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Biomedical subjects

B Lunghi

Publications and source records attributed to B Lunghi.

35 records · Page 2Linked to original sources
PubMed1996

Effect of 1,25-dihydroxyvitamin D3 on proliferation in senescent IMR-90 human fibroblasts.

The response of IMR-90 human fetal lung fibroblasts at high population doubling level (PDL > 42) to 1,25-dihydroxyvitamin D3[1,25(OH)2D3] was investigated to clarify whether some metabolic and molecular parameters of senescent cells are affected by the hormone treatment. Pyruvate kinase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity significantly increased after treatment of confluent-phase cells with 10 nM 1,25(OH)2D3 for 24 h. Steroid specificity was established by the failure of 10 nM levels of 25-hydroxyvitamin D3 to affect the enzyme activities, while estradiol-17 beta and progesterone produced a slight increase in glucose-6-phosphate dehydrogenase and lactate dehydrogenase levels, respectively. 1,25(OH)2D3 also affected fibroblast proliferation, protein content/cell and DNA synthesis. The cell number significantly decreased after a 48 h incubation with 1,25(OH)2D3 at various concentrations (0.01-1 nM) when compared with control fibroblasts, while an increase in the protein content/cell was demonstrated. The same experiment, carried out by protracting the incubation with the hormone for 72 h, showed a similar trend, but 10 nM 1,25(OH)2D3 was also able to inhibit cell proliferation and to stimulate protein synthesis. The incorporation of [3H]thymidine into DNA increased after the treatment of high PDL fibroblasts with 0.01-1 nM of hormone for 48 h in comparison with controls.

Aging↗
PubMed1996

Mutation pattern in clinically asymptomatic coagulation factor VII deficiency.

A total of 122 subjects, referred after presurgery screening or checkup for prolonged prothrombin time, were characterized for the presence of coagulation factor VII deficiency. Fourteen subjects carried a partial and asymptomatic deficiency, and in half of them dysfunctional molecules were detected in plasma. In nine subjects we found five missense mutations differing from those previously found in factor VII deficient patients. The others were homozygous for a common polymorphism (R353Q) that affects factor VII levels. A new codon dimorphism (A330) was also found in exon 8. Four mutations (R223W, M298I, R304Q, and R353Q) located at FVII-specific residues point out protein regions that are important for coagulation factor evolution, and two mutations (G342E and E265K) affect generic or partially generic residues. The newly reported mutations were combined with those we previously found, totalling 17 independent mutations responsible for FVII deficiency in 27 Italian pedigrees. We observed several similarities with the mutation pattern determined in factor IX, which include a high percentage of transitions at CpG doublets, the presence of hot spot sites affected by multiple substitutions, and of several topologically equivalent mutations.

Factor VII Deficiency↗
PubMed1996

Resistance to activated protein C, associated with oral contraceptives use; effect of formulations, duration of assumption, and doses of oestro-progestins.

Resistance to activated protein C (APC-R) is at present considered the most frequent laboratory abnormality in patients with deep vein thrombosis. An increased risk for venous thrombosis is associated with the use of oral contraceptives (OCs). We recently described a statistically significant association between APC-R status and oral contraceptives use in a healthy group of women. We re-evaluated 50 healthy women taking low-dose combination OCs in order to consider a possible correlation between the APC sensitivity ratio (APC-SR) and different oral contraceptive formulations. Seven women showed an APC ratio < or = 2 (APC-resistant). Only one of the seven women was found to be heterozygous for Leiden factor V mutation. We observed no significant differences between normally sensitive and APC-resistant women in terms of duration of OC use, amount of estrogenic or progestogenic dose, or type of formulation. We conclude that APC-resistance associated with oral contraceptives use seems to occur only in predisposed subjects (in our results, about 12% of the healthy population).

Adolescent↗
PubMed1996

A novel mutation (Leu817Pro) causing type 2A von Willebrand disease.

