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Biomedical subjects

M Conti

Publications and source records attributed to M Conti.

At least 109 records · Page 6Linked to original sources
PubMed1997

Selective activation of rolipram-sensitive, cAMP-specific phosphodiesterase isoforms by phosphatidic acid.

In rat thymic lymphocytes, accumulation of phosphatidic acid (PA) occurs at the same time as decrease in cAMP levels and activation of a cAMP-specific phosphodiesterase (PDE) [type 4, EC 3.1.4.17 (PDE4)]. We investigated the nature of the PDE activated by PA and the mechanism of activation by using recombinant cAMP-specific PDE4 isoforms derived from three different genes (PDE4A, PDE4B, and PDE4D). The "long" variants expressed from each gene (PDE4A5, PDE4B1, and PDE4D3) were activated by PA, whereas the "short" variants (PDE4A1, PDE4B2, PDE4D1, and PDE4D2) were not. Phosphatidylserine was an activator that was as effective as PA, whereas phosphatidylcholine was ineffective, indicating that activation was restricted to anionic phospholipids. PA caused an increase in the Vmax value of PDE4D3 without affecting the Km value of the enzyme for the cAMP substrate. PA also caused a change in the Mg2+ requirement for hydrolysis. Half-maximal stimulation of the PDE was obtained with approximately 10 microg/ml PA. Although protein kinase A-mediated phosphorylation of PDE4D3 produces effects similar to those elicited by PA, the mechanism of PA-induced activation was not found to involve a phosphorylation. Instead, several observations suggest that PA may directly interact with the enzyme. The stimulation of cAMP PDEs by PA and other acidic phospholipids may be a mechanism by which growth factors and hormones modulate the cAMP-dependent signal transduction pathway during cell stimulation.

Animals↗
PubMed1997

Characterization of an intronic promoter of a cyclic adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase gene that confers hormone and cAMP inducibility.

In the Sertoli cell, FSH stimulates transcription of a cAMP-phosphodiesterase (PDE) gene (PDE4D) and accumulation of corresponding mRNA and PDE protein. The regulation of this PDE gene is an important component of the desensitization state induced by this hormone. Given the ubiquitous nature of this regulation controlling cAMP levels, the molecular basis for the PDE4D induction was further investigated. FSH stimulation of the Sertoli cell causes the accumulation of only two of the four known PDE4D mRNAs (PDE4D1 and PDE4D2). The promoter controlling the expression of these two messages was identified and characterized. An EcoRI fragment containing a coding exon as well as 5'-upstream sequence of the PDE4D1/2 mRNA was isolated from rat genomic libraries and sequenced. No TATA box was identified, but GC-rich regions were present upstream of the putative translation start site. RNAse protection and PCR analysis indicated the presence of at least two distinct cap sites. This genomic region had promoter activity when transfected both in Sertoli and MA-10 cells. Deletion mutation indicated that basal promoter activity was contributed by regions upstream of both cap sites. Transcription from this promoter was activated by FSH and (Bu)2cAMP, and elements responsible for cAMP regulation were present upstream from the second cap site. These data demonstrate that an intronic promoter that is cAMP- and hormone-inducible directs the expression of these truncated PDE proteins.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

[Comparative, randomized, parallel clinical study of the effectiveness and safety of aceclofenac vs. paracetamol in the treatment of viral pharyngoamygdalitis].

The purpose of this open, controlled, and randomized study was to evaluate the effectiveness of aceclofenac compared with paracetamol in improving the signs and symptoms of viral pharyngoamygdalitis and to evaluate the safety of both drugs. Thirty outpatients (age range 18-65 years) with acute viral pharyngoamygdalitis received either aceclofenac 100 mg/12 h or paracetamol 650 mg/ 12 h per os. Patients were evaluated at baseline and on days 1, 3, and 7 after beginning treatment. The parameters of effectiveness evaluated were: severity of pharyngoamygdalitis, spontaneous pharyngeal pain, saliva swallowing, and duration of pharyngeal pain. The results showed that the aceclofenac group had a significant decrease in the severity of pharyngoamygdalitis and spontaneous pharyngeal pain after day 1 of treatment (p < 0.001), but the paracetamol group showed no improvement in these parameters until day 3 of treatment (p < 0.001). There were no adverse reactions in either group. Therefore, patients treated with aceclofenac showed an earlier improvement in the signs and symptoms of acute viral pharyngoamygdalitis than those treated with paracetamol.

Acetaminophen↗
PubMed1997

[Accident during retinal fluorescein angiography].

