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1. [Quality of life, depression and cognitive functions 3. The main characteristics of the QDF study population]

Author

Rete Infermieri GISSI HF, Di Giulio P, Pera C, Scarano M, Ferri B, Lepore V, Miani D, Tognoni G, Rosson V, Serafini C, Zumino MT, Partemi L, Falleroni ML, Moretti L, Porta L, Santorsola A, Tenerelli T, Bertoncelli B, Mengoli G, Stagnitto M, Puddu GM, Masini E, Pasini A, Boni S, Valsesia E, Cornacini G, Melis D, Mulina A, Giurlanda A, Fava M, Bellini P, Vallucci S, Cuomo A, Campanale S, Monatti G, Bianco T, Milli V, Antonelli L, Fanin L, Faiola L, Stefanizzi R, Mariani M, Rossi A, Regnani ST, Loreni F, Cianfrocca R, Pisani A, Faraolfi V, Ponzalli P, Savli T, Cresci M, Guardalben S, Belluzzo B, Ruberti R, Zizzi M, Tonda C, Cadrobbi MG, Gilardi R, Mondì A, Costa A, Milan S, Zottarelli A, Parodi B, Sartorio N, Rossin B, Santi A, Cagnan P, Maradei E, Cauteruccio A, Carpino C, Addeo R, Izzo A, Amendola G, Prizzitano AM, Piluso GM, Iadevaio AL, Cinà MT, Cirrincione V, Gumina S, Codato G, Raimondo F, Cappiello MG, Creta F, Barbieri M, Chiarella L, Abbondio L, Piccin M, Spillone V, Cutrupi G, Ruggeri A, Sicilia G, Provengano A, Marcari P, Ricci S, Pazzagli E, La Grotteria M, Orsini R, Leandri L, Vetere NA, Braggion E, Giordanino S, Armando S, Zampieri E, Danese MC, Valentini A, Ferrante MG, Santacroce C, Crocetti I, Macchi F, Bernacchi M, Pini D, Varì M, Lucente M, Scaramastra F, Reichardt S, Gabasio S, Zocchi C, Studhika M, Budzowska E, Cescon M, Lazzari D, Prizzon A, Di Biaggio P, Buda C, Mastrangelo V, Avigliano M, Dell'Aquila L, Bonanomi E, Mezzani A, Porcini F, Girardi G., PERRONE FILARDI, PASQUALE, Rete Infermieri GISSI, Hf, Di Giulio, P, Pera, C, Scarano, M, Ferri, B, Lepore, V, Miani, D, Tognoni, G, Rosson, V, Serafini, C, Zumino, Mt, Partemi, L, Falleroni, Ml, Moretti, L, Porta, L, Santorsola, A, Tenerelli, T, Bertoncelli, B, Mengoli, G, Stagnitto, M, Puddu, Gm, Masini, E, Pasini, A, Boni, S, Valsesia, E, Cornacini, G, Melis, D, Mulina, A, Giurlanda, A, Fava, M, Bellini, P, Vallucci, S, Cuomo, A, Campanale, S, Monatti, G, Bianco, T, Milli, V, Antonelli, L, Fanin, L, Faiola, L, Stefanizzi, R, Mariani, M, Rossi, A, Regnani, St, Loreni, F, Cianfrocca, R, Pisani, A, Faraolfi, V, Ponzalli, P, Savli, T, Cresci, M, Guardalben, S, Belluzzo, B, Ruberti, R, Zizzi, M, Tonda, C, Cadrobbi, Mg, Gilardi, R, Mondì, A, Costa, A, Milan, S, Zottarelli, A, Parodi, B, Sartorio, N, Rossin, B, Santi, A, Cagnan, P, Maradei, E, Cauteruccio, A, Carpino, C, Addeo, R, Izzo, A, Amendola, G, PERRONE FILARDI, Pasquale, Prizzitano, Am, Piluso, Gm, Iadevaio, Al, Cinà, Mt, Cirrincione, V, Gumina, S, Codato, G, Raimondo, F, Cappiello, Mg, Creta, F, Barbieri, M, Chiarella, L, Abbondio, L, Piccin, M, Spillone, V, Cutrupi, G, Ruggeri, A, Sicilia, G, Provengano, A, Marcari, P, Ricci, S, Pazzagli, E, La Grotteria, M, Orsini, R, Leandri, L, Vetere, Na, Braggion, E, Giordanino, S, Armando, S, Zampieri, E, Danese, Mc, Valentini, A, Ferrante, Mg, Santacroce, C, Crocetti, I, Macchi, F, Bernacchi, M, Pini, D, Varì, M, Lucente, M, Scaramastra, F, Reichardt, S, Gabasio, S, Zocchi, C, Studhika, M, Budzowska, E, Cescon, M, Lazzari, D, Prizzon, A, Di Biaggio, P, Buda, C, Mastrangelo, V, Avigliano, M, Dell'Aquila, L, Bonanomi, E, Mezzani, A, Porcini, F, and Girardi, G.

