Ennio Maccari

In vitro effects of gonadotropin-releasing hormone (GnRH) on Leydig cells of adult alpaca (Lama pacos) testis: GnRH receptor immunolocalization, testosterone and prostaglandin synthesis, and cyclooxygenase activities.

The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α).

Different modulation of aromatase activity in frog testis in vitro by ACE and ANG II

The aim of the present research was to study the role of angiotensin-converting enzyme (ACE) and ANG II in amphibian ( Rana esculenta) testicular steroidogenesis and prostaglandin production. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog ANG I, and [Val5]ANG II were determined in frog testis of prereproductive period. Production of 17β-estradiol, progesterone, androgens, and PGE2 and PGF2α was determined by incubating frog testes with ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val5]ANG II (1 μM), and synthetic bullfrog ANG I (1 μM). The analysis of the data showed an independent modulation of 17β-estradiol and androgen production by ACE and ANG II. The ACE pathway caused a decrease of 17β-estradiol production and an increase of androgen production in frog testes; on the other hand, the ANG II pathway increased 17β-estradiol production and decreased androgen production. The determination of testicular aromatase activity showed a positive regulation by ANG II and a negative regulation by ACE. As for prostaglandin production, only ANG II influenced PGF2α. These results suggest a new physiological role of ACE and ANG II in modulating steroidogenesis and prostaglandin production.The aim of the present research was to study the role of angiotensin-converting enzyme (ACE) and ANG II in amphibian (Rana esculenta) testicular steroidogenesis and prostaglandin production. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog ANG I, and [Val(5)]ANG II were determined in frog testis of prereproductive period. Production of 17beta-estradiol, progesterone, androgens, and PGE(2) and PGF(2alpha) was determined by incubating frog testes with ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val(5)]ANG II (1 microM), and synthetic bullfrog ANG I (1 microM). The analysis of the data showed an independent modulation of 17beta-estradiol and androgen production by ACE and ANG II. The ACE pathway caused a decrease of 17beta-estradiol production and an increase of androgen production in frog testes; on the other hand, the ANG II pathway increased 17beta-estradiol production and decreased androgen production. The determination of testicular aromatase activity showed a positive regulation by ANG II and a negative regulation by ACE. As for prostaglandin production, only ANG II influenced PGF(2alpha). These results suggest a new physiological role of ACE and ANG II in modulating steroidogenesis and prostaglandin production.

Different modulation of steroidogenesis and prostaglandin production in frog ovary in vitro by ACE and ANG II

Our aim was to study the role of angiotensin-converting enzyme (ACE) and angiotensin II (ANG II) on ovarian steroidogenesis and prostaglandin production of amphibian. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog angiotensin I (ANG I), and [Val5]ANG II were compared on frog ovaries of postreproductive and prereproductive periods. Very high ACE activity was found in ovary of water frog ( Rana esculenta) compared with other frog tissues, and this activity was inhibited by the typical ACE inhibitors, captopril and lisinopril. Frog ovary tissue in postreproductive and prereproductive periods was incubated in vitro in the presence of ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val5]ANG II (1 μM), and synthetic bullfrog ANG I (1 μM). Production of 17β-estradiol, progesterone, androgens, and prostaglandins E2 and F2α was determined. The data showed a modulation of 17β-estradiol, progesterone, and prostaglandin E2 production by ovary ACE; on the other hand, [Val5]ANG II modulated the production of progesterone and prostaglandin F2α, whereas androgen production was not influenced. The present in vitro studies suggest the existence of two pathways independently regulated by ACE and ANG II modulating ovarian steroidogenesis and prostaglandin production.

Degradation of thymic humoral factor γ2 by human plasma: involvement of angiotensin converting enzyme

The degradation of thymic humoral factor-gamma2 (THF-gamma2), an immunoregulatory octapeptide important for T-lymphocyte regulation, by enzymes present in human plasma, was investigated. THF-gamma2 was metabolized through two steps that involved the detaching of N-terminal amino acid leucine followed by hydrolysis of the Lys(6)-Phe(7) bond. The THF-gamma2 cleavages were sensitive to aminopeptidase and metalloproteinase inhibitors. The degradation was completely blocked by amastatin and specific inhibitors of angiotensin converting enzyme (ACE). The cleavages occurred independently, with two different kinetics, faster for the N-terminal hydrolysis than for that of the Lys(6)-Phe(7) bond. Purified human plasma ACE was used to characterize the hydrolysis of Lys(6)-Phe(7) bond. The K(m) and K(cat) values for THF-gamma2 hydrolysis were 0.273 mM and 107 s(-1), respectively. The optimum of chloride concentration was 300 mM, while that of pH was 7.6. The presence of ACE in circulating mononuclear cells raises the possibility that it may play a role in modulating the THF-gamma2 activity.

Purification of swine serum angiotensin converting enzyme with high recovery of activity using lisinopril coupled to epoxy-activated sepharose 6B.

