75 results on '"Petronini, P G"'
Search Results
2. The effect of