Ninety-six hours subzero nonfrozen storage of rat kidneys: preliminary report.
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Animals↗
Biomedical subjects
M Conti
Publications and source records attributed to M Conti.
Characterization of a hormone-inducible, high affinity adenosine 3'-5'-cyclic monophosphate phosphodiesterase from the rat Sertoli cell.
In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100,000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps. The major purified form of cAMP-PDE had a specific activity of 1-2 mumol/(min.mg of protein). Polyacrylamide gel electrophoresis and silver staining analysis showed that a 67-68 kDa polypeptide comigrated with the major peak of cAMP hydrolytic activity. The molecular weight of the crude or purified enzyme determined by gel filtration and sucrose density gradients was 150,000, suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a Km of 1.97 +/- 0.26 microM. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 microM. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The activity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxanthine > cilostamide = milrinone. Because mRNAs derived from two distinct PDE4B and PDE4D genes are present in the Sertoli cell, selective and nonselective PDE antibodies were used to determine the origin of the inducible PDE protein.(ABSTRACT TRUNCATED AT 250 WORDS)
3',5'-Cyclic-AMP Phosphodiesterases↗
Screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries.
Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, Fac, A) by capillary isoelectric focusing (IEF) in a pH 6-8 gradient is reported. Even in coated capillaries (with covalently bound chains of linear acrylamido derivatives, notably N-acryloylaminoethoxyethanol), no base line separation is obtained between Hb F and A, although this is routinely achieved in gel slab IEF. However, when the carrier ampholyte buffers were added to 3% short-chain liquid linear polyacrylamide, base line resolution and stabilization of peak transit times were obtained. This suggests that even in the best coating procedures, patches of the inner capillary surface could still be naked, so that the static coating is complemented by a dynamic coating on the unoccupied sites. An additional improvement in separation occurs if the above mixture, comprising 5% carrier ampholytes in the pH 6-8 range and 3% soluble polyacrylamide, is made to contain 50 mM beta-alanine, a "separator" known to flatten the pH gradient around pH 7. In the normal newborns analyzed (n = 30), the following average values were obtained: Hb F, 70.1% (range 65-75%); Hb A, 20.2% (range 15-25%); and HbFac, 9.5% (range 7-11%).
Acetylation↗
Effect of aging on left ventricular filling in untreated hypertensive patients.
The aim of this study was to evaluate the influence of age and other clinical and echocardiographic parameters on left ventricular filling in a group of 174 untreated patients with mild to moderate hypertension (aged 20-82 years; mean 51.2 years) and in 189 age-matched normotensive subjects. All subjects underwent an echocardiographic study with pulsed Doppler evaluation of left ventricular filling. Left ventricular dimensions and indexes of systolic function were similar and within normal limits in both groups. Left ventricular filling was altered in hypertensive subjects < 65 years with a decrease of peak early velocity (peak E), an increase of peak atrial velocity (peak A) and a reduced E/A ratio. However in subjects > or = 65 years, we did not observe any differences in transmitral flow velocity pattern between hypertensive and normotensive subjects. The stepwise regression analysis showed that age alone explains up to 8% of peak E variance, 14% of peak A and 26% of E/A ratio in hypertensives, while in normotensives it explains up to 18% of peak E variance, 50% of peak A and 61% of E/A ratio. The other variables entered into the regression slightly improved the predictive power. In conclusion, age is the major independent factor affecting left ventricular filling in both groups, even if its predictive power was smaller in the hypertensive group. The similarity of diastolic filling pattern in elderly hypertensive and normotensive subjects suggests that the 'aging factor' plays an important role in influencing left ventricular filling pattern so as to mask the effect of hypertension in the elderly patients.
Adult↗
Plasma from patients exposed to ischemia reperfusion contains clastogenic factors and stimulates the chemiluminescence response of normal leukocytes.
