Biomedical subjects

A Miano

Publications and source records attributed to A Miano.

At least 19 recordsLinked to original sources

PubMed2003

Degradation of thymic humoral factor gamma2 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.

Amino Acid Sequence↗

PubMed2003

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 degrees C and the optimum temperature was 50 degrees 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 17beta-estradiol, progesterone, and prostaglandins E2 and F2alpha was determined. The data showed a modulation of 17beta-estradiol, progesterone and prostaglandin E2 production by ovary ACE.

Angiotensin I↗

PubMed2002

Bradykinin is not involved in angiotensin converting enzyme modulation of ovarian steroidogenesis and prostaglandin production in frog Rana esculenta.

Angiotensin converting enzyme (ACE) was demonstrated to modulate the production of 17beta-estradiol, progesterone and prostaglandin E2 (PGE2) in frog ovary of Rana esculenta. However, the activity was not mediated by angiotensin II (Ang II). In an attempt to identify the peptide involved in the pathway modulated by ACE, bradykinin, another physiological substrate of ACE, was chosen and incubated in the presence of the membrane suspension purified from the frog ovary homogenate. The hydrolytic products were analysed by reverse-phase high-pressure liquid chromatography (HPLC) analysis and the results showed that bradykinin was metabolized by membrane suspension. The presence of the protease inhibitors in the incubation mixture indicated ACE and neutral endopeptidase as being responsible for the bradykinin hydrolysis. Frog ovary was incubated in vitro in the presence of bradykinin (10 microM), bradykinin receptor antagonist NPC 567 (1 mg mL-1), bradykinin fragment (1-7) (10 microM), ACE (2.5 mU mL-1), captopril (0.1 mM) and lisinopril (0.1 mM). The results showed no modulating activity by bradykinin on ovarian 17beta-estradiol and PGE2 production, thus demonstrating that it was not involved in the ACE-modulated pathway.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2001

Synthesis and characterization of tetramethylrhodaminethiocarbamoyl-(Glu(1))-epidermal mitosis-inhibiting pentapeptide.

A fluorescent analog of epidermal mitosis-inhibiting pentapeptide (pGlu-Glu-Asp-Ser-Gly) was synthesized by reacting tetramethylrhodamine isothiocyanate with ring-opened epidermal mitosis-inhibiting pentapeptide. The ring-opening reaction of the pyrrolidone moiety was performed with mild acidic hydrolysis and the product purified by reversed-phase high-performance liquid chromatography. Tetramethylrhodaminethiocarbamoyl-(Glu(1))-epidermal mitosis-inhibiting pentapeptide was purified by chromatography on Sephadex G-25 and reversed-phase high-performance liquid chromatography. After characterization by amino acid analysis, the analog was incubated in presence of A431 cell line to visualize the cellular localization of the epidermal mitosis-inhibiting pentapeptide. The data gave negative results.

Cell Adhesion↗

PubMed2001

Neonatal screening for cystic fibrosis: long-term clinical balance.

BACKGROUND/AIMS: Very few studies have been performed on the long-term clinical advantages of neonatal screening programs for cystic fibrosis (CF) and these have been inconclusive. This is a preliminary report of two observational cohort studies on this subject. METHODS: In the first study, CF patients born between 1973 and 1981 in northeastern Italy were split into 4 groups according to the modality of diagnosis: screening by meconium test (58 patients); meconium ileus (45 patients); symptoms and pancreatic insufficiency (PI; 75 patients), or symptoms and pancreatic sufficiency (PS; 19 patients). The patients were followed for up to 26 years by three CF centers sharing common treatment protocols. In the second study, two cohorts of CF patients born between 1983 and 1992 were compared. Patients from one cohort (126 patients) were born in the Veneto region, where a neonatal screening program had been established based on immunoreactive trypsinogen. Patients from the other cohort (152 patients) were born in Sicily, where an intensive program of early diagnosis by symptoms was implemented. The cohorts were comparable for CF incidence, CFTR genotypes, gender proportion and common treatment protocols. Statistical analyses were performed by Kaplan-Meier survival curves, a Cox proportional hazard model for survival and cross-sectional comparisons by 2-year periods for weight z score, height z score and body mass index. RESULTS: In the first study, the patients detected by newborn screening (PI) showed better survival and nutritional status compared to patients diagnosed through meconium ileus or symptom presentation with PI. PS patients diagnosed by symptoms showed the best outcome, but most of them had a mild genotype. In the second study, the Veneto cohort showed better outcome with regard to survival and nutritional status over 16 years of follow-up. CONCLUSIONS: Observational cohort studies cannot give definitive evidence of the clinical benefit of neonatal CF screening; however, data have been accumulated which strongly suggest a better clinical outcome for CF patients born in an area where a screening program is performed.