We studied a patient affected by von Willebrand disease type 2A who experienced several mild bleeding episodes and was characterized by markedly reduced haemostatic parameters. In the exon 28 of von Willebrand factor (vWF) gene a T to C transition at nucleotide 8680, resulting in the missense mutation Leu817Pro, was found in the heterozygous form in the patient and in two affected relatives. As suggested by the presence in platelets of a complete spectrum of vWF multimers as well as by the increased vWF antigen levels and improved haemostasis after DDAVP treatment, the mutation is compatible with normal multimerization, and could be responsible for a reduced stability or an impaired physiological secretion of vWF.

Base Sequence↗
PubMed1996

Detection of new polymorphic markers in the factor V gene: association with factor V levels in plasma.

Three novel polymorphisms were found in the repeated region of the large exon 13 of factor V gene, one giving rise to a codon dimorphism (Ser1240) and two causing aminoacid substitutions (His1299Arg, Leu1257Ile). An increasing frequency of the Arg1299 (R2 allele) correlated with a decreasing mean plasma factor V activity in the groups of subjects under study, which included 26 unrelated subjects with partial factor V deficiency. Family studies supported the co-inheritance both of low factor V activity and of R2 allele. The reduction of factor V activity associated with the R2 allele was not clinically symptomatic even in the homozygous condition and was characterized by a parallel reduction of antigen in plasma, in which abnormal molecules were not detected. Data suggest that the R2 allele represents a marker in linkage with an unknown defect rather than a functional polymorphism. These studies provide the first evidence of a genetic component in determining factor V levels in plasma and of a genetic linkage between the factor V gene and factor V deficiency. They also define specific haplotypes which are associated with factor V deficiency or with APC resistance (Arg506Gln) and are valuable tools for the study of factor V defects.

Base Sequence↗
PubMed1996

A heparin cofactor II mutation (HCII Rimini) combined with factor V Leiden or type I protein C deficiency in two unrelated thrombophilic subjects.

305 patients with juvenile thromboembolic episodes were screened for the presence of heparin cofactor II deficiency. The heterozygous deletion of two bases was found in the exon 5 of the heparin cofactor II gene in two unrelated patients, very likely due to a founder effect. This molecular lesion, causing a frameshift and elongated translation, affects the core of the molecule and should cause the complete unfolding of the protein, which is in accordance with the observed type I deficiency. The corresponding region of antithrombin III gene is affected by a cluster of frameshift mutations suggesting that heparin cofactor II and antithrombin III could share similar mutational patterns. The heparin cofactor II gene alteration was associated with, in one patient, the factor V Leiden mutation and, in the other, type I protein C deficiency. The tracing of the single defects in several family members indicated that the mutations became clinically manifest only when present in the doubly heterozygous condition. This study provides two examples, based on molecular findings, of the interplay of risk factors which is potentially useful to define a role for heparin cofactor II deficiency in inherited thrombophilia.

Adult↗
PubMed1995

1,25-dihydroxyvitamin D3 inhibits proliferation of IMR-90 human fibroblasts and stimulates pyruvate kinase activity in confluent-phase cells.

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a role in regulating some aspects of metabolism in IMR-90 normal human fetal lung fibroblasts. Among the enzymes studied, only pyruvate kinase showed a significant increase after treatment of confluent-phase cells with 1,25(OH)2D3 at various concentrations (0.1-100 nM range) for 24 h. A parallel increase in lactate output was observed. Steroid specificity was established by the failure of 10 nM levels of 25-hydroxyvitamin D3, estradiol-17 beta and progesterone to affect pyruvate kinase activity. The determination of the time course of [3H]-2-deoxy-D-glucose transport indicated that the hormone did not influence the transmembrane transport system of D-glucose. The addition of the inhibitors cycloheximide and actinomycin D to the culture medium abolished, at least in part, the 1,25(OH)2D3 stimulation of pyruvate kinase activity, suggesting the probable dependence of the hormone effect on cellular RNA and protein synthesis. 1,25(OH)2D3 also affected fibroblast growth and DNA synthesis. Cell number significantly decreased after 2-5 days treatment with 10 nM hormone in comparison with control fibroblasts, and also the incorporation of [3H]thymidine into DNA decreased after treatment of the cells with 1 and 10 nM hormone for 48 h. In conclusion, these data demonstrate that 1,25(OH)2D3 stimulates pyruvate kinase activity in confluent-phase IMR-90 human fibroblasts by a mechanism probably dependent on de novo protein synthesis, and also affects cell growth and DNA synthesis in sub-confluent-phase cells.