Clastogenic factors (CFs) are released by cells exposed to superoxide radicals and are found in various situations of oxidative stress. Certain of their components stimulate further superoxide production by competent cells, as shown with cytochrome c assay in previous work. In the present study, we report CF formation after ischemia reperfusion in patients undergoing coronary bypass surgery. Plasma ultrafiltrates, collected 20 min after reperfusion, had clastogenic properties in contrast to those collected before ischemia. We also show that the luminol-enhanced chemiluminescence response of neutrophils from healthy persons is increased when these cells are exposed to CF-containing postreperfusion samples from patients. Light emission was reduced to control values in the presence of superoxide dismutase. The burst of oxyradicals upon reperfusion is probably the initiating event of CF formation, which in turn leads to further oxyradical generation. This amplification process may explain why detectable levels of CF need a delay of at least 10 min. The activated state of neutrophils in ischemia reperfusion is at once a consequence and a source of CFs. Individual variation in the persistence of this clastogenic and leukocyte-activating material was observed. Therefore, antioxidants for prevention of ischemia reperfusion injury should be continued during the postoperative course.
Antioxidants↗
Chromosomal localization of the human and rat genes (PDE4D and PDE4B) encoding the cAMP-specific phosphodiesterases 3 and 4.
Through the use of somatic cell hybrids segregating either human or rat chromosomes, we determined the chromosome localizations of two genes encoding cAMP-specific phosphodiesterases (cAMP-PDEs). PDE4D, the gene encoding the cAMP-PDE isoform 3 (IVd), was assigned to human chromosome 5 and rat chromosome 2, and PDE4B, the gene encoding the cAMP-PDE isoform 4 (IVb), was assigned to human chromosome 1 and rat chromosome 5. These localizations extend the homology between rat chromosome 2 and human chromosome 5, on the one hand, and between rat chromosome 5 and human chromosome 1, on the other hand.
3',5'-Cyclic-AMP Phosphodiesterases↗
Recent progress in understanding the hormonal regulation of phosphodiesterases.
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Animals↗
Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium.
Four genes (ratPDE1/IVc, ratPDE2/IVa, ratPDE3/IVd, and ratPDE4/IVb) encoding different isoforms of phosphodiesterase that specifically hydrolyze the second messenger cAMP (cAMP-PDEs) are present in the rat. Previous data from our laboratory indicated that these genes are differentially expressed in the somatic and germ cells of the seminiferous epithelium of the testis. To further characterize their spatial and temporal expression in the seminiferous tubules, in situ hybridization was used to monitor the expression of the four cAMP-PDE messenger RNAs (mRNAs). The signals corresponding to ratPDE1/IVc and ratPDE2IVa mRNAs were localized in two restricted layers of the seminiferous epithelium. The ratPDE1/IVc mRNA was present in a region of the epithelium corresponding to the location of middle-late pachytene spermatocytes. Conversely, the ratPDE2/IVa signal was confined to a more adluminal area corresponding to the location of maturing round spermatids. The ratPDE3/IVd and ratPDE4/IVb mRNAs were distributed throughout the span of the seminiferous epithelium, indicating a localization in the Sertoli cell cytoplasm. Although the intensity of the signal corresponding to ratPDE4/IVb was similar in all seminiferous tubule stages, the ratPDE3/IVd signal varied in intensity in tubules at different stages of the seminiferous cycle. Maximal expression was present in tubules at stages I-V and XI-XIII of the cycle and minimal at stages VIII-IX of the cycle. The expression of the ratPDE3/IVd mRNA positively correlated with the ability of specific inhibitors of the cAMP-PDEs to potentiate the FSH-dependent cAMP accumulation in tubules at different stages of the seminiferous cycle, with maximal potentiation observed at stages II-VI of the cycle. These data demonstrate that different cAMP-PDE genes are active in different cells of the seminiferous tubules and that the ratPDE3/IVd gene is expressed in the Sertoli cell in a cyclical fashion during the seminiferous cycle.
3',5'-Cyclic-AMP Phosphodiesterases↗
Activation of cyclic nucleotide phosphodiesterases in FRTL-5 thyroid cells expressing a constitutively active Gs alpha.
