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Biomedical subjects

M Conti

Publications and source records attributed to M Conti.

At least 91 records · Page 5Linked to original sources
PubMed1999

Mediation of the hepatic effects of growth hormone by its lipolytic activity.

The aim of the study was to investigate the acute effect of GH per se, independent from its lipolytic activity, on glucose and lipid oxidation and glucose turnover in seven healthy subjects. Five tests lasting 360 min were performed. Each test consisted of a 4-h equilibration period followed by a euglycemic hyperinsulinemic (25 mU/kg x h) clamp lasting 2 h. In test 1 (control experiment) saline was infused, leaving GH and FFA at basal levels. In tests 2, 3, and 4, GH was infused (80 ng/kg x min) to increase GH levels. Whereas in test 2 FFA levels were free to increase due to GH lipolytic activity, in test 3 FFA elevation was prevented by using an antilipolytic compound (Acipimox) that allowed evaluation of the effect of GH at low FFA levels. In test 4 (GH+Acipimox+heparin) GH infusion was associated with the administration of Acipimox and heparin to maintain FFA at the basal level to evaluate the effect of GH per se independent from GH lipolytic activity. In test 5 Acipimox and a variable heparin infusion were given to evaluate possible effects of Acipimox other than the inhibition of lipolysis. During the euglycemic hyperinsulinemic clamp in the presence of high GH and FFA levels (test 2), glucose oxidation was significantly lower and lipid oxidation was significantly higher than in tests 1, 3, 4, and 5. During the same period, hepatic glucose production was completely suppressed in the control study (test 1; 94%) and in test 5 (99.6%), whereas it was significantly less inhibited (65%, 74%, and 73%) when GH was administered in tests 2, 3, and 4. In conclusion, these results suggest that GH directly mediates the reduction of insulin's effect on the liver. In addition, the effect of GH on glucose and lipid oxidation is not direct, but is mediated by its lipolytic activity.

Adult↗
PubMed1999

[Latex allergy. An not rare intraoperative event].

Latex allergy has become a real problem among both surgical staff (paramedics and physicians) and patients especially pediatric patients with urogenital malformations and spina bifida. Latex allergy is produced from both natural molecules which compose the substance produced from Hevea brasiliensis (rubber tree) and industrial additives contents in latex devices. Diagnosis of latex allergy may be carried out through a preoperative Prik-test. A characteristic of latex allergy reaction is the starting of symptoms (more than 15 minutes after allergen contact). Pathophysiology of latex allergy is the same of all allergic reactions; it is an antigen-antibody reaction and type I or II reaction may occur. There are a lot of devices that surgeons and anesthesists use in the operative theatre and that should not be used in presence of a patient with latex allergy. Guaranteed latex-free devices should always be present in store.

Humans↗
PubMed1999

In situ detection of lipid peroxidation by-products as markers of renal ischemia injuries in rat kidneys.

PURPOSE: Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes. In renal transplantation, this mechanism is triggered by ischemia/reperfusion and may be of relevance in graft failure. MATERIALS AND METHODS: Using specific antibodies directed against malondialdehyde (MDA) and 4-hydroxynonenal (HNE) adducts, major aldehydic metabolites of lipid peroxidation, we investigated, in situ, by means of an immunohistochemical procedure, the occurrence of lipid peroxidation during different warm ischemic periods of 0, 15, 30, 45 and 60 minutes in rat kidneys prior to reperfusion. The same experiments included followup of the rats after nephrectomy and reperfusion for 10 days. RESULTS: We observed superficial and deep cortex immunostaining with both antibodies against MDA and HNE after 30 minutes of warm ischemia. This immunostaining was observed in the absence of any histological lesions, as assessed by routine staining. After 45 and 60 minutes of warm ischemia, lipid peroxidation byproducts were detected both in the cortex and in the medulla, which is associated with 33% and 66% of rat deaths respectively. CONCLUSIONS: This study confirms the involvement of the lipid peroxidation process in kidney damage during anoxia before reperfusion, and its extension to the whole organ. Lipid peroxidation byproducts were detectable in warm ischemic kidney, and the presence of medulla immunostaining was associated with the animals' death. Lipid peroxidation immunostaining might thus be useful as a sensitive tool to detect ischemic damage after warm ischemia prior to reperfusion, as well as in the decision to carry out kidney transplantation in humans.

