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Biomedical subjects

M Conti

Publications and source records attributed to M Conti.

At least 289 records · Page 16Linked to original sources
PubMed1984

The B----Z transition in two synthetic oligonucleotides: d(C-2-amino-ACGTG) and d(m5CGCAm5CGTGCG) studied by IR, NMR and CD spectroscopies.

The sequences CA'CGTG (where A' = 2-aminodeoxyadenosine) and m5CGCAm5CGTGCG are prepared and studied by IR, CD and 1H-NMR. Infrared spectra demonstrate the capacity of the modified hexamer and decamer to adopt a Z conformation. The influence of the NH2 substitution on the adenine or of the methylated terminal part of the decamer acting with the increase of the DNA concentration stabilizes the Z conformation at room temperature in low humidity films. Very weak proportion of Z conformation is detected in UV dilute solutions. In more concentrated NMR solutions, the Z proportion induced by high salt content is only 20-25%. The effects of the concentration and of the covalent modification of the bases are discussed.

5-Methylcytosine↗
PubMed1984

Characterization of a calmodulin-dependent high-affinity cyclic AMP and cyclic GMP phosphodiesterase from male mouse germ cells.

Two cyclic nucleotide phosphodiesterase activities were separated by ion-exchange chromatography of cytosol from male mouse germ cells. A form eluted at low salt concentration showed high affinity (Km congruent to 2 microM) and low affinity (Km congruent to 20 microM) for cyclic AMP, and high affinity (Km congruent to 3.5 microM) for cyclic GMP. A second form, eluted at high salt concentration, showed high affinity (Km congruent to 5 microM) for cyclic AMP and was similar to a phosphodiesterase activity described in rat germ cells. The present study was performed to characterize the first form, which represents most of the phosphodiesterase activity in mouse germ cells. The enzyme was sensitive to Ca2+ and calmodulin stimulation, which increased its activity 3-4-fold. Calmodulin stimulation depended on direct interaction of the activator with the enzyme, as indicated by the reversible changes in the chromatographic elution pattern in the presence of Ca2+, as well as by the increase in the sedimentation coefficient in the presence of calmodulin. Reciprocal inhibition kinetics between cyclic AMP and cyclic GMP for the calmodulin-dependent form demonstrated a non-competitive inhibition between the two substrates, suggesting the presence of separate catalytic sites. This is in agreement with kinetic parameters and different thermal stabilities of cyclic AMP- and cyclic GMP-hydrolysing activities. Furthermore, the relevant change in s value, depending on the absence or presence of Ca2+ and calmodulin, suggested that the enzyme is composed of subunits, which aggregate in the presence of the activator. A model for catalytic site composition and reciprocal interaction is also proposed.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Hormonal regulation of 3',5'-adenosine monophosphate phosphodiesterases in cultured rat granulosa cells.

The effect of gonadotropins on phosphodiesterase activity of rat granulosa cells was studied in an in vitro model. Granulosa cells were prepared from hypophysectomized or intact, estrogen-primed immature female rats and treated with FSH, hCG, or (Bu)2cAMP in vitro. Phosphodiesterase activity was determined in cell homogenates. FSH treatment for 2 days produced a marked increase in phosphodiesterase activity, while hCG was ineffective. FSH stimulation was potentiated by the addition of 1-methyl-3-isobutylxanthine, while treatment with the cAMP analog, (Bu)2cAMP by itself also markedly stimulated enzyme activity. FSH stimulated cAMP, but not cGMP, hydrolysis, suggesting that a phosphodiesterase specific for cAMP was stimulated by the gonadotropin. Time-course studies showed that an increase in phosphodiesterase activity was apparent after 1 h of incubation and was maximal at 48 h. FSH stimulation of phosphodiesterase was dose-dependent, with an ED50 of 30 ng/ml FSH and a maximal increase at 100-300 ng/ml. Treatment with cycloheximide (1 or 10 micrograms/ml) completely blocked the gonadotropin stimulation, suggesting that on-going protein synthesis is required for the FSH action. DEAE-cellulose chromatography of soluble extracts of control and FSH-treated cells indicated that two forms of phosphodiesterase were present in unstimulated granulosa cells. The first form, eluting at 0.17 M Na-acetate, hydrolyzed both cAMP and cGMP and was stimulated by Ca++ and calmodulin; the second form, eluting at 0.48 M Na-acetate, was insensitive to Ca++ or calmodulin and hydrolyzed mainly cAMP. FSH treatment markedly stimulated cAMP hydrolysis by the calmodulin-dependent first form as well as that by the second form. Double reciprocal analysis indicated that the FSH-stimulated enzymes are of high affinity for cAMP. In agreement with the data on total homogenate, the cGMP hydrolysis was not affected by the hormone treatment. These data demonstrate that FSH stimulates cAMP, but not cGMP, phosphodiesterase activity in rat granulosa cells in vitro. This stimulation might represent a mechanism for termination of the FSH primary stimulus and regulation of granulosa cell responsiveness to the gonadotropin.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Purine modulation of the hormonal response of the rat Sertoli cell in culture.