The Authors describe the purification of swine serum ACE to molecular homogeneity with high recovery of activity (40 %) in a few stepsl. The purification procedure consists of affinity chromatography, using commercial activated resin (epoxy‐activated sepharose 6B) and two steps of anion exchange chromatography (Resource Q) performed at different pH (pH 9.0 and pH 6.0). Furthermore, a specific and sensitive method for the accurate quantitation of ACE activity in biological fluids was developed, based on the hydrolysis of synthetic FAPGG (N‐[3‐(2‐furyl) acryloyl] L‐phenylalanyl glycyl glycine), as substrate and following the separation of prodcts by reversed‐phase HPLC. Some kinetic parameters were determined. The Km and Kcat values for FAPGG were 0.793±0.052 mM and 5830 s‐1, respectively, and the I50 values for captopril and lisinopril, two specific ACE inhibitors, are 5.7±0.67 nM and 1.0±0.29 nM, respectively.

Purification and characterisation of swine serum proteinase which hydrolyses epidermal inhibitory pentapeptide

In this paper we describe the purification to molecular homogeneity of the enzyme that cleaves the synthetic epidermal mitosis-inhibiting pentapeptide (pyroGlu-Glu-Asp-Ser-Gly; EPP) from swine serum. Biochemical characterisation of the enzyme shows a glycoprotein with apparent molecular mass of 200 kDa. The Km and Kcat values for EPP hydrolysis are 0.624 mM and 694 s-1, respectively. Use of proteinase inhibitors shows the enzymes metalloendopeptidase character. Moreover, captopril and lisinopril prevent the cleavage of EPP. The N-terminal amino-acid sequence of the purified protein corresponds to the N-terminal amino-acid sequence of swine kidney angiotensin-converting enzyme, a dipeptidyl carboxypeptidase (EC 3.4.15.1).

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro.

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg−1 and 326 U mg−1 respectively. The optimum pH range was from 7–8.5 at 37°C and the optimum temperature was 50 °C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608±0.07 mM and 249 sec−1 respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68±12.55 nM and 6.763±0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17β-estradiol, progesterone, and prostaglandins E2 and F2α was determined. The data showed a modulation of 17β-estradiol, progesterone and prostaglandin E2 production by ovary ACE.ResumenLa finalidad de este estudio es la purificación y caracterización de la enzima convertidora de angiotensina (ACE) contenida en el ovario de rana (Rana esculenta). La enzima, extraída tanto con detergente como con tripsina, se purificó mediante un proceso único, consistente en una cromatografía de afinidad con lisinopril ligado a Sepharose 6B. El peso molecular de la enzima resultó ser de 150 kDa, tanto por la extracción con detergente como con tripsina. La actividad específica de la ACE extraída con detergente y tripsina fue de 294 U mg−1 y 326 U mg−1, respectivamente. El intervalo de pH óptimo fue de 7–8,5 a 37°C y la temperatura óptima, de 50 °C. La concentración molar óptima de cloruro fue sobre 200 mM para el sustrato sintético FAPGG (N-[3-(2-furyl) acryloyl] L-phenylalanyl glycyl glycine) y angiotensina I, y 10 mM para la bradiquinina. Los valores de Km y Kcat para FAPGG fueron 0,608±0,07 mM y 249 sec−1, respectivamente, y los valores de I50 para captopril y lisinopril, dos inhibidores específicos de ACE, fueron 68±12,55 nM y 6,763±0,66 nM, respectivamente. Tejido ovárico de rana en períodos prerre-productivos se incubó in vitro en presencia de ACE de ovario de rana (2,5 mU/ml), captopril (0,1 mM), y lisinopril (0,1 mM), determinán-dose la producción de 17β-estradiol, progesterona y de prostaglandinas E2 y F2α. Los resultados muestran modulación de la producción de 17β-estradiol, progesterona y prostaglandinas E2 por ACE ovárica.

Synthetic seminal plasma peptide inhibits testosterone production in frog testis in vitro

The role of synthetic seminal plasma peptide, designed using biochemical and mass spectroscopy analyses of native peptides extracted from seminal plasma, was studied in amphibian (Rana esculenta) testicular steroidogenesis. Production of testosterone and prostaglandin F(2alpha) was determined by incubating frog testes with synthetic peptide in vitro. Analysis of the data showed a dose-dependent inhibition of testosterone production (43% at 10(-5) M concentration) without prostaglandin F(2alpha) synthesis being affected. Determination of the peptide activity during the annual R. esculenta reproductive cycle showed inhibition of testosterone production in post-reproductive and recovery periods, suggesting a possible involvement of peptide in gonad steroidogenesis.

In vitro phosphorylation of proteins tightly bound to DNA by protein kinase NII.

1. Highly purified DNA from calf thymus was phosphorylated with protein kinase NII. 2. Digestion with proteinase K of this DNA demonstrates proteins as phosphorylated component. 3. Gel filtration chromatography on Bio-Gel A-0.5m gel column shows a major protein peak between 50 and 70 kDa. 4. SDS gel electrophoresis, after hydrolysis, to digest completely DNA, shows three major phosphorylated bands corresponding to polypeptides of M(r) between 31 and 21 kDa. 5. After high voltage electrophoresis on TLC plates tryptic digested polypeptides show very similar phosphopeptides patterns.