The expression of a constitutively activated Gs alpha protein in the rat thyroid cell line FRTL-5 causes an increase in the hormone-independent adenylyl cyclase activity and promotes TSH-independent growth of the cells. In spite of the constitutive activation of the adenylyl cyclase, the basal cAMP levels in these cells are only marginally increased. To define the role of phosphodiesterases (PDEs) in the genesis of this phenotype, cyclic nucleotide hydrolysis was determined in two cell lines expressing a mutated Gs alpha (Q227L). In these cells, the hydrolysis of both cAMP and cGMP was markedly increased in comparison with normal cells. This increase is the result of the activation of different forms of PDEs. Analysis of the cGMP hydrolysis and Ca++/calmodulin stimulation of the PDE activity indicated that the activity of a Ca++/calmodulin-stimulated PDE is increased in both cell lines. In addition, an increase in high-affinity, rolipram-sensitive cAMP-PDE activity was associated in both cell lines with the appearance of a 67-68 kilodalton (kDa) protein that cross-reacts with two antibodies against cAMP-PDEs. This form had the properties of ratPDE3.2/PDE4D2, a cAMP-PDE that is inducible by TSH in wild type cells. That an increase in cAMP-specific, rolipram-sensitive PDE plays a role in the phenotype induced by Q227L Gs alpha was confirmed by measurements of the mitogenic activity. Incubation with rolipram, which had no effect on wild type cells, caused an increase in cAMP levels and further stimulated TSH-independent proliferation in both cell lines carrying the mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
3',5'-Cyclic-AMP Phosphodiesterases↗
A novel insulin secretagogue is a phosphodiesterase inhibitor.
The arylpiperazine L-686,398 was described as an oral hypoglycemic agent and is shown to be an insulin secretagogue in vitro. The characteristics of its activity were similar to those of the incretin glucagon-like peptide I (GLP-I). We demonstrate that both the peptide and L-686,398 increase the accumulation of cAMP in isolated ob/ob mouse pancreatic islet cells, but by different mechanisms. Although GLP-I activates adenylate cyclase, the arylpiperazine has no effect on this enzyme or on the binding of 125I-labeled GLP-I to its receptor on RINm5F rat insulinoma cell membranes. However, L-686,398 inhibits the total cAMP phosphodiesterase (PDE) activity in homogenates of ob/ob mouse pancreatic islets with an EC50 of approximately 50 mumol/l. To determine the mechanism of PDE inhibition by the arylpiperazine and to examine its specificity, we studied the kinetics of arylpiperazine inhibition of two recombinant PDEs. The arylpiperazine is a competitive inhibitor of both a human heart type III PDE and a rat type IV-D PDE. Inhibition of the type III and IV isozymes are characterized by Ki values of 27 and 5 mumol/l, respectively. Although not extremely potent, the arylpiperazine does exhibit modest selectivity between these PDEs. The observation that L-686,398 acts as a PDE inhibitor suggests that exploration for beta-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.
Animals↗
Activation and selective inhibition of a cyclic AMP-specific phosphodiesterase, PDE-4D3.
Prostaglandin E2 produces a transient increase in the intracellular concentration of cAMP in a human promonocytic cell line (U937). The temporal pattern consists of a rapid increase followed by a gradual decline to a new steady state. The decline phase coincides with an increase in the activity of a high affinity form of cAMP phosphodiesterase (PDE). Immunoprecipitation with specific antibodies revealed that the activated enzyme is a variant of PDE-4D. To confirm this observation, three isoforms of human PDE-4 (A, B, and D) were cloned and expressed in Sf9 cells with recombinant baculovirus infection. The activity of only one of the isoforms (PDE-4D3) increased after incubation with the catalytic subunit of protein kinase A and Mg-ATP. Hydrolytic activity of human PDE-4D3 was dependent on Mg2+. Before phosphorylation, the concentration-response curve for Mg2+ was biphasic and ranged from 0.1 to 100 mM. Phosphorylation of PDE-4D3 by protein kinase A produced a monophasic Mg2+ response curve (0.5 Vmax = 0.2 mM). Phosphorylation of PDE-4D3 increased the sensitivity of the enzyme to inhibition by RS-25344 (approximately 100-fold) and RS-33793 (approximately 330-fold). Thus, phosphorylation of PDE-4D3 induces an apparent conformation change that increases maximum velocity and sensitivity to inhibition by some analogues of nitraquazone. These observations provide the basis for a novel pharmacological strategy that targets an activated form of PDE in human leukocytes. Selective PDE-4D3 inhibitors may have useful anti-inflammatory properties with fewer adverse side effects than other PDE-4 inhibitors.
3',5'-Cyclic-AMP Phosphodiesterases↗
[Spontaneous transmural rupture of the esophagus (Boerhaave's syndrome)].
A case is presented of spontaneous transmural rupture of the lower third of the esophagus, penetrating the left pleural space. The patient underwent repair under 7 hours by combined approach (laparotomy and left thoracotomy). High mortality and morbidity of Boerhaave's syndrome can be lowered by prompt and careful evaluation of symptoms and radiological signs, so avoiding incorrect or late diagnosis. Results appear to be related more to the time interval between perforation and operation than to the specific technique used.