Biological Transport↗
PubMed1995

Molecular bases of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glu102Lys) in the second EGF-like domain.

The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in asymptomatic subjects with FX deficiency characterized by the presence of dysfunctional molecules in plasma, as demonstrated by the discrepancy between clotting activity and antigen level. A missense mutation (Ser334Pro) in the catalytic domain was found in three unrelated families in both the homozygous and the heterozygous conditions, and also in the compound heterozygous form with the substitution of Lys for 102 Glu. None of the mutations was detected in 40 unrelated subjects from the same geographic area. The Ser334Pro mutation affects a serine protease region characterized by extensive variation in the coagulation factors but conserved in mammalian factor X molecules. The Glu102Lys mutation affects a residue of the second EGF-like module also conserved in protein C. Both mutated residues are surface-exposed and found in protein regions suggested to be involved in macromolecular interactions which are impaired in the dysfunctional molecules.

Base Sequence↗
PubMed1995

Resistance to activated protein C in healthy women taking oral contraceptives.

Resistance to activated protein C (APC) is at present considered the most frequent laboratory abnormality in patients with deep-vein thrombosis. An increased risk for venous thrombosis is associated to the use of oral contraceptives (OC). We studied APC sensitivity in 50 healthy women taking OC and in 50 healthy controls, matched for age, smoking habit, educational and social levels, and the main biochemical routinary parameters. Subjects with a personal or familial history of thrombosis and also with chronic or acute diseases were excluded. Protein C, protein S, antithrombin III and lupus anticoagulant activity (LAC) were also evaluated. Increased fibrinogen and protein C levels, decreased protein S. and shortened PT and APTT were also observed in women taking OC. APC sensitivity ratio (APC-SR) was significantly lower in the OC group than in a control group (2.6 +/- 0.38 v 2.81 +/- 0.35, P < 0.01). Seven of eight women with APC ratio < or = 2 (APC resistant) were OC users: the difference of prevalence was statistically significant (chi-squared test, P < 0.05). Only two out of eight women were found heterozygous for the Leiden factor V mutation. Two APC-resistant women without the Leiden mutation subsequently discontinued OC and both then normalized their APC-SR. We conclude that acquired factors, i.e. oral contraceptives, may play an important role in determining plasma APC resistance.

Adult↗
PubMed1995

Vitamin D--related modification of enzyme activities in synaptosomes and mitochondria isolated from rat cerebral cortex.

We previously demonstrated that feeding rats the Steenbock and Black rickets-inducing diet produces remarkable changes in the metabolic pattern of intestinal mucosa, kidney, liver, cerebral cortex and heart. We have now determined the levels of calcium, phosphorus and citrate in cerebral cortex and the activity of some enzymes in synaptosomes and cerebral cortex mitochondria of three rat groups: control (Group A), fed a vitamin D-deficient diet (Group B), fed a vitamin D-deficient diet and treated with 1,25-dihydroxyvitamin D3 (Group C). While calcium content increased in Groups B and C, phosphorus concentration increased only in Group C and citrate in Group B in comparison with control. The increase in acetylcholinesterase and citrate synthase registered in Group B was restored to control values by 1,25-dihydroxyvitamin D3 treatment, while, neither the decrease in cytochrome c oxidase, nor the increase in glucose-6-phosphate dehydrogenase, acid phosphatase and NADP+(-)isocitrate dehydrogenase observed in Group B were corrected by 1,25-dihydroxyvitamin D3 supply. Acyl phosphatase showed a remarkable increase in consequence of 1,25-dihydroxyvitamin D3 administration.

Animals↗
PubMed1994

Effect of vitamin D deficiency and 1,25-dihydroxyvitamin D3 on rat heart metabolism.