To investigate the possibility that purines modulate the response of testicular cells to gonadotropin, binding of adenosine analogs and biological responses to adenosine were evaluated in Sertoli cell-enriched cultures. The adenosine analog cyclohexyladenosine bound specifically to a crude particulate fraction prepared from such cultures. Binding was saturable, and steady state studies showed the presence of a high affinity binding site (Kd = 2.1 +/- 0.3 nM; n = 4) and a receptor density of 200-300 fmol/mg protein. The bound radioactive ligand was displaced by N6-phenylisopropyladenosine (PIA), adenosine, and methylisobutylxanthine. In addition to the presence of a specific binding site, purines modulated the biological function of the Sertoli cell. The adenosine analog PIA inhibited both the FSH-dependent cAMP response and the FSH-stimulated androgen aromatization. Under all experimental conditions, the IC50 of PIA was 1-3 nM, and maximal effects were observed at 10-100 nM PIA. Adenosine itself inhibited the FSH-dependent response of the Sertoli cell, but was less potent than PIA. In addition, purine inhibition of the FSH response was antagonized by methylisobutylxanthine, while the nonxanthine phosphodiesterase inhibitor Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone] was without effect. Purine modulation was evident not only when cells were stimulated with FSH, but also when the androgen aromatization was augmented by the beta-adrenergic agonist isoproterenol, cholera toxin, and forskolin. On the contrary, the purines had no effect when cells were stimulated with (Bu)2cAMP. The data reported are consistent with the presence of purine inhibitory receptors in Sertoli cell-enriched cultures and show that purines can regulate the response of the immature Sertoli cell in vitro.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Computer-aided evaluation of gastric proteoglycans by high-resolution gas chromatography.

A convenient procedure is described for the simultaneous separation and identification of monosaccharides and aminomonosaccharides constituting the proteoglycans of gastric mucosa. Computer assistance was used to obtain significant information from gas chromatographic data on proteoglycan modifications induced by anti-inflammatory drugs. Glass capillary chromatography was used for the separation and identification of saccharides and aminomonosaccharides of hydrolyzed mucus samples scraped from the stomachs of control and treated rats. The data, transmitted to a 48 K personal computer, were processed to obtain three-dimensional probability density functions or subjected to factor analysis to evaluate the suitability of the method to evidence modifications at the proteoglycan level induced by drugs, orally administered to rats. Indomethacin, zolimidine and acetylsalicylic acid were used as models for the applications.

Administration, Oral↗
PubMed1983

Fever, chills, and hypotension following cardiac catheterization with single- and multiple-use disposable catheters.

Recognition of pyrogen reactions in patients studied with multiple-use cardiac catheters led to recommendations regarding their cleaning and ultimately to the expensive practice of discarding catheters after a single use. Primarily because of cost considerations, our laboratory continued to clean and reuse catheters through 1981. This afforded an opportunity to assess the endemic rate of adverse reactions associated with this practice. We prospectively evaluated 341 patients who were studied with single-use or multiple-use catheters. The overall incidence of adverse reactions was: hypotension 27%, fever 3%, chills 3%, and all three 0.6%. There were no statistically significant increases in these reactions associated with the reuse of catheters. Bacterial infection did not appear responsible for these reactions, and it is possible that they were due to angiographic dye. We conclude that careful cleaning and reuse of catheters does not create an obvious increase in the risk of infection, and it appears to play a minor role, if any, in the development of other adverse reactions.