Aged↗
Spontaneous involution of optic pathway lesions in neurofibromatosis type 1: serial contrast MR evaluation.
PURPOSE: To evaluate with contrast MR the evolution in size, signal, and contrast enhancement of optic pathway lesions in four patients with neurofibromatosis type 1. METHODS: The four reported patients are children with ages ranging from 21 months to 13 years affected by neurofibromatosis type 1 and optic pathway lesions. No treatment of the optic pathway lesions was carried out in these patients. They have been followed by serial contrast MR. RESULTS: In all patients a change in size, signal, and enhancement of optic pathways lesions was noted with time, and in the last follow-up study a marked reduction in size and enhancement of optic pathway lesions was observed in all cases. CONCLUSIONS: Modification and regression of optic pathway lesions with spontaneous disappearance of the enhancement is demonstrated. This finding could have a crucial influence on the therapeutic approach of the optic pathway lesions.
Adolescent↗
The ratPDE3/IVd phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein kinase.
Several mRNAs coding for a cAMP-specific phosphodiesterase (ratPDE3/IVd) with divergent 5' regions have been detected in mammalian cells. To determine the physiological significance of these differences, the expression of these mRNAs and the properties of the corresponding proteins were investigated. At least three mRNA species derived from the ratPDE3/IVd gene (ratPDE3.1, ratPDE3.2, and ratPDE3.3 mRNAs) are present in Sertoli and thyroid cells and in brain. Expression of ratPDE3.1 and ratPDE3.2 but not ratPDE3.3 mRNAs was dependent on hormone stimulation. The ratPDE3.2 and ratPDE3.3 mRNA variants were translated into polypeptides with immunochemical and biochemical properties identical to the native cAMP phosphodiesterases (PDEs) found in the Sertoli cell and thyroid FRTL-5 cells. Incubation of the recombinant PDEs with the catalytic subunit of the cAMP-dependent protein kinase in a cell-free system caused the phosphorylation and activation of the ratPDE3.3 protein variant. Under the same experimental conditions, ratPDE3.1 and ratPDE3.2 protein variants were neither phosphorylated nor activated by the cAMP-dependent protein kinase. Similar results were obtained by stimulating cells expressing the three recombinant PDE variants with dibutyryl cAMP. These findings demonstrate that the ratPDE3/IVd gene codes for PDE forms subject to different regulations.
Alternative Splicing↗
The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation.
To elucidate the mechanism causing the transient accumulation of intracellular cAMP in the FRTL-5 thyroid cell line, the short-term effect of thyroid-stimulating hormone (TSH) on phosphodiesterase (PDE) activity was studied. Together with an increase in cAMP levels, TSH produced a significant increase in total PDE activity as early as 3 min, with a maximal stimulation reached after 15 min. This short-term increase in PDE activity was dependent on the TSH concentration (ED50 = 4 x 10(-11) M TSH). Forskolin and dibutyryl cAMP produced an even larger stimulation than that produced by TSH, suggesting that the effect of TSH is mediated by cAMP. To determine the properties of the PDE forms activated by TSH, antibodies specific for the cAMP-PDEs were used to immunoprecipitate the PDEs present in control cells, and cells incubated for 15 min in the presence of 10 nM TSH. Comparison of the activity recovered in the immunoprecipitation pellets demonstrated that TSH produced more than a 2.5-fold increase in the cAMP-PDE form(s) recognized by this antibody. Conversely, the activity remaining in the supernatants was not affected by the TSH treatment. Most of the activity recovered in the immunoprecipitation pellets (90%) was inhibited by 10 microM Rolipram, an inhibitor specific for the high affinity cAMP-PDEs. No TSH stimulation of the Rolipram-insensitive PDE activity could be observed under these conditions. Western blot analyses with two different cAMP-PDE specific antibodies showed that a 15-min stimulation with TSH induced the appearance of a new band with electrophoretic mobility slower than the polypeptide present in unstimulated cells. The appearance of this band did not require ongoing protein synthesis because it occurred in the presence of cycloheximide. Metabolic [32P]orthophosphate labeling of intact FRTL-5 cells indicated that the TSH treatment caused an increased 32P incorporation into a polypeptide that co-purified with the stimulated PDE activity and had an electrophoretic mobility identical to that of the cAMP-PDE. Okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A, elicited a potentiation of the TSH-stimulated PDE activity. The stimulating of a PDE with the same immunological properties and Rolipram sensitivity as the cAMP-PDE stimulated by TSH in the intact cells was reproduced, in a cell-free system, by incubating soluble extracts from FRTL-5 cells with the catalytic subunit of cAMP-dependent protein kinase. These data provide evidence that TSH produces a rapid activation of a cAMP-PDE in the FRTL-5 cells through a cAMP-dependent phosphorylation.