OBJECTIVE: To evaluate adverse reactions frequency following fluorescein i.v. administration to perform retinoic angiography (ARF). DESIGN: Retrospective study. SETTING: Eye Clinic of the University of Florence. MATERIALS AND METHODS: 6524 patients undergoing 10,003 ARF. PARAMETERS EVALUATED: For each patient age, sex, concomitant diseases, previous ARF, allergic history and any adverse reaction were evaluated. RESULTS: Adverse experiences were described in 7.5% of 6524 patients. Most of the observed reactions were mild: nausea (3.8%), vomiting (0.43%), vasovagal phenomena (1.18%), anaphylactoid reactions (1.84%). A major rate of adverse reactions were observed in patients previously submitted to ARF who reported in the anamnesis a previous adverse reaction, and in those with a positive allergic history. Severe reactions occurred in 0.27% of patients, but only in 2 of 6524 patients a rianimatory treatment (0.03) was necessary. CONCLUSION: From the data retrospective analysis it was observed that the manifestation of mild adverse effects was to be expected in many cases. It was therefore possible to point out classes of patients at risk (patients who reported a previous adverse reaction after ARF and patients with positive allergic history). Unfortunately it was not possible to expect and so to prevent the severe effects. Thus it is recommended to perform ARF in an adequately equipped room.

Adolescent↗
PubMed1996

Effects of low-dose heparin infusion on arterial endothelin-1 release in humans.

BACKGROUND: The aim of this study was to evaluate the effect of low-dose heparin infusion on arterialized endothelin-1 (ET-1) release in the presence of fasting or high insulin levels in healthy humans. METHODS AND RESULTS: Eleven normal subjects underwent two tests in random order lasting 240 minutes. A primed (250 IU), continuous heparin (600 IU/h) infusion was performed in test 1; saline was infused in test 2 as control. At 120 minutes, a euglycemic hyperinsulinemic clamp (25 mU.kg-1.h-1) was started that lasted 2 hours in both tests. Two hours after heparin infusion (test 1), ET-1 levels decreased by 32% (3.52 +/- 0.60 to 3.02 +/- 0.73 pg/mL), while nitric oxide (NO) and forearm blood flow increased by 29% and 14%, respectively. During saline infusion, ET-1, nitric oxide, and forearm blood flow remained unchanged. There was a significant interaction between the effect of decreasing ET-1 levels and the heparin treatment (F, 4.06; df, 3.30; P < .01). The decrease in ET-1 levels was significantly correlated with the increase in forearm blood flow in test 1 (r = .74; P < .01) but not in test 2. During the heparin/insulin period, ET-1 increased by 25%, returning to fasting values; nitric oxide levels increased by 12%; and forearm blood flow remained unchanged. CONCLUSIONS: The present study showed that it is possible to decrease ET-1 levels by use of low-dose heparin infusion in humans. This effect seems mediated by a simultaneous increase in nitric oxide levels and is completely reversed by a mild increase in insulin concentrations.

Adult↗
PubMed1996

Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors.

The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.

Animals↗
PubMed1996

Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation.

A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Identification of cyclic AMP-phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells.

To determine whether the expression of different PDE4D variants is unique to the rat or conserved through evolution, we have characterized the different PDE4D mRNAs expressed in human peripheral blood mononuclear cells. RT-PCR was performed using primers based on rat sequences and mRNAs from mononuclear cells. The specifically amplified fragments had a size identical to that predicted for rat PDE4D1, PDE4D2 and PDE4D3. Sequencing confirmed that these fragments are derived from the human PDE4D gene. Their sequence was highly homologous to that reported for the rat variants. cDNAs corresponding to the entire ORF of human PDE4D2 and PDE4D3 were expressed in mammalian cells, causing a large increase in PDE activity. Western blot analysis of human peripheral blood mononuclear cell extracts demonstrated the presence of proteins corresponding to the recombinant PDE4D1 and PDE4D2. The pattern of splicing and different promoter usage of the PDE4D gene is therefore conserved during evolution, which indicates an important physiological role.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Quantitation of glycated hemoglobins in human adult blood by capillary isoelectric focusing.

A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), two species which differ by minute delta pI values, < 0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6-8, 0.5% Pharmalyte, pH 3-10, 3% short-chain liquid polyacrylamide and an equimolar mixture of two "separators", 0.33 M beta-alanine and 0.33 M 6-aminocaproic acid. The last two compounds flatten the pH gradient in the pI region of the two Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse-loaded, are uniformly distributed in the background electrolyte. A longer capillary life-time is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (+/- 6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.

Acrylic Resins↗
PubMed1996

Effects of an acute decrease in non-esterified fatty acid levels on muscle glucose utilization and forearm indirect calorimetry in lean NIDDM patients.