Subjects
Aged, 80 and over, Male, Depression, Age Factors, Cardiology, Prognosis, Education, Nursing Research, Cognition, Sex Factors, Italy, Surveys and Questionnaires, Quality of Life, Humans, Female, Cardiomyopathies, Aged, Randomized Controlled Trials as Topic
Abstract

This contribution describes the patients included for the QDF (Quality of life, Depression and Cognitive functions) study and explores the distribution and time trends of scores of the three dimensions.The large majority of patients are males (78.3%), and mean age is 67.2 +/- 10.7 years (9.2%80 years). Patients in NYHA class III-IV experience more depression and a worse QoL as compared to the less severly clinically affected counterparts in NYHA class. Although QoL and depression are influenced by the same variables, not all patients with bad QoL are depressed and viceversa. Mean scores of cognitive functioning are not influenced by the same variables that influence QoL and depression.The regular measurement of QoL, depression and cognitive function is recommended by some authors. It seems to be confirmed that the three dimensions need to be explored to disentangle the specific informative value that each of them could add to clinical measures.

Published
2009

2. [Quality of life, depression and cognitive functions 6. Cognitive functions]

Author

Rete Infermieri GISSI HF, Di Giulio P, Pera C, Scarano M, Ferri B, Lepore V, Miani D, Tognoni G, Rosson V, Serafini C, Zumino MT, Partemi L, Falleroni ML, Moretti L, Porta L, Santorsola A, Tenerelli T, Bertoncelli B, Mengoli G, Stagnitto M, Puddu GM, Masini E, Pasini A, Boni S, Valsesia E, Cornacini G, Melis D, Mulina A, Giurlanda A, Fava M, Bellini P, Vallucci S, Cuomo A, Campanale S, Monatti G, Bianco T, Milli V, Antonelli L, Fanin L, Faiola L, Stefanizzi R, Mariani M, Rossi A, Regnani ST, Loreni F, Cianfrocca R, Pisani A, Faraolfi V, Ponzalli P, Savli T, Cresci M, Guardalben S, Belluzzo B, Ruberti R, Zizzi M, Tonda C, Cadrobbi MG, Gilardi R, Mondì A, Costa A, Milan S, Zottarelli A, Parodi B, Sartorio N, Rossin B, Santi A, Cagnan P, Maradei E, Cauteruccio A, Carpino C, Addeo R, Izzo A, Amendola G, Prizzitano AM, Piluso GM, Iadevaio AL, Cinà MT, Cirrincione V, Gumina S, Codato G, Raimondo F, Cappiello MG, Creta F, Barbieri M, Chiarella L, Abbondio L, Piccin M, Spillone V, Cutrupi G, Ruggeri A, Sicilia G, Provengano A, Marcari P, Ricci S, Pazzagli E, La Grotteria M, Orsini R, Leandri L, Vetere NA, Braggion E, Giordanino S, Armando S, Zampieri E, Danese MC, Valentini A, Ferrante MG, Santacroce C, Crocetti I, Macchi F, Bernacchi M, Pini D, Varì M, Lucente M, Scaramastra F, Reichardt S, Gabasio S, Zocchi C, Studhika M, Budzowska E, Cescon M, Lazzari D, Prizzon A, Di Biaggio P, Buda C, Mastrangelo V, Avigliano M, Dell'Aquila L, Bonanomi E, Mezzani A, Porcini F, Girardi G., PERRONE FILARDI, PASQUALE, Rete Infermieri GISSI, Hf, Di