3',5'-Cyclic-AMP Phosphodiesterases↗
Structure of two rat genes coding for closely related rolipram-sensitive cAMP phosphodiesterases. Multiple mRNA variants originate from alternative splicing and multiple start sites.
The products of two phosphodiesterase (PDE) genes (ratPDE3/IVd and ratPDE4/IVb) are present in the rat Sertoli cell in culture, and their expression is under the control of the gonadotropin follicle-stimulating hormone (Swinnen, J.V., Tsikalas, K.E., and Conti, M. (1991) J. Biol. Chem. 266, 18370-18377). To understand the basis of the sequence heterogeneity found in the 5'-region of the different cDNAs thus far characterized, the structure of the coding region of these two cAMP PDE genes was investigated. Analysis of five ratPDE3/IVd and ratPDE4/IVb genomic clones showed that the coding region of these genes expressed in the Sertoli cell is divided into 11 exons distributed over 35-45 kilobases of genomic DNA. The intron/exon boundaries agreed, with some exceptions, with the established consensus sequences and were located in the same position in the coding region of the two genes. Also present were similarities to the exon composition of the Drosophila melanogaster "dunce" gene, the ancestor of these mammalian cAMP PDEs. Multiple AUG codons and short open reading frames were present at the 5'-untranslated end of the ratPDE4/IVb mRNA, but not in the ratPDE3 mRNA. By using polymerase chain reaction amplification or Northern analysis, it was determined that at least two forms of ratPDE3/IVd mRNA are present in rat Sertoli and FRTL-5 thyroid cells, but not in the brain. These mRNA variants are generated by inclusion or removal of an intron sequence that produces a frameshift affecting the position of the initiation AUG codon. Both mRNA species were efficiently translated into cAMP PDE proteins with different molecular masses in a transient transfection assay in COS cells. Polymerase chain reaction amplification demonstrated that heterogeneity of ratPDE4/IVb mRNAs was present in the same location as in the ratPDE3/IVd mRNA. Two ratPDE4/IVb mRNAs with different 5'-ends were expressed in Sertoli and FRTL-5 cells and in the brain. This heterogeneity is caused by the presence of an intron promoter that controls the transcription of this mRNA in Sertoli and FRTL-5 cells, but not in the brain. Upstream exons and additional promoters are probably present and necessary to generate the brain-specific mRNAs. These findings demonstrate that the cAMP-specific PDE genes have complex structure and that cAMP PDE proteins with different amino termini are derived from these genes.
3',5'-Cyclic-AMP Phosphodiesterases↗
Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan.
A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.
Animals↗
Long latency evoked potentials in a case of corpus callosum agenesia.
Following monoaural stimulation, long latency auditory evoked potentials (LLAEPs) recorded from contralateral temporal areas have a shorter latency and larger amplitude than those recorded from the ipsilateral temporal areas. This observation agrees with the operational model drawn up in 1967 by Kimura, which assumes that only anatomically prevailing crossed auditory pathways are active during dichotic hearing, while direct pathways are inhibited. The inputs may then be conveyed to the contralateral cortex, from where they finally reach the ipsilateral temporal areas by means of interhemispheric commissures. It is this mechanism which may underline the right ear advantage for verbal stimuli and the left ear advantage for melodies observed when administering dichotic listening tasks. With the aim of verifying this hypothesis, we recorded temporal LLAEPs in a 21 year-old woman suffering from complex partial seizures, whose CT scan and MRI showed corpus callosum agenesia. Our data support the hypothesis that ipsilateral pathways are greatly inhibited by the contralateral pathways, and therefore auditory stimuli can be supposed to reach the contralateral auditory cortex from where they are transferred through the corpus callosum to the ipsilateral auditory cortex.
Acoustic Stimulation↗