The aim of the study was to evaluate an acute decrease in NEFA levels during an oral glucose tolerance test and its effects on glucose tolerance, muscle glucose uptake and muscle indirect calorimetry in ten lean non-insulin-dependent diabetic subjects. Two 75-g oral glucose tolerance tests were performed in random order. Placebo or 250 mg acipimox (to inhibit lipolysis) were administered orally 2 h before the start of the oral glucose tolerance test. Two hours after acipimox administration (time 0), non-esterified fatty acid, glycerol and 3-hydroxybutyrate levels decreased by 84, 68 and 77% respectively, compared to basal levels. Concomitantly, muscle lipid oxidation and non-oxidative glycolysis also decreased significantly. After placebo administration, non-esterified fatty acids, glycerol and 3-hydroxybutyrate and lipid oxidation increased by 29, 28, 106 and 33%, respectively (NS vs basal levels; p < 0.001 vs acipimox). There was a negative rate of net glucose storage (interpreted as glycogenolysis) during post-absorptive conditions and at time 0 after administration of both drugs. After oral glucose tolerance test, the incremental areas of blood glucose and insulin were significantly decreased by 18 and 19% after acipimox compared to placebo. In addition, the ratio between the incremental area of forearm muscle glucose uptake and the insulin levels was significantly increased by 45% during acipimox compared to placebo administration. Glucose oxidation and non-oxidative glycolysis were significantly higher while lipid oxidation was significantly lower after acipimox than after placebo. In conclusion, our study found that in lean non-insulin-dependent diabetic subjects, an acute decrease in non-esterified fatty acid levels improves glucose tolerance, muscle glucose uptake, glucose oxidation and non-oxidative glycolysis, but is unable to normalize glucose storage.

3-Hydroxybutyric Acid↗
PubMed1996

Extinction of peroxisomal functions in hepatoma cell-fibroblast hybrids.

Although peroxisomes are ubiquitous, differences in the number of organelles and in the expression of associated metabolic activities are observed, depending on the cell type. To investigate the control of peroxisomal activity in connection with cell differentiation, we constructed hybrids between two types of cells whose histogenetic origins dictate significant differences in peroxisomal activities: hepatoma cells and fibroblasts, with high and low expression, respectively, of peroxisomal functions. In these hybrids, extinction of the elevated activities that characterize liver cells is observed, in parallel with the well-documented extinction of differentiated functions. This suggests the existence in fibroblasts of a negative trans-acting regulation.

Acetyl-CoA C-Acetyltransferase↗
PubMed1996

Translocation (3;3) in a patient with thrombocytopenia and erythroid dysplasia.

We describe a case with a translocation between the two chromosomes 3 with breakpoints q21 and q26, associated with a unique constellation of hematologic abnormalities. Megakaryocytic dysplasia and peripheral thrombocytosis are the most common abnormalities associated with this chromosome abnormality. Our patient had acute myeloid leukemia (AML), thrombocytopenia, dyserythropoiesis, and an increased myeloid/erythroid ration but no mekakaryocytic dysplasia. Although multilineage involvement has been reported, erythrocyte dysplasia associated with thrombocytopenia and without megakaryocyte dysplasia appears to be extremely rare. Our case supports the speculation that #3 abnormalities with breakpoints q21 and q26 affect a pluripotent stem cell and suggests that megakaryocytic involvement may not be pathognomonic in hematologic abnormalities with t(3;3)(q21;26).

Chromosomes, Human, Pair 3↗
PubMed1996

Human quadriceps cross-sectional area, torque and neural activation during 6 months strength training.

Quadriceps muscle and fibre cross-sectional areas (CSA), torque and neural activation were studied in seven healthy males during 6 months of weight training on alternate days with six series of eight unilateral leg extensions at 80% of one repetition maximum. After training, the quadriceps cross-sectional area increased by 18.8 +/- 7.2% (P < 0.001) and 19.3 +/- 6.7% (P < 0.001) in the distal and proximal regions respectively, and by 13.0 +/- 7.2% (P < 0.001) in the central region of the muscle. Hypertrophy was significantly different between and within the four constituents of the quadriceps. Biopsies of the vastus lateralis at mid-thigh did not show any increase in mean fibre cross-sectional area. Maximum isometric voluntary torque increased by 29.6 +/- 7.9%-21.1 +/- 8.6% (P < 0.01-0.05) between 100 degrees and 160 degrees of knee extension, but no change in the optimum angle (110 degrees-120 degrees) for torque generation was found. A 12.0 +/- 10.8% (P < 0.02) increase in torque per unit area together with a right shift in the IEMG-torque relation and no change in maximum IEMG were observed. Time to peak isometric torque decreased by 45.8% (P < 0.03) but no change in time to maximum IEMG was observed. In conclusion, strength training of the quadriceps results in a variable hypertrophy of its components without affecting its angle-torque relation. The increase in torque per unit area, in the absence of changes in IEMG, may indicate changes in muscle architecture. An increase in muscle-tendon stiffness may account for the decrease in time to peak torque.

Adult↗