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M Conti

Publications and source records attributed to M Conti.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

The olfactory adenylyl cyclase III is expressed in rat germ cells during spermiogenesis.

To identify the adenylyl cyclase (AC) genes expressed in mammalian germ cells, RT-PCR of testis and germ cell RNA was performed using degenerated primers based on the homologous region of the AC catalytic domain. This strategy yielded high-frequency amplification of a complementary DNA (cDNA) identical to type III AC (ACIII), a form previously identified as the major adenylyl cyclase expressed in the olfactory system. Ribonuclease protection studies confirmed that ACIII transcripts are present in germ cells, appear during the meiotic prophase, and accumulate during spermiogenesis. A Northern blot analysis performed on total testis RNA demonstrated the presence of a predominant transcript of 7.5 kb, suggesting that the ACIII expressed in germ cells may derive from a splicing variant different from the 4.5 kb transcripts expressed in somatic cells. To determine whether these RNAs are translated into a protein, Western blot analysis was performed using an antibody specific for the carboxyl terminus of ACIII. An immunoreactive protein of 170 kDa was detected in extracts from total testis and from germ cells. Immunofluorescence localization of this protein in the seminiferous tubules showed that ACIII was predominantly expressed in postmeiotic germ cells from round spermatids in the cap phase to maturing elongating spermatids. The ACIII antigen was located mostly on the acrosomal membrane rather than on the plasma membrane of developing spermatids. The spatial and temporal expression of ACIII in germ cells indicates a role of this AC in the acrosome formation. Together with the observation that members of the olfactory receptor family and an olfactory phosphodiesterase are expressed in spermatids, these findings suggest that a signal transduction system used in olfaction is also used during gamete development.

Adenylyl Cyclases↗
PubMed1998

[Diagnostic imaging of gastrointestinal leiomyosarcoma. Experience with 12 cases].

PURPOSE: Gastrointestinal (GI) leiomyosarcoma is an uncommon malignant cancer arising in the smooth muscle of the alimentary tract. It is known for its widely variable patterns and aspecific symptoms and signs preventing correct clinical assessment in the majority of cases. We will illustrate the key role of diagnostic imaging in the detection and staging of this lesion, describing the most suggestive imaging findings for the correct diagnosis. MATERIAL AND METHODS: January, 1990, to June, 1998, we examined 12 patients with GI leiomyosarcoma; they were 10 men and 2 women whose age ranged 42 to 85 years (mean: 63.7 years). Four lesions were found in the stomach, 3 in the jejunum and ileum, and 2 in the rectum. Due to the difficult clinical assessment of this type of lesion and to the development of emergency conditions, we could plan no diagnostic protocol in advance; thus, the most suitable diagnostic imaging approach was decided on the spot for studying the supposedly involved GI portions. Double contrast studies, US, CT and endoscopy were performed and each patient underwent at least two examinations. RESULTS: Barium contrast studies were performed in 9 patients: the lesion was detected in 7 cases and tumor site and extent were defined in 5, while the double contrast study of the colon allowed to exclude large bowel involvement in 2 ileal tumors. In all 9 cases US and US-guided endoscopy permitted better assessment of extra-luminal spread and involvement of adjacent organs. CT, which is essential to staging, provided useful information suggesting the lesion nature: a round, inhomogeneous mass in continuity with the intestinal wall, with irregular margins, peripheral enhancement after i.v. injection of contrast material and a central necrotic area. Histology confirmed CT diagnosis in 7/9 cases while an aspecific diagnosis of large retroperitoneal and abdominal lesion was made in 2 cases. CT did not allow to define the origin of 2 large exophytic lesions in the stomach and jejunum and missed peritoneal metastases in 3 cases. CONCLUSIONS: Although the aspecific and quite variable clinical patterns make it extremely difficult to plan a correct diagnostic protocol, in our experience all diagnostic imaging techniques played a fundamental role in identifying and staging alimentary tract leiomyosarcoma. Particularly, CT showed high sensitivity and specificity in characterizing and staging this lesion but exhibited rather poor sensitivity in recognizing peritoneal spread.

Adult↗
PubMed1998

A short history of plant virology. III. The thirties.

The Thirties testified on the outstanding development of plant virology: the new discoveries formalized the concept of virus on a physicochemical background. Plant viruses, which had received their own taxonomical position at the end of the Twenties, were no longer considered as simple "infective pathogens" as their size, shape and chemical nature were determined, particularly for one of them--tobacco mosaic virus (TMV). This paramount contribution was achieved as a consequence of a functional interaction between biology on one side, and chemistry and physics on the other side, from the development of which molecular biology was born. The chemical characterization of TMV developed from the first determination of nitrogen presence in purified virus, performed by Carl Vinson, through the identification of TMV as Wendell Stanley's infective, autoreplicative protein macromolecule, to the final discovery of its nucleoprotein nature by the British group of Frederick Bawden. Thorough analytical techniques--in particular electron microscopy--led to disclose the exact shape and size of TMV particle. These discoveries, that opened a new era of virology, were corroborated by new knowledge that, although less explosive, can be considered of great importance for the development of plant virology. The methodologies to estimate viral activity; the study of the relationships between viruses and insect vectors; the studies on virus spread within plants; the identification of non-sterile type of resistance and of correlation between single plant genes and viral pathogenesis benefited plant virology of a set of knowledge that, together with the discoveries on the physico-chemical properties of TMV, raised plant virology from a secondary branch of plant pathology to a new independent science by itself.

History, 20th Century↗
PubMed1997

Current trends in capillary isoelectric focusing of proteins.