Giulio, P, Pera, C, Scarano, M, Ferri, B, Lepore, V, Miani, D, Tognoni, G, Rosson, V, Serafini, C, Zumino, Mt, Partemi, L, Falleroni, Ml, Moretti, L, Porta, L, Santorsola, A, Tenerelli, T, Bertoncelli, B, Mengoli, G, Stagnitto, M, Puddu, Gm, Masini, E, Pasini, A, Boni, S, Valsesia, E, Cornacini, G, Melis, D, Mulina, A, Giurlanda, A, Fava, M, Bellini, P, Vallucci, S, Cuomo, A, Campanale, S, Monatti, G, Bianco, T, Milli, V, Antonelli, L, Fanin, L, Faiola, L, Stefanizzi, R, Mariani, M, Rossi, A, Regnani, St, Loreni, F, Cianfrocca, R, Pisani, A, Faraolfi, V, Ponzalli, P, Savli, T, Cresci, M, Guardalben, S, Belluzzo, B, Ruberti, R, Zizzi, M, Tonda, C, Cadrobbi, Mg, Gilardi, R, Mondì, A, Costa, A, Milan, S, Zottarelli, A, Parodi, B, Sartorio, N, Rossin, B, Santi, A, Cagnan, P, Maradei, E, Cauteruccio, A, Carpino, C, Addeo, R, Izzo, A, Amendola, G, PERRONE FILARDI, Pasquale, Prizzitano, Am, Piluso, Gm, Iadevaio, Al, Cinà, Mt, Cirrincione, V, Gumina, S, Codato, G, Raimondo, F, Cappiello, Mg, Creta, F, Barbieri, M, Chiarella, L, Abbondio, L, Piccin, M, Spillone, V, Cutrupi, G, Ruggeri, A, Sicilia, G, Provengano, A, Marcari, P, Ricci, S, Pazzagli, E, La Grotteria, M, Orsini, R, Leandri, L, Vetere, Na, Braggion, E, Giordanino, S, Armando, S, Zampieri, E, Danese, Mc, Valentini, A, Ferrante, Mg, Santacroce, C, Crocetti, I, Macchi, F, Bernacchi, M, Pini, D, Varì, M, Lucente, M, Scaramastra, F, Reichardt, S, Gabasio, S, Zocchi, C, Studhika, M, Budzowska, E, Cescon, M, Lazzari, D, Prizzon, A, Di Biaggio, P, Buda, C, Mastrangelo, V, Avigliano, M, Dell'Aquila, L, Bonanomi, E, Mezzani, A, Porcini, F, and Girardi, G.

Subjects
Aged, 80 and over, Heart Failure, Male, Time Factors, Depression, Cardiology, Prognosis, Education, Hospitalization, Nursing Research, Sex Factors, Italy, Risk Factors, Prevalence, Quality of Life, Humans, Female, Mortality, Cognition Disorders, Aged, Randomized Controlled Trials as Topic
Abstract

The prevalence of cognitive impairment (CI) is reported to be higher in heart failure patients than in the general population.To describe the prevalence of CI and to assess its prognostic value on the increased risk of hospitalizations and death over the medium term (3 years).MMSE was administered to 620/745 heart failure patients70 years. Up to 35% of the tested population were classified as mild CI and 12% as moderate. The severity of baseline clinical conditions, as expressed as NYHA classes, do not appear to be associated to different degrees of CI. The subgroup of patients with moderate CI includes higher proportion of people with the lowest educational level, of feminine gender, of higher age (80). Even in a population closely monitored as the one included in the GISSI-HLF trial, it is worth to be underlined that the presence of moderate CI appears to be strongly prognostic of an increased burden of care (hospitalizations), and of death over the whole observation period of the study (3 years).The presence of even mild or moderate CI is prognostic for increased mortality and readmissions for patients of the same NYHA class. In the absence of effective intervention strategies, research should concentrate on the yield, if any, of a systematic screening for CI in heart failure patients.