Aldehydes↗
PubMed1999

Plasmodesmata and plant viruses. A centenary story.

The passage of plant viruses from a cell to adjacent ones remained for a long time an unexplained event. Only during the thirties did Samuel and other plant virologists put forward the hypothesis that the passage occurred through plasmodesmata, i.e. those protoplasmic connections between plant cells described since the late 19th century. A direct relation between viruses and plasmodesmata was first demonstrated by electron microscopy during the late 1960s by Esau and co-workers, and then widely confirmed. The mechanism of the passage was investigated in depth starting from the 1970s, and research received a remarkable impulse after that a well-defined model of plasmodesmata had been obtained thank, in particular, to work of the Robards' and Gunning's groups. In this context, the discovery of the polycystronic functionality of the viral genomes was fundamental. A protein coded by tobacco mosaic virus, discovered in 1982 independently by the Soviet group of Atabekov and the American group of Zaitlin, was demonstrated to be indispensable for the transport of virus infection from cell to cell through plasmodesmata. Elegant investigations on this 'movement protein' demonstrated that it actually increases the permeability of plasmodesmata. The relation between viruses and plasmodesmata is one of the most interesting and investigated theme of research, which is receiving much attention from plant virologists, physiologists and molecular biologists. The current status of knowledge still presents unsolved questions, and the story is far from over.

History, 20th Century↗
PubMed1998

Role of cyclic nucleotide phosphodiesterases in resumption of meiosis.

In the follicles of the mammalian and amphibian ovary, oocyte maturation is arrested at the prophase of the first meiotic division. Prior to ovulation, oocytes reenter the cell cycle, complete the meiotic division, and extrude the first polar body. Work from several laboratories including ours has provided evidence that the cAMP-mediated signal transduction pathway plays an important role in regulation of meiosis, the cyclic nucleotide acting as a negative regulator of maturation. Since cAMP can be regulated both at the level of synthesis and degradation, our laboratory is investigating the role of phosphodiesterases (PDE) in the control of cAMP levels of oocytes. Using pharmacological and molecular tools, we have determined that a PDE3 is the enzyme involved in the control of cAMP levels in the oocytes. In vitro and in vivo studies have established that inhibition of the oocyte PDE3 blocks resumption of a PDE is per se sufficient to cause resumption of meiosis in an amphibian oocyte model. The pathways regulating this PDE isoform expressed in the oocyte is under investigation, as they may uncover the physiological signals controlling meiosis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents.

During each reproductive cycle, a preovulatory surge of gonadotropins induces meiotic maturation of the oocyte in the preovulatory follicle followed by ovulation. Although gonadotropins stimulate cAMP production in somatic cells of the follicle, a decrease in intra-oocyte cAMP levels is required for resumption of meiosis in oocytes. Based on the observed compartmentalization of the cAMP-degrading enzyme, phosphodiesterase, in follicular somatic and germ cells, inhibitors of phosphodiesterase 3 were used to block meiosis in ovulating oocytes in rodents. By this strategy, we demonstrated that fertilization and pregnancy could be prevented without disturbing follicle rupture and normal estrous cyclicity. In contrast to conventional contraceptive pills that disrupt ovarian steroidogenesis and reproductive cycles, the present strategy achieves effective contraception by selective blockage of oocyte maturation and development without alterations in ovulation and reproductive cyclicity.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Subcellular localization of rolipram-sensitive, cAMP-specific phosphodiesterases. Differential targeting and activation of the splicing variants derived from the PDE4D gene.