Adolescent↗
PubMed1983

Meiotic resumption and intracellular cAMP levels in mouse oocytes treated with compounds which act on cAMP metabolism.

We have studied the effect of agents known to stimulate adenylate cyclase on spontaneous meiotic resumption in vitro by mouse oocytes. We have found that cholera toxin (CT) (up to 1 microM) and prostaglandin E1 (PGE1) (up to 160 microM) are not able to prevent meiotic resumption, but a clear dose-dependent delay in meiosis resumption was observed during the first 3 h of incubation in medium containing CT or PGE1. The effect became clearer when a small concentration of isobutylmethylxanthine (MIX) (1 microM) was added to the medium. We have also measured the cAMP content of the oocyte: the basal content is 2.1 fmol; this value drops to 0.9 fmol during a 2-h culture period. The decrease is partially prevented if CT is present in the incubation medium, while total cAMP content increases to 3.1 fmol in the presence of 1 mM MIX. The results suggest that isolated mouse oocytes contain toxin and prostaglandin sensitive adenylate cyclase and also an active phosphodiesterase system.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Calmodulin in mouse male germ cells: a qualitative and quantitative study.

Isolated male germ cells of the mouse possess a heat-stable stimulatory activity of Ca2+-dependent, calmodulin-free phosphodiesterase. Ionic exchange chromatography allowed partial purification of the activator and the isolation of multiple forms of phosphodiesterase stimulation inhibitor. The activator has been identified as calmodulin on the basis of chromatographic behaviour and electrophoretic mobility. Quantitative analysis showed variations of calmodulin levels at different stages of spermatogenesis. Quantitative analysis of cyclic nucleotide hydrolysis in germ cell cytosol showed that the activity of Ca2+-dependent phosphodiesterase is different in meiotic and post-meiotic mouse male germ cells. These data suggest that calcium-dependent pathway and a Ca2+-dependent regulation of cyclic nucleotides are present in developing germ cells.

Animals↗
PubMed1983

Follicle-stimulating hormone regulates in vivo testicular phosphodiesterase.

The effect of FSH on testicular phosphodiesterase was studied in immature rats in order to verify that the regulation of response to hormone in the gonad involves an increased cyclic AMP catabolism. Hydrolysis of cyclic AMP and cyclic GMP was measured in the homogenates of seminiferous tubules and interstitium of control animals and animals injected i.p. with 50 micrograms ovine FSH twice, 24 h and 12 h before necroscopy. After hormonal treatment, cyclic AMP phosphodiesterase activity in the seminiferous tubules was markedly increased whether results were expressed per testis or per mg protein, while cyclic GMP phosphodiesterase present in the same compartment was apparently unaffected. In the interstitium, cyclic nucleotide hydrolysis was usually decreased after FSH injection. The stimulation of phosphodiesterase, a slow process reaching maximal stimulation after 12 h, was dependent on the dose of FSH injected. In addition, DEAE-cellulose chromatography of cytosol prepared from control and treated seminiferous tubules confirmed that stimulation was restricted to a cyclic AMP hydrolysing enzyme while the activity of the cyclic GMP hydrolysing form was not modified. Thus it is demonstrated that testicular phosphodiesterase is under FSH control. It is proposed that this in vivo regulation is a relevant phenomenon in the modulation of Sertoli cell function and contributes to the refractoriness that follows gonadotropin treatment.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Involvement of phosphodiesterase in the refractoriness of the Sertoli cell.