Published
2009

3. [Quality of life, depression and cognitive functions 5. Depression]

Author

Rete Infermieri GISSI HF, Di Giulio P, Pera C, Scarano M, Ferri B, Lepore V, Miani D, Tognoni G, Rosson V, Serafini C, Zumino MT, Partemi L, Falleroni ML, Moretti L, Porta L, Santorsola A, Tenerelli T, Bertoncelli B, Mengoli G, Stagnitto M, Puddu GM, Masini E, Pasini A, Boni S, Valsesia E, Cornacini G, Melis D, Mulina A, Giurlanda A, Fava M, Bellini P, Vallucci S, Cuomo A, Campanale S, Monatti G, Bianco T, Milli V, Antonelli L, Fanin L, Faiola L, Stefanizzi R, Mariani M, Rossi A, Regnani ST, Loreni F, Cianfrocca R, Pisani A, Faraolfi V, Ponzalli P, Savli T, Cresci M, Guardalben S, Belluzzo B, Ruberti R, Zizzi M, Tonda C, Cadrobbi MG, Gilardi R, Mondì A, Costa A, Milan S, Zottarelli A, Parodi B, Sartorio N, Rossin B, Santi A, Cagnan P, Maradei E, Cauteruccio A, Carpino C, Addeo R, Izzo A, Amendola G, Prizzitano AM, Piluso GM, Iadevaio AL, Cinà MT, Cirrincione V, Gumina S, Codato G, Raimondo F, Cappiello MG, Creta F, Barbieri M, Chiarella L, Abbondio L, Piccin M, Spillone V, Cutrupi G, Ruggeri A, Sicilia G, Provengano A, Marcari P, Ricci S, Pazzagli E, La Grotteria M, Orsini R, Leandri L, Vetere NA, Braggion E, Giordanino S, Armando S, Zampieri E, Danese MC, Valentini A, Ferrante MG, Santacroce C, Crocetti I, Macchi F, Bernacchi M, Pini D, Varì M, Lucente M, Scaramastra F, Reichardt S, Gabasio S, Zocchi C, Studhika M, Budzowska E, Cescon M, Lazzari D, Prizzon A, Di Biaggio P, Buda C, Mastrangelo V, Avigliano M, Dell'Aquila L, Bonanomi E, Mezzani A, Porcini F, Girardi G., PERRONE FILARDI, PASQUALE, Rete Infermieri GISSI, Hf, Di Giulio, P, Pera, C, Scarano, M, Ferri, B, Lepore, V, Miani, D, Tognoni, G, Rosson, V, Serafini, C, Zumino, Mt, Partemi, L, Falleroni, Ml, Moretti, L, Porta, L, Santorsola, A, Tenerelli, T, Bertoncelli, B, Mengoli, G, Stagnitto, M, Puddu, Gm, Masini, E, Pasini, A, Boni, S, Valsesia, E, Cornacini, G, Melis, D, Mulina, A, Giurlanda, A, Fava, M, Bellini, P, Vallucci, S, Cuomo, A, Campanale, S, Monatti, G, Bianco, T, Milli, V, Antonelli, L, Fanin, L, Faiola, L, Stefanizzi, R, Mariani, M, Rossi, A, Regnani, St, Loreni, F, Cianfrocca, R, Pisani, A, Faraolfi, V, Ponzalli, P, Savli, T, Cresci, M, Guardalben, S, Belluzzo, B, Ruberti, R, Zizzi, M, Tonda, C, Cadrobbi, Mg, Gilardi, R, Mondì, A, Costa, A, Milan, S, Zottarelli, A, Parodi, B, Sartorio, N, Rossin, B, Santi, A, Cagnan, P, Maradei, E, Cauteruccio, A, Carpino, C, Addeo, R, Izzo, A, Amendola, G, PERRONE FILARDI, Pasquale, Prizzitano, Am, Piluso, Gm, Iadevaio, Al, Cinà, Mt, Cirrincione, V, Gumina, S, Codato, G, Raimondo, F, Cappiello, Mg, Creta, F, Barbieri, M, Chiarella, L, Abbondio, L, Piccin, M, Spillone, V, Cutrupi, G, Ruggeri, A, Sicilia, G, Provengano, A, Marcari, P, Ricci, S, Pazzagli, E, La Grotteria, M, Orsini, R, Leandri, L, Vetere, Na, Braggion, E, Giordanino, S, Armando, S, Zampieri, E, Danese, Mc, Valentini, A, Ferrante, Mg, Santacroce, C, Crocetti, I, Macchi, F, Bernacchi, M, Pini, D, Varì, M, Lucente, M, Scaramastra, F, Reichardt, S, Gabasio, S, Zocchi, C, Studhika, M, Budzowska, E, Cescon, M, Lazzari, D, Prizzon, A, Di Biaggio, P, Buda, C, Mastrangelo, V, Avigliano, M, Dell'Aquila, L, Bonanomi, E, Mezzani, A, Porcini, F, and Girardi, G.