Biochemical and immunofluorescence analyses revealed that phosphodiesterase variants encoded by the PDE4D gene are targeted to discrete subcellular structures. In quiescent FRTL-5 thyroid cells, the rolipram-sensitive phosphodiesterase (PDE) activity (cAMP-PDE) was recovered both in the soluble and particulate fractions of the homogenate. Although an immunoreactive 93-kDa PDE (PDE4D3) variant was recovered in both compartments, a 105-kDa variant with the properties of PDE4D4 was recovered mostly in the particulate fraction. The PDE4D3 form was readily solubilized with nonionic detergents. Conversely, the PDE4D4 form required buffers containing ionic detergents for extraction, suggesting that different mechanisms target these variants to insoluble structures. A 15-min stimulation with thyroid-stimulating hormone (TSH) led to an activation of the cAMP-PDE in both compartments and was correlated with a shift in electrophoretic mobility of the PDE4D3 polypeptide. Long term incubation with TSH caused an increase of the PDE activity in the soluble fraction and the appearance of a 68-kDa immunoreactive polypeptide with the properties of PDE4D2. Immunofluorescence analysis showed, in addition to diffuse staining, a signal localized on regions adjacent to the plasma membrane on cytoskeletal structures and in a perinuclear region of quiescent cells. Long term incubation with TSH caused an increase in the immunofluorescence signal in the soluble compartment. These data demonstrate that three PDE4D splicing variants are targeted to discrete subcellular compartments and that hormones cause the activation of these isoforms in a temporally and spatially dependent manner.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Protein kinase B/Akt induces resumption of meiosis in Xenopus oocytes.

The activation of protein kinase B/Akt is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. Because insulin-like growth factor 1 stimulates the resumption of meiosis in Xenopus laevis oocytes via phosphoinositide 3-kinase activation, we investigated the Akt involvement in this process. Injection of mRNA coding for a constitutively active Akt in Xenopus oocytes induced germinal vesicle breakdown (GVBD) to the same extent as progesterone or insulin treatment. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a lysine mutation in the catalytic domain that abolishes the kinase activity had no effect. A mutant Akt lacking a membrane-targeting sequence did not induce GVBD, despite high levels of expression and activity. As previously reported for insulin, induction of GVBD by Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for the type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE is required for Akt action. That an increase in PDE activity in the oocyte is sufficient to induce meiotic resumption was demonstrated by expression of an active PDE protein. In addition, the constitutively active Akt caused a 2-fold increase in the activity of the endogenous PDE. These data demonstrate that Akt is in the pathway controlling resumption of meiosis in the Xenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Heterologous expression and purification of recombinant rolipram-sensitive cyclic AMP-specific phosphodiesterases.

With the cloning of cDNAs coding for the different phosphodiesterase 4 (PDE4) isoenzymes present in mammals, homogeneous preparations of these forms have become readily available. This strategy has greatly facilitated the understanding of the properties of the myriad of isoforms derived from the four PDE4 genes found in mammals, and has opened a new avenue to develop inhibitors with a different degree of selectivity for each isoform. Here we describe the strategies and methods used to express PDE4 in bacterial, yeast, insect, and mammalian cell heterologous systems, and review the advantages and disadvantages of each of these expression strategies. In addition, procedures to purify the recombinant proteins are described. The recently developed purification of a PDE4 by immunoaffinity chromatography provides a rapid and efficient method to prepare large quantities of PDE4. This method should be very useful for structural and kinetic studies on the PDE4D isoforms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Anticonvulsant properties of BIBP3226, a non-peptide selective antagonist at neuropeptide Y Y1 receptors.

Several lines of evidence indicate that neuropeptide Y (NPY)-mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. We have investigated the role of NPY Y1 receptor subtypes, which are enriched in the dentate area of the hippocampus, on EEG seizures induced by a local injection of 0.04 microg kainic acid in rats. Intrahippocampal administration of 10 microg BIBP3226 (N2- (diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]D-arginamide), a non-peptide selective antagonist at the NPY Y1 receptors, increased threefold on average (P < 0.01) the time to onset of seizures and reduced the number of seizures and the total time in seizures three- and fourfold, respectively (P < 0.01). Its inactive S-enantiomer BIBP3435 was ineffective on seizure activity. One microgram [Leu31,Pro34]NPY, an agonist at Y1 receptors, did not modify per se the EEG sequelae induced by kainic acid but it antagonized the anticonvulsant effect of BIBP3226. These results indicate that NPY Y1 receptors in the hippocampus are involved in epileptic phenomena and suggest that selective Y1 receptor antagonists may be of value for attenuating limbic seizures.