The purpose of this study was to investigate whether vitamin D3 deficiency and 1,25-dihydroxyvitamin D3 treatment affect some aspects of heart metabolism in the rat. To this end, five experimental groups were studied: (1) the control group of the vitamin D3 supplemented rats (Group A); (2) rachitic rats (Group B); (3) rachitic rats treated with 1,25-dihydroxyvitamin D3 (Group C); (4) rats fed a vitamin D-deficient diet (Group D); (5) rats fed a vitamin D-deficient diet and treated with 1,25-dihydroxyvitamin D3 (Group E). The five groups were compared by checking in the heart some metabolic parameters, i.e. citrate content, and enzyme activities in cytosol and mitochondria. Citrate content was higher in the heart of treated animals when compared with the control. As regards the enzymatic activities in heart mitochondria, NAD(+)-dependent isocitrate dehydrogenase remarkably decreased in Group B rats and 1,25-dihydroxyvitamin D3 restored quite normal values. NADP(+)-dependent isocitrate dehydrogenase decreased in Group B and Group D animals, and 1,25-dihydroxyvitamin D3 treatment was effective in restoring control values. Cytochrome c oxidase activity did not change, while citrate synthase showed an increase in all the treated rats. As regards the cytosolic enzymes, fructose-6-phosphate kinase increased in the two groups of vitamin D-deplete rats in comparison with the control. Glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase showed a similar trend: an increase in all the treated animals. In heart homogenate, acylphosphatase and acid phosphatase activities were also determined. Acylphosphatase increased in the treated rats, while acid phosphatase decreased in the rats injected with 1,25-dihydroxyvitamin D3. These results support the hypothesis of a participation of 1,25-dihydroxyvitamin D3 in some aspects of heart metabolism.

Animals↗
PubMed1993

Effect of vitamin D deficiency and 1,25-dihydroxyvitamin D3 on metabolism and D-glucose transport in rat cerebral cortex.

We previously demonstrated that feeding rats Steenbock and Black's rickets-inducing diet produces remarkable changes in the metabolic pattern of the intestinal mucosa, kidney, and liver and in some membrane transport systems of intestinal mucosa and kidney. 1,25-Dihydroxyvitamin D3 administration to rachitic rats did not always prove to be effective in restoring normal values. We have now investigated the effect of 1,25-dihydroxyvitamin D3 on the levels of some metabolites in rat cerebral cortex, on the activity of some enzymes, and on the transport of 2-deoxy-D-glucose and D-glucose in synaptosomes. Our experiments were carried out on three rat groups: control, rachitic, and rachitic treated with 1,25-dihydroxyvitamin D3. The decrease in phosphorus content and the increase in citrate concentration observed in rachitic rat cerebral cortex were corrected by 1,25-dihydroxyvitamin D3 treatment. The activity of acetylcholinesterase, glucose-6-phosphate dehydrogenase, and acyl phosphatase significantly increased in rachitic rat synaptosomes, as well as NAD(+)-dependent isocitrate dehydrogenase in cerebral cortex mitochondria; the administration of 1,25-dihydroxyvitamin D3 to rachitic rats restored enzyme levels to normal. The transport of 2-deoxy-D-glucose and D-glucose in rachitic rat synaptosomes was lower than in the control group and returned to control values in consequence of 1,25-dihydroxyvitamin D3 treatment. The results reported here support the hypothesis of a participation of 1,25-dihydroxyvitamin D3 in some aspects of cerebral cortex metabolism.

Acetylcholinesterase↗
PubMedDate not supplied

Protection of rat heart from ischaemia-reperfusion injury by the 21-aminosteroid U-74389G.