Subjects
Male, Time Factors, Depression, Health Status, Cardiology, Patient Readmission, Hospitalization, Nursing Research, Cognition, Sex Factors, Italy, Risk Factors, Data Interpretation, Statistical, Prevalence, Quality of Life, Humans, Female, Mortality, Geriatric Assessment, Aged, Randomized Controlled Trials as Topic
Abstract

Depression is common in patients with heart failure but its causal and/or prognostic role with respect to the cardiac condition in still a matter of research.To describe the prevalence of depression and to assess its specific prognostic value in terms of increased risk of hospitalizations or death.Overall, 27.5% of 1495 patients experience symptoms of depression. Risk factors for depression are female sex, age70, higher NYHA class, previous hospitalizations and comorbidities. Depressed patients of the same NYHA class are at higher risk for 1 and 3 years mortality and all causes hospital admissions.Depression, assessed with a simple and easy to administer instrument, can add information to clinical variables in the identification of patients at increased risk of mortality and readmissions within the same NYHA class.

Published
2009

4. Molecular epidemiology by ribotyping and PCR-ribotyping of Enterococcus faecium strains isolated from intercontinental areas

Author

Sechi, La, Zanetti, S, Duprè, I, Cappiello, Mg, Delogu, Giovanni, Mortensen, Je, Daneo Moore, L, Fadda, Giovanni, Delogu, Giovanni (ORCID:0000-0003-0182-8267), Sechi, La, Zanetti, S, Duprè, I, Cappiello, Mg, Delogu, Giovanni, Mortensen, Je, Daneo Moore, L, Fadda, Giovanni, and Delogu, Giovanni (ORCID:0000-0003-0182-8267)

Abstract

In this study classical ribotyping based on hybridization of an enteroccocal ribosomal operon previously cloned from Enterococcus hirae (Sechi and Daneo-Moore, 1993) with XbaI cut chromosomal DNA and PCR-ribotyping were used to characterize the molecular epidemiology of 131 Enterococcus faecium, with high-level resistance to gentamicin, isolated from different hospitals in Italy and the United States. The ribotyping was able to differentiate all 131 clinical isolates into 96 family patterns. These family patterns appeared to be useful in establishing epidemiological spread. The results obtained were in agreement with those previously published, suggesting the presence of five to six operons in the Enterococcus genus (Sechi et al., 1994). We performed PCR-ribotyping, based on conserved sequences at the 3' end of the enterococcal 16S rrn and the 5' end of the 23S rrn, on 131 clinical isolates as well as on several enterococcal ATCC strains tested. The results were then compared with those obtained with the classical ribotyping method. The results suggest the presence of at least four classes of intergenic spacers among enterococci, but these classes are not helpful in differentiating between Enterococci or among Enterococcal isolates.

