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M Conti

Publications and source records attributed to M Conti.

At least 55 records · Page 3Linked to original sources

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

[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↗

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↗