Adolescent↗

PubMed2000

Validation of the 13C-urea breath test for the diagnosis of Helicobacter pylori infection in children: a multicenter study.

OBJECTIVE: The 13C-urea breath test (13C-UBT) is a safe, noninvasive, and accurate test for the detection of Helicobacter pylori (H. pylori) infection in adults. The aim of this study was to evaluate sensitivity and specificity of 13C-UBT in children using different types of test meal, doses of 13C-urea and breath sampling intervals. As yet, a validated, standardized 13C-UBT protocol for children has not been formulated. METHODS: 13C-UBT was performed in 115 children and repeated within 3 days, modifying the test meal or the dose of 13C-urea. H. pylori status was assessed by histology and rapid urease test. 13C-UBT was performed using 100 mg or 50 mg of 13C-urea and a fatty test meal (100 FA; 50 FA), 50 mg of 13C-urea, and a carbohydrate test meal (50 CA). Breath samples were collected every 10 min for 60 min. RESULTS: The 13C-UBT in children was highly sensitive and specific with all three protocols used. The best combination of sensitivity (97.92%) and specificity (97.96%) was obtained with Protocol 50 FA at 30 min with a cut-off of 3.5 per mil. CONCLUSIONS: The 13C-UBT is an accurate test for the detection of H. pylori infection also in children. Administration of 50 mg of 13C-urea, a fatty test meal, and breath sampling at 30 min appears to be the most convenient protocol.

Adolescent↗

PubMed1999

Presence and comparison of angiotensin converting enzyme in commercial cell culture sera.

This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes were able to hydrolyze, with different efficiency, angiotensin I, bradykinin and epidermal mitosis inhibiting pentapeptide. The heat inactivation of commercial sera at 56 degrees C for 30 min showed a reduction of ACE activity of about 35-80%. Therefore, the presence of ACE activity in commercial sera can influence the activity of biological peptides tested on cell lines cultured "in vitro."

Angiotensin II↗

PubMed1999

Shwachman's syndrome: pathomorphosis and long-term outcome.

BACKGROUND: Shwachman's syndrome is the second most common cause of inherited/congenital pancreatic insufficiency after cystic fibrosis. The main associated features are usually cyclic neutropenia, metaphyseal dysostosis, and growth retardation. Other organs or functions may be involved in this syndrome, showing a wide range of abnormalities and symptoms. There are reports of Shwachman's syndrome in childhood, but little is known about the long-term clinical course of these patients. This article reports on the pathomorphosis and long-term follow-up of 13 patients with Shwachman's syndrome diagnosed in infancy focusing, in particular, on modifications of the exocrine pancreatic function over time. METHODS: Exocrine pancreatic function was evaluated by duodenal intubation followed by a pancreatic stimulation test. Nutritional, biochemical, hematologic, radiologic, and psychological evaluations were performed at various intervals. Six patients were included in long-term follow-up evaluation. RESULTS: At diagnosis, growth retardation was present in all patients, and all subjects showed pancreatic insufficiency. Hematologic features (intermittent neutropenia, anemia and thrombocytopenia), respiratory infection during the first years of life, and skeletal abnormalities were also frequently observed. Other associated features at diagnosis included hepatic involvement and occasional renal dysfunction. In the six patients followed up, a significant growth improvement was observed. In five of them the pancreatic stimulation test showed values of lipase within reference range outputs, whereas fat balance or fecal fat losses were normal in all but one subject. Of seven subjects assessed by psychological evaluation, IQ test results were markedly abnormal in one and bordered on abnormality in the others. CONCLUSIONS: The present data on Shwachman's syndrome diagnosed in infancy underline the possibility of improvement or normalization of exocrine pancreatic function with age, suggesting the need for periodic checks on pancreatic activity in these subjects. It also indicates the possibility of diagnosis of this syndrome in the absence of pancreatic insufficiency; decreasing frequency of infections over time; and the usefulness of early neuropsychological evaluation.

Child, Preschool↗

PubMed1999

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 [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.