Published
1998

5. Optimization of orally bioavailable alkyl amine renin inhibitors.

Author

Xu Z, Cacatian S, Yuan J, Simpson RD, Jia L, Zhao W, Tice CM, Flaherty PT, Guo J, Ishchenko A, Singh SB, Wu Z, McKeever BM, Scott BB, Bukhtiyarov Y, Berbaum J, Mason J, Panemangalore R, Cappiello MG, Bentley R, Doe CP, Harrison RK, McGeehan GM, Dillard LW, Baldwin JJ, and Claremon DA

Subjects
Administration, Oral, Amines chemical synthesis, Amines pharmacokinetics, Animals, Binding Sites, Blood Pressure drug effects, Carbamates chemical synthesis, Carbamates pharmacokinetics, Crystallography, X-Ray, Drug Design, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacokinetics, Haplorhini, Humans, Piperidines chemical synthesis, Piperidines pharmacokinetics, Rats, Rats, Transgenic, Renin blood, Renin metabolism, Structure-Activity Relationship, Amines chemistry, Carbamates chemistry, Enzyme Inhibitors chemistry, Piperidines chemistry, Renin antagonists & inhibitors
Abstract

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Published
2010
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6. Design and optimization of renin inhibitors: Orally bioavailable alkyl amines.

Author

Tice CM, Xu Z, Yuan J, Simpson RD, Cacatian ST, Flaherty PT, Zhao W, Guo J, Ishchenko A, Singh SB, Wu Z, Scott BB, Bukhtiyarov Y, Berbaum J, Mason J, Panemangalore R, Cappiello MG, Müller D, Harrison RK, McGeehan GM, Dillard LW, Baldwin JJ, and Claremon DA

Subjects
Administration, Oral, Amino Acid Sequence, Animals, Antihypertensive Agents chemical synthesis, Antihypertensive Agents pharmacokinetics, Blood Pressure, Computer Simulation, Crystallography, X-Ray, Drug Design, Humans, Methylamines chemical synthesis, Methylamines pharmacokinetics, Rats, Rats, Transgenic, Renin metabolism, Structure-Activity Relationship, Antihypertensive Agents chemistry, Methylamines chemistry, Renin antagonists & inhibitors
Abstract

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

Published
2009
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7. Purification and characterization of recombinant human renin for X-ray crystallization studies.

Author

Wu Z, Cappiello MG, Scott BB, Bukhtiyarov Y, and McGeehan GM

Subjects
Amino Acid Sequence, Cell Line, Crystallization, Crystallography, X-Ray, Humans, Hydrogen-Ion Concentration, Hydrolysis, Molecular Structure, Recombinant Proteins chemistry, Recombinant Proteins genetics, Renin chemistry, Renin genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Renin isolation & purification, Renin metabolism
Abstract

Background: The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies., Results: A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained., Conclusion: This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.

Published
2008
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8. Purification and characterization of recombinant human cathepsin E expressed in human kidney cell line 293.

Author

Cappiello MG, Wu Z, Scott BB, McGeehan GM, and Harrison RK

Subjects
Amino Acid Sequence, Cathepsin E chemistry, Cathepsin E genetics, Cathepsins chemistry, Cathepsins genetics, Cell Line, Cloning, Molecular, Enzyme Activation, Enzyme Precursors chemistry, Enzyme Precursors genetics, Humans, Hydrogen-Ion Concentration, Molecular Sequence Data, Molecular Structure, Molecular Weight, Recombinant Proteins chemistry, Recombinant Proteins genetics, Cathepsin E isolation & purification, Cathepsin E metabolism, Cathepsins isolation & purification, Cathepsins metabolism, Enzyme Precursors isolation & purification, Enzyme Precursors metabolism, Kidney cytology, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism
Abstract

A cDNA encoding human prepro-cathepsin E was introduced into the adenovirus-transformed HEK-293 (human embryonic kidney) cell line. The construct contained both a V5 peptide epitope and histidine tags at the carboxy terminus. Transfected cells efficiently secreted recombinant pro-cathepsin E into the culture medium. The secreted pro-cathepsin E was purified in a single step using Ni affinity chromatography yielding a protein of about 92 kDa under non-reducing conditions. The amino-terminal sequence of the purified protein began at Ser20, suggesting human cathepsin E accumulated in the culture supernatant as the pro-enzyme. The purified protein was rapidly and completely converted to the active form by treatment at pH 4.0 or below. Steady state kinetic parameters for hydrolysis of the fluorogenic peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 (cleavage at the Phe-Phe bond) were consistent with previously reported values for purified human enzyme (kc/Ki= 53 x 10(6) M(-1) s(-1), Km= 6.3 microM, and kcat= 3 x 10(2) s(-1)). The activated protein was potently inhibited by pepstatin with Ki= 0.2 nM, as well as a reported beta secretase inhibitor. This work demonstrates the potential for producing large quantities of highly purified human cathepsin E from HEK-293 cells in quantities to support both biochemical and structural characterization of the enzyme.