Animals↗
PubMed1998

Enhancement of motility and acrosome reaction in human spermatozoa: differential activation by type-specific phosphodiesterase inhibitors.

Inhibition of sperm phosphodiesterase (PDE) has been shown to increase cAMP concentrations and stimulate motility and the acrosome reaction. While several PDE genes exist in mammals, little is known about the physiological role of PDE forms expressed in human spermatozoa. Using type-selective inhibitors, we identified two of the PDE forms expressed in human spermatozoa and studied their involvement in sperm function. Selective inhibitors of calcium-calmodulin-regulated PDE1 (8-methoxy-isobutyl-methylxanthine) and cAMP-specific PDE4 (RS-25344, Rolipram) were used to study PDE forms in human sperm extracts. 8-MeIBMX and Rolipram/RS-25344 inhibited sperm PDE activity by 35-40 and 25-30% respectively. Subcellular fractionation of the sperm homogenate suggests these pharmacologically distinct forms may be located in separate cellular regions. To evaluate the functional significance of different PDE forms, the effect of type-specific PDE inhibition on sperm motility and the acrosome reaction was examined. PDE4 inhibitors enhanced sperm motility over controls without affecting the acrosome reaction, while PDE1 inhibitors selectively stimulated the acrosome reaction. These data indicate at least two distinct PDE types exist in human spermatozoa. Our findings also support the hypothesis that PDE subtypes affect sperm function by regulating separate pools of cAMP and may ultimately offer novel treatments to infertile couples with abnormal semen parameters.

1-Methyl-3-isobutylxanthine↗
PubMed1998

CCU patient survey tool: all-out recovery.

Studies show that patients' recall of their CCU stays is extremely limited due to various factors. To monitor patient satisfaction in this area, a team of CCU managers developed a survey and began an "All-Out Recovery Program."

Coronary Care Units↗
PubMed1998

Characterization of the rolipram-sensitive, cyclic AMP-specific phosphodiesterases: identification and differential expression of immunologically distinct forms in the rat brain.

To determine the properties of the cAMP-specific, rolipram-sensitive phosphodiesterases (cAMP-PDEs) that are expressed in different organs, monoclonal and polyclonal antibodies were raised against different epitopes present in the cAMP-PDE sequences. Of the several antibodies generated against peptides and fusion proteins, one monoclonal and four polyclonal antibodies recognized both the native cAMP-PDEs as well as the denatured proteins on Western immunoblot analysis. An immunoprecipitation assay demonstrated that these antibodies recognized the recombinant rat PDE4A, PDE4B, and PDE4D proteins with different avidity. The polyclonal antibody K118 and the monoclonal M3S1 were most specific for rat PDE4B and PDE4D forms, respectively, whereas the AC55 antiserum displayed the highest affinity for PDE4A forms. This selectivity was confirmed by Western blot analysis using recombinant rat PDE4A, PDE4B, and PDE4D proteins expressed in a heterologous system. These antibodies were used to characterize the cAMP-PDEs expressed in the rat brain. An immunoblot of extract of cortex and cerebellum demonstrated that at least seven different polypeptides specifically cross-reacted with the different antibodies, indicating that multiple cAMP-PDEs are expressed in this tissue. On the basis of cross-reactivity with PDE4D but not PDE4A or PDE4B antibodies, 93- and 105-kDa PDE4D species were detected in the cortex and cerebellum extract. These forms are different from the 68-kDa PDE4D form expressed in endocrine cells after hormonal stimulation. Although the 93-kDa form was recovered in both the soluble and particulate fractions, the 105-kDa polypeptide was mostly particulate in the cortex and cerebellum extracts. PDE4B forms of 90-87 kDa were recovered in both soluble and particulate compartments of the brain extract. These forms were different from the previously identified PDE4A variants of 110 and 75 kDa. These data demonstrate that the presence of multiple cAMP-PDE genes is translated into cAMP-PDE proteins of different sizes and distinct immunological properties and that multiple variants derived from these cAMP-PDE genes are expressed in different regions of the brain and different subcellular compartments. These immunological tools will be useful to identify different cAMP-PDE forms expressed in organs targeted for pharmacological intervention with PDE4 inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Role of phosphodiesterases in the regulation of gonadotropin- releasing hormone secretion in GT1 cells.

