabetic patients on endothelial cells obtained from human umbilical vein.

The aim of the present work was to analyze the effect of LDL obtained from type 1 diabetic patients in good metabolic control on human umbilical vein endothelial cells (HUVECs) after a short incubation period to detect possible atherogenic modifications of endothelial properties. Cultured HUVECs were incubated for 3 h with culture medium alone (control HUVEC), with native LDL from 12 healthy men (control LDL), or with native LDL from 12 type 1 diabetic men (type 1 LDL) (100 pg/ml). After the incubation, the following parameters were evaluated: nitric oxide synthase (NOS) activity, cytoplasmic Ca2+ levels, Na+-K+-ATPase activity, plasma membrane fluidity determined by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and plasma membrane conjugated diene (CD) content. The same experiments were repeated after bradykinin stimulation or in the presence of the antioxidant butylated hydroxytoluene (BHT), and nitric oxide (NO) production in intact HUVECs was also evaluated. HUVECs incubated with control LDL in comparison with control HUVECs showed a decreased fluidity of the membrane surface evaluated by TMA-DPH and a higher CD content. These alterations were prevented by the presence of BHT. HUVECs incubated with type 1 LDL in comparison with both control HUVECs and cells incubated with control LDL showed 1) increased NOS and Na+-K+-ATPase activity, cytoplasmic Ca2+ levels, and CD content, and 2) decreased fluidity of the membrane surface evaluated by TMA-DPH. These modifications were blunted--but not abolished--by the presence of BHT. After bradykinin stimulation either in the absence or in the presence of BHT, both cytoplasmic Ca2+ levels and NO production were increased in control HUVECs and in HUVECs incubated with control LDL, while a reduced response was observed in HUVECs incubated with type 1 LDL. The alterations observed in the endothelial function after the cell-LDL interaction might play a central role in the atherogenic process in diabetes.

Adult↗
PubMed1999

Changes in the composition of human unstimulated whole saliva with age.

The aim of this study was to evaluate changes in the concentration of certain components of human unstimulated whole saliva during aging, in order to better understand the role played by aging in oral health. In particular, we studied total protein concentration, alpha-amylase activity, sialic acid content and calcium and phosphorus concentrations in 100 healthy subjects of both genders, aged between 10 and 80 years, who were subdivided into four groups according to their age: 10-25 years, 26-40 years, 41-65 years, and 66-80 years. Other than sialic acid, the concentrations of the components studied were not affected by age. There was a significant negative correlation between sialic acid content and age. Our data indicate the presence of a decreased submandibular/sublingual function with aging, thus suggesting the possibility of a concomitant reduction in the modulating action of unstimulated whole saliva on the oral flora.

Adolescent↗