In ischaemia-reperfusion syndromes lipid peroxidation appears an important factor contributing to tissue damage. The 21-aminosteroids (lazaroids) exhibit beneficial effects in various pathological conditions, especially in post-traumatic lesions of the central nervous system, where a peroxidative injury seems to be involved. The aim of our study was to ascertain if one of these compounds, U-74389G, plays a significant role in protecting heart muscle from ischaemia-reperfusion damage. Rat hearts used for heterotopic transplantation represented the experimental model in this investigation. Animals (Wistar rats weighing 200-250 g) were divided into five groups: controls, untreated and treated donors, untreated and treated recipients. Donors were anaesthetized and heparinized, and the heart was excised through a bilateral thoracotomy, arrested with St Thomas solution and stored in cold saline for 2 hours. For the recipient preparation, a modified Ono's technique was used, and heart reimplantation was performed with a termino-lateral aorto-aortic anasthomosis and a termino-lateral pulmonary-cava anasthomosis. After the anasthomoses were completed hearts were reperfused for 30 min; then hearts were excised and specimens were taken for biochemical and morphological studies. These were conducted on three groups of hearts: (A) hearts reimplanted and reperfused without treatment of the donor or of the recipient animal; (B) hearts subjected to the same procedure but in the presence of U-74389G treatment of donors and recipient rats; (C) control hearts rapidly excised from normal, non-operated animals. Electron microscopy studies showed, in hearts transplanted without treatment, the typical morphological aspects of lipoperoxidative injury: swollen mitochondria with disrupted cristae, damaged endothelial cells with the nucleous bulging into the lumen and a discontinued endothelial lining with diffuse oedema among the fibers. Lazaroid treatment attenuated most of these damages in hearts of group B. As for the biochemical findings, the hearts transplanted in the presence of U-74389G treatment had significantly higher ATP and creatine phosphate levels (P < 0.01) and lower malondialdehyde concentrations (P < 0.05) with respect to the hearts transplanted without treatment. Furthermore, serum creatine kinase activity was lower in treated than in untreated recipient animals (P < 0.05). Taken together, all these results indicate that U-74389G treatment is effective in protecting cardiac muscle from structural and functional ischaemia-reperfusion injuries, at least from those arising during a heart transplantation procedure.

Adenosine Triphosphate↗
PubMedDate not supplied

Relationship between age and GSH metabolism in synaptosomes of rat cerebral cortex.

A comprehensive analysis on glutathione metabolism in rat cerebral cortex synaptosomes as a function of age was performed. All different glutathione system components (GSH, GSSG, total GSH, and GSH redox index) changed significantly only during aging. GSH, total GSH, and GSH redox index decreased by about 40%, 24%, and 52%, respectively, while GSSG showed a remarkable increase of about 60%. On the contrary, some GSH-related enzyme activities showed characteristic changes both during growth and aging. GSH peroxidase and GSH-S-transferase activities significantly increased both during growth and aging, GSH reductase and gamma-glutamylcysteine synthetase activities showed lower levels only during aging, while glucose-6-phosphate dehydrogenase activity did not change throughout the life of the rat. The results obtained suggest an increase of the oxidative status due to a reduced antioxidant capacity of the GSH system in the synaptosomal compartment during aging. The main cause of these metabolic modifications is a lowering of the rates of both GSSG reduction to GSH and GSH synthesis. Moreover, an irreversible loss of GSH as GSH-S-conjugates due to a high detoxification mechanism during aging is also possible. These alterations in glutathione metabolism, found mainly during aging in rat cerebral cortex synaptosomes may contribute to clarify some aspects of cerebral diseases.

Aging↗
PubMedDate not supplied

Glutathione metabolism in heart and liver of the aging rat.

A comprehensive study on glutathione metabolism in rat heart and liver as a function of age was performed. In the heart, reduced glutathione, total glutathione, and the glutathione redox index showed a decrease during aging, while oxidized glutathione levels increased in 5-month-old rats with respect to the young animals and remained quite constant in 14- and 27-month-old rats. In the liver, the highest levels of reduced glutathione were found in the 2-month-old rats, while oxidized glutathione reached a peak at 5 months. Glutathione-associated enzymes showed age-related changes. Glutathione peroxidase, unaffected by aging in the heart, decreased in the liver of the 27-month-old rats. In the heart and the liver, the highest values of glutathione S-transferase were found at 5 months and 27 months, respectively. Glucose-6-phosphate dehydrogenase followed a similar trend in both heart and liver. Glutathione reductase also showed the same behaviour in heart and in liver, increasing in old rats with respect to the other age groups. A decrease in gamma-glutamylcysteine synthetase was found in the heart and liver of 27-month-old rats in comparison with the 2-month-old ones. In conclusion, a decreased antioxidant capability has been demonstrated in both heart and liver of old rats.

Aging↗