Increases in the level of cAMP stimulate the secretion of GnRH from GT1 GnRH neuronal cells. We hypothesized that cyclic nucleotide phosphodiesterases (PDEs), the enzymes that hydrolyze cAMP, may constitute a negative feedback signaling mechanism for GnRH regulation by decreasing the level of cAMP. GT1 cells were shown to express three PDEs by RT-PCR analysis: the cAMP-specific PDE4B and PDE4D and the calmodulin-dependent PDE1B. A splice variant of PDE4D, PDE4D3, which is activated when phosphorylated by cAMP-dependent protein kinase (PKA), was identified in GT1 cells by Western analysis. Consistent with PDEs negatively regulating GnRH secretion, treatment with the nonselective PDE inhibitor, IBMX, stimulated GnRH secretion 137% in 30-min static cultures. Furthermore, treatment with the PDE4-specific inhibitors Rolipram and RS-25344 increased GnRH secretion 48 and 125%, while treatment with the PDE1-specific inhibitor 8-MeoM-IBMX only caused a modest increase of 28%. In perifusion studies a rapid multi-fold stimulation of GnRH secretion was observed following treatment with IBMX, Rolipram or RS-25344. In conclusion, the level of PDE activity appears to be an important negative feedback signal for GnRH secretion. We hypothesize that activation of PDE4D3 by PKA may constitute a negative feedback signaling pathway which participates in the regulation of cAMP levels.

Animals↗
PubMed1998

Enhanced blood pressure sensitivity to deoxycorticosterone in mice with disruption of bradykinin B2 receptor gene.

The renal kallikrein-kinin system is activated under conditions of mineralocorticoid excess. To evaluate whether endogenous kinins exert a protective role against the development of mineralocorticoid-induced hypertension, we studied the cardiovascular effects induced by long-term administration of deoxycorticosterone (DOC; 0.3 micromol/g body wt s.c. once per week for 6 weeks) or vehicle in transgenic mice (Bk2r-/-) lacking the bradykinin B2 receptor gene and in wild-type controls (Bk2r+/+). Under basal conditions, Bk2r-/- mice showed higher systolic blood pressure (tail-cuff plethysmography) than wild-type Bk2r+/+ and heterozygous Bk2r+/- mice (121+/-2 versus 114+/-2 and 115+/-2 mm Hg, respectively; P<0.05 for both comparisons). Heart rate was higher in Bk2r-/- and Bk2r+/- than in Bk2r+/+ (459+/-12 and 418+/-7 versus 390+/-7 bpm; P<0.05 for both comparisons). Systolic blood pressure was increased by DOC in transgenic as well as in wild-type mice, whereas no change was induced by the vehicle. The pressor response to DOC was more rapid and pronounced in Bk2r-/- than in Bk2r+/+ and Bk2r+/- (30+/-5 versus 15+/-4 and 6+/-3 mm Hg, respectively, at 3 weeks; P<0.01 for both comparisons). The difference in systolic blood pressure was consistent with that detected by direct intra-arterial measurements of mean blood pressure. Neither DOC nor its vehicle altered heart rate or gain in body weight over time. Under basal conditions, urinary sodium excretion did not differ between strains. During DOC administration, cumulative urinary sodium excretion was lower in Bk2r-/- than in Bk2r+/+ (2.59+/-0.15 versus 3.31+/-0.22 mmol, respectively, during the first week; P<0.05). Urinary kinin excretion was increased by DOC in both Bk2r-/- (from 0.65+/-0.17 to 4.27+/-0.80 pmol/24 h; P<0.01) and Bk2r+/+ (from 0.55+/-0.09 to 6.27+/-1.48 pmol/24 h; P<0.05). The increase in urinary kinin excretion was similar between strains. These results show that integrity of the bradykinin B2 receptor is essential for regulation of blood pressure and heart rate under basal conditions. In addition, they indicate that activation of the kallikrein-kinin system represents a compensatory response against the development of hypertension induced by mineralocorticoid excess.

Analysis of Variance↗