Gonadotropin treatment of the Sertoli cell produces a marked refractory state of the cell to subsequent hormonal stimulation. Because FSH also stimulates the phosphodiesterase activity of these cells, the possible involvement of an altered cAMP catabolism during refractoriness was investigated in an in vitro model. Sertoli cells, after 3 days of culture in a defined medium, were exposed to FSH or isoproterenol for 1-24 h. After this pretreatment, cells were stimulated for 1 h with a maximal FSH dose, and the responsiveness was measured in terms of cAMP accumulation. Sertoli cells previously treated with hormone entered a refractory state, a second exposure being ineffective in elevating intracellular or extracellular cAMP. Addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine in the second incubation partially restored the ability of the cell to accumulate cAMP in the presence of hormone. This phosphodiesterase inhibitor also caused an apparent decrease in the potency of FSH to induce the refractory state. Such an impairment of response developed in the intact cell in 4 h, and was accompanied by a partial desensitization of the adenylate cyclase and an increase in phosphodiesterase activity. The stimulation of phosphodiesterase activity, but not the desensitization of adenylate cyclase, was inhibited by cycloheximide. The inhibition of protein synthesis also prevented the onset of the refractory state of the intact Sertoli cell. Pretreatment of the Sertoli cells with either FSH or isoproterenol rendered the cell refractory to a second stimulation with either agonist; in contrast, the adenylate cyclase desensitization in the homogenate was apparent only for the agonist employed in the preincubation. These results indicate that phosphodiesterase regulation is involved in the control of Sertoli cell responsiveness to hormone. Thus, the net decrease in cAMP production of the FSH-treated cells is the result of a decreased adenylate cyclase stimulation and an increased cAMP catabolism mediated by phosphodiesterase. The latter phenomenon appears to be the predominant cause of the partial refractoriness induced by low doses of gonadotropin.

Adenylyl Cyclases↗
PubMed1982

Progressive resistance in a single strain of Acinetobacter calcoaceticus recovered during a nosocomial outbreak.

A study was undertaken to investigate a nosocomial outbreak of acinetobacter calcoaceticus. The outbreak occurred in a shock-trauma intensive care unit (ICU) during a 6-month period and involved a total of 37 cases. Although no deaths were attributable to Acinetobacter, 60% of patients from whom Acinetobacter was isolated were determined to have a lower respiratory tract infection. A significant difference (p less than 0.05) was noted between the length to stay in the unit of patients who acquired Acinetobacter (mean 23.9 days) and of those who remained free of the organism (mean 2.58 days). The average day on which cultures first became positive was 14.85. During the last 3 months of the outbreak, the ICU Acinetobacter isolates developed an unusual pattern of aminoglycoside resistance that differed significantly (p less than 0.001) from the sensitivities of non-ICU Acinetobacter isolates. No plasmids could be detected in selected aminoglycoside-resistance Acinetobacter. Pseudomonas aeruginosa isolated concurrently from patients with Acinetobacter showed no abnormal variation in antimicrobial sensitivities. These findings were interpreted to mean that the outbreak was the result of the persistence of a single strain of A. calcoaceticus within the ICU. Termination of the outbreak was attributed to initiation of control measures.

Acinetobacter↗
PubMed1982

Cyclic nucleotide phosphodiesterase in developing rat testis. Identification of somatic and germ-cell forms.

Several forms of phosphodiesterase are present in the male gonad, and their relative concentrations vary during testicular development. On the basis of kinetic analysis and chromatography on DEAE cellulose, 3 forms were separated: (a) a high-affinity cGMP phosphodiesterase, regulated by Ca2+ and calmodulin, similar to a form described in different tissues (peak I); (b) a high-affinity cAMP form insensitive to Ca2+, which cannot be readily compared with forms described elsewhere (peak II); and (c) a high-affinity cAMP phosphodiesterase, Ca2+- and calmodulin-insensitive, corresponding to the "hormone-regulated" form described in several systems (peak III). The elution pattern of peak I and calmodulin stimulation were dependent on free calcium concentration during cytosol preparation and chromatography. This datum and rechromatography in the presence or absence of excess calmodulin suggested that the enzyme complexes calmodulin in a Ca2+-dependent manner and is therefore activated. Moreover, whereas peak I was observed in all cell compartments of the testis, peak II was present in germ cells and peak III was found mainly in somatic cells. During development of the testis, a relevant enhancement in the ratio between cAMP and cGMP hydrolytic activity was observed together with an overall increase of phosphodiesterase activity, thus suggesting that the previously described increase in cAMP phosphodiesterase activity during testis maturation should be ascribed to forms present in both germ cells and somatic cells.

3',5'-Cyclic-AMP Phosphodiesterases↗