Published
2004
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9. An array of target-specific screening strains for antibacterial discovery.

Author

DeVito JA, Mills JA, Liu VG, Agarwal A, Sizemore CF, Yao Z, Stoughton DM, Cappiello MG, Barbosa MD, Foster LA, and Pompliano DL

Subjects
Alkyl and Aryl Transferases metabolism, Alleles, Escherichia coli metabolism, Inhibitory Concentration 50, Kinetics, Models, Genetic, Plasmids metabolism, Protein Conformation, Recombination, Genetic, Time Factors, Anti-Bacterial Agents pharmacology, Drug Evaluation, Preclinical methods, Oligonucleotide Array Sequence Analysis
Abstract

As the global threat of drug- and antibiotic-resistant bacteria continues to rise, new strategies are required to advance the drug discovery process. This work describes the construction of an array of Escherichia coli strains for use in whole-cell screens to identify new antimicrobial compounds. We used the recombination systems from bacteriophages lambda and P1 to engineer each strain in the array for low-level expression of a single, essential gene product, thus making each strain hypersusceptible to specific inhibitors of that gene target. Screening of nine strains from the array in parallel against a large chemical library permitted identification of new inhibitors of bacterial growth. As an example of the target specificity of the approach, compounds identified in the whole-cell screen for MurA inhibitors were also found to block the biochemical function of the target when tested in vitro.

Published
2002
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10. Physical and genetic map of Streptococcus mutans GS-5 and localization of five rRNA operons.

Author

Cappiello MG, Hantman MJ, Zuccon FM, Peruzzi F, Amjad M, Piggot PJ, and Daneo-Moore L

Subjects
Blotting, Southern, Chromosomes, Bacterial, Electrophoresis, Gel, Pulsed-Field, Genome, Bacterial, Physical Chromosome Mapping, rRNA Operon, Streptococcus mutans genetics
Abstract

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

Published
1999
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11. Molecular epidemiology by ribotyping and PCR-ribotyping of Enterococcus faecium strains isolated from intercontinental areas.

Author

Sechi LA, Zanetti S, Duprè I, Cappiello MG, Delogu G, Mortensen JE, Daneo-Moore L, and Fadda G

Subjects
DNA, Bacterial genetics, Drug Resistance, Microbial, Enterococcus faecium classification, Enterococcus faecium isolation & purification, Humans, Italy epidemiology, Molecular Epidemiology, Operon genetics, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S analysis, RNA, Ribosomal, 16S genetics, RNA, Ribosomal, 23S analysis, RNA, Ribosomal, 23S genetics, United States epidemiology, DNA, Bacterial analysis, Enterococcus faecium genetics
Abstract

In this study classical ribotyping based on hybridization of an enteroccocal ribosomal operon previously cloned from Enterococcus hirae (Sechi and Daneo-Moore, 1993) with XbaI cut chromosomal DNA and PCR-ribotyping were used to characterize the molecular epidemiology of 131 Enterococcus faecium, with high-level resistance to gentamicin, isolated from different hospitals in Italy and the United States. The ribotyping was able to differentiate all 131 clinical isolates into 96 family patterns. These family patterns appeared to be useful in establishing epidemiological spread. The results obtained were in agreement with those previously published, suggesting the presence of five to six operons in the Enterococcus genus (Sechi et al., 1994). We performed PCR-ribotyping, based on conserved sequences at the 3' end of the enterococcal 16S rrn and the 5' end of the 23S rrn, on 131 clinical isolates as well as on several enterococcal ATCC strains tested. The results were then compared with those obtained with the classical ribotyping method. The results suggest the presence of at least four classes of intergenic spacers among enterococci, but these classes are not helpful in differentiating between Enterococci or among Enterococcal isolates.

Published
1998

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