Androgens↗

PubMed1997

Presence of angiotensin converting enzyme (ACE) activity in serum of amphibian: comparison with ACE activity of mammalian serum.

The occurrence of angiotensin converting enzyme (EC 3.4.15.1; ACE) was demonstrated for the first time in serum of newt (Triturus carnifex) and frog (Rana esculenta). The enzymatic activity was evidenced following hydrolysis of N-[3-(2-furyl) acryloyl]L-phenylalanyl glycyl glycine (FAPGG), a synthetic substrate of ACE. The serum enzyme liberated N-[3-(2-furyl) acryloyl]L-phenylalanine (FAP) from FAPGG. The properties of the amphibian serum enzymes were compared with those of swine. The amphibian serum FAPGG hydrolysing activities were inhibited by typical ACE inhibitors, captopril and lisinopril. The optimum of pH was 8.3 at 10 and 37 degrees C and the temperature optimum was 45 degrees C. The values were similar to those of swine serum. The FAPGG Michaelis-Menten constants (K(m)) at 37 degrees C of amphibian serum enzymes (0.337 mM and 0.282 mM for frog and newt, respectively) were lower than that of swine (1.305 mM), but close to human serum enzyme. The K(m) values obtained at 10 degrees C were lower than those at 37 degrees C (0.152, 0.086, and 1.029 mM for frog, newt, and swine serum, respectively). Amphibian sera hydrolysed bullfrog synthetic angiotensin I to produce angiotensin II. Captopril (50 microM) inhibited the production of angiotensin II.

Angiotensin I↗

PubMed1997

Genetic history of cystic fibrosis mutations in Italy. I. Regional distribution.

Earlier analysis of the Italian population showed patterns of genetic differentiation that were interpreted as being the result of population settlements going back to pre-Roman times. DNA disease mutations may be a powerful tool in further testing this hypothesis since the analysis of diseased individuals can detect variants too rare to be resolved in normal individuals. We present data on the relative frequencies of 60 cystic fibrosis (CF) mutations in Italy and the geographical distribution of the 12 most frequent CF mutations screened in 3492 CF chromosomes originating in 13 Italian regions. The 12 most frequent mutations characterize about 73% of the Italian CF chromosomes. The most common mutation, delta F508, has an average frequency of 51%, followed by N1303K and G542X, both with average frequencies around 5%. Multivariate analyses show that the relative frequencies of CF mutations are heterogeneous among Italian regions, and that this heterogeneity is weakly correlated with the geographical pattern of non-DNA 'classical' genetic markers. The northern regions are well differentiated from the central-southern regions and within the former group the western and eastern regions are remarkably distinct. Moreover, Sardinia shows the presence of mutation T338I, which seems absent in any other European CF chromosome. The north-western regions of Italy, characterized by the mutation 1717-1G-->A, were under Celtic influence, while the north-east regions, characterized by the mutations R1162X, 2183AA-->G and 711 + 5G-->A, were under the influence of the Venetic culture.

Cystic Fibrosis↗

PubMed1997

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 microM), and synthetic bullfrog ANG I (1 microM). Production of 17 beta-estradiol, progesterone, androgens, and prostaglandins E2 and F2 alpha was determined. The data showed a modulation of 17 beta-estradiol, progesterone, and prostaglandin E2 production by ovary ACE; on the other hand, [Val5]ANG II modulated the production of progesterone and prostaglandin F2 alpha, 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.

Androgens↗

PubMed1997

Presence of pituitary adenylate cyclase-activating polypeptide 38-immuno-like material in the brain and ovary of the female crested newt, Triturus carnifex: its involvement in the ovarian synthesis of prostaglandins and steroids.

The presence of pituitary adenylate cyclase-activating peptide (PACAP) 38-immuno-like material (PACAP 38-IL) in the brain and ovary of the crested newt, Triturus carnifex, and its action on ovarian steroidogenesis and prostaglandin synthesis were evaluated. The HPLC, brain and ovary extract peaks that eluted like PACAP 38 were considered PACAP 38-like material. The concentrations of PACAP 38-II in the HPLC extracts were measured by RIA. T. carnifex ovary was incubated with PACAP 38, brain and ovary PACAP 38-IL, and inhibitors of cyclooxygenase (COX), adenylate cyclase (AC) and phospholipase C (PLC) for 30 and 60 min. PACAI 38, and brain and ovary PACAP 38-IL increased prostaglandin E2 (PGE2) (30 and 60 min), and progesterone and corticosterone (60 min), but decreased oestradiol-17 beta (60 min). COX and PLC inhibitors counteracted the increases in PGE2, progesterone and corticosterone and the decrease in oestradiol-17 beta, and the AC, inhibitor also counteracted them except for PGE2. These results suggest that PACAP 38-IL, present in T. carnifex brain and ovary, acts on PLC, inducing the increase of PGE2 which, in turn, acting on AC, induces increases in progesterone and corticosterone and a decrease in oestradiol-17 beta.