Isoelectric focusing (IEF) in thin capillaries is reviewed here. After an introduction on the genesis and chemistry of the carrier ampholyte buffers, different approaches to IEF are discussed and evaluated. The classical approach consists on IEF under conditions of suppressed electroosmotic (EOF) flow, usually obtained by covalently bonding hydrophilic polymers to the inner capillary wall. The other approach consists of IEF in dynamically (and partially) coated capillaries, so as to allow a reduced EOF flow to coexist with the IEF process, so that focusing and transport of the train of stacked bands occurs simultaneously. The various experimental parameters: focusing, elution and detection steps, pI measurements, as well as typical drawbacks, such as isoelectric precipitation are evaluated. The review ends with some examples of analytical separations, at the moment mostly limited to focusing of native hemoglobins (normal and point mutants). These separations are compared with those obtained by slab-gel IEF and in immobilized pH gradients.

Animals↗
PubMed1997

Co-expression of defective luteinizing hormone receptor fragments partially reconstitutes ligand-induced signal generation.

Gonadotropin receptors are unique members of the seven-transmembrane (TM), G protein-coupled receptor family with a large extracellular (EC) sequence forming the high-affinity ligand binding domain. In a patient with Leydig cell hypoplasia, we identified a mutant LH receptor that is truncated at TM5. This protein retains limited ligand binding ability but cannot mediate cAMP responses. To study interactions between receptor fragments defective in either ligand binding or signal transduction, we co-expressed this truncated receptor together with a chimeric receptor containing the EC region of the FSH receptor and the TM region of the LH receptor. Although the chimeric receptor could not respond to human chorionic gonadotropin in producing cAMP, co-expression with the truncated LH receptor allowed partial restoration of ligand signaling through intermolecular interactions. In addition, co-expression of the same truncated LH receptor with an N-terminally truncated LH receptor that lacked the EC ligand binding domain also partially restored ligand signaling. Further shortening of the TM region in the mutant receptor found in the patient indicated that the EC domain and TM1 were sufficient for interactions with the N terminally truncated receptor. In contrast, co-expression of the N terminally truncated receptor together with cell-associated or soluble EC region of the LH receptor did not allow ligand signaling. Unlike thrombin receptors, co-expression of the anchored EC region of the LH receptor together with the N-terminally truncated receptor did not allow ligand signaling despite moderate levels of human chorionic gonadotropin binding in transfected cells. These studies demonstrate that the co-expression of binding (+)/signaling (-) and binding (-)/signaling (+) receptor fragments partially restores ligand-induced signal generation and indicate the importance of TM1 of the LH receptor in the proper orientation of the EC ligand binding domain.

Amino Acid Sequence↗
PubMed1997

Selective stimulation of a cAMP-specific phosphodiesterase (PDE4A5) isoform by phosphatidic acid molecular species endogenously formed in rat thymocytes.

We have previously reported that concanavalin A (ConA) stimulation of rat thymocytes induces an increase in the cellular phosphatidic acid mass as well as a change in its fatty acid composition. An increase in phosphodiesterase (PDE) activity, mostly due to cAMP-specific (PDE4) isoforms, has also been observed in thymocytes stimulated by ConA. Furthermore, phosphatidic acid was able to stimulate PDE4 activity in vitro. In the present study, cAMP levels have been shown to decrease upon ConA stimulation of thymocytes. Decreasing phosphatidic acid level using diacylglycerol kinase inhibitors induced a parallel decrease of the ConA-stimulated cAMP-specific PDE activity in these cells. Analyses of phosphatidic acid molecular species in cells stimulated for 5 min by ConA revealed a significant increase in 1-stearoyl-2-arachidonoyl-sn-glycerol-3-phosphate and a relative decrease in the other molecular species of phosphatidic acid, mainly species containing palmitate. On the other hand, phosphatidic acid extracted from ConA-stimulated cells activated more efficiently the recombinant PDE4A5 isoform in vitro, as compared to phosphatidic acid extracted from unstimulated cells. In addition, phosphatidic acid species containing unsaturated fatty acids were stimulatory, while those containing two saturated fatty acids had only a marginal effect on the enzyme activity. Taken together, these data suggest that the mitogenic stimulation of thymocytes is accompanied by the synthesis of peculiar phosphatidic acid molecular species able to activate a PDE4 isoform. This activation might be of physiological relevance since cAMP is a major negative effector of the mitogenic response.

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

Study of haptoglobin-hemoglobin complexes by titration curves, capillary electrophoresis and capillary isoelectric focusing.

A novel method is described for monitoring complex formation between macromolecules, based on combined isoelectric focusing-electrophoresis in capillaries. The example studied is the binding of serum haptoglobin (Hp) to hemoglobin (Hb). A known amount of Hb is focused in a capillary in a pH 6-8 range (pI of Hb = 7.0) and thus kept temporarily "immobilized" in the electrophoretic chamber. Subsequently, increasing amounts of ligand (Hp) are loaded cathodically and allowed to sweep past the focused Hb zone. As the complex formed has a pI value well-outside the bounds of such a pH gradient (the 1:1 molar Hb-Hp complex has a pI of 5.5, the 1 to 1/2 molar Hp-Hb complex has a pI of 5.0) it escapes immobilization and moves past the detector window, where it is monitored and quantified. Since the detector is set at 416 nm, where only Hb absorbs, and since the molar extinction coefficient of Hb is well known, it is quite easy to calculate the molar amount of Hb bound to the complex. As an additional check, the amount of unreacted Hb can now be mobilized by disrupting the pH gradient and allowing this residual free Hb to also reach the detector and be quantified. The method is easy, fast, simple and fully automated and thus could represent a valid alternative to existing methods in clinical chemistry for quantifying the amount of Hp in human sera in pathological conditions, such as hemolytic anemias and transfusion reactions.

Ampholyte Mixtures↗