Adenosine↗

PubMed1996

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 enzyme's 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).

Amino Acid Sequence↗

PubMed1996

The coeliac iceberg in Italy. A multicentre antigliadin antibodies screening for coeliac disease in school-age subjects.

BACKGROUND: Recent studies suggest that coeliac disease (CD) is one of the commonest, life-long disorders in Italy. The aims of this multicentre work were: (a) to establish the prevalence of CD on a nationwide basis; and (b) to characterize the CD clinical spectrum in Italy. PATIENTS AND METHODS: Fifteen centres screened 17,201 students aged 6-15 years (68.6% of the eligible population) by the combined determination of serum IgG- and IgA-antigliadin antibody (AGA) test; 1289 (7.5%) were IgG and/or IgA-AGA positive and were recalled for the second-level investigation; 111 of them met the criteria for the intestinal biopsy: IgA-AGA positivity and/or AEA positivity or IgG-AGA positivity plus serum IgA deficiency. RESULTS: Intestinal biopsy was performed on 98 of the 111 subjects. CD was diagnosed in 82 subjects (75 biopsy proven, 7 not biopsied but with associated AGA and AEA positivity). Most of the screening-detected coeliac patients showed low-grade intensity illness often associated with decreased psychophysical well-being. There were two AEA negative cases with associated CD and IgA deficiency. The prevalence of undiagnosed CD was 4.77 x 1000 (95% CI 3.79-5.91), 1 in 210 subjects. The overall prevalence of CD, including known CD cases, was 5.44 x 1000 (95% CI 4.57-6.44), 1 in 184 subjects. The ratio of known to undiagnosed CD cases was 1 in 7. CONCLUSIONS: These findings confirm that, in Italy, CD is one of the most common chronic disorders showing a wide and heterogeneous clinical spectrum. Most CD cases remain undiagnosed unless actively searched.

Adolescent↗

PubMed1994

Molecular models of small phosphorylated chromatin peptides. Structure-function relationship and regulatory activity on in vitro transcription and on cell growth and differentiation.

We previously reported the isolation of low molecular weight phosphorylated peptides from the chromatin of several tissues. The chromatin peptides show a regulatory activity on DNA in vitro transcription and on cell growth and differentiation. In this paper, we report a molecular model of the native peptides designed according to the structural information obtained by means of biochemical and mass spectrometry analysis: pyroGlu-Ala-Gly-Glu-Asp-Ser(P)-Asp-Glu-Glu-Asn. This or very similar sequences are present in many transcription factors; on the basis of the structural model we presented and of related protein sequences, we have synthesized the peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn. This peptide affects transcription rate in reconstituted systems in vitro and in isolated nuclei; moreover, it inhibits the growth of HL60 cells with a parallel stimulus of differentiation.

Amino Acid Sequence↗

PubMed1993

Phosphorylation of synthetic acidic peptides by casein kinase II: evidence for competition with phosphorylation of proteins involved in transcription.

Phosphorylation of several synthetic acidic peptides by biochemically isolated casein kinase II (CKII) and by cellular and nuclear extracts containing CKII-like activity has been investigated. Especially the synthetic peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn comprising the carboxy-terminal acidic hepta-peptide of the largest subunit of RNA polymerase II was found to serve as an excellent substrate for purified CKII. Moreover, this peptide reduces the rate of 'in vitro' ATP-dependent stimulation of DNA transcription induced by the proteins in the extracts. Since the peptide itself is also significantly phosphorylated in such assays, it is supposed that it serves as a competitive substrate for the phosphorylation of proteins in the extracts whose phosphorylation seems to be a prerequisite for their activity in the transcription process. This points to the involvement of CKII and substrate(s) of CKII in the process of transcription.

Acids↗

PubMed1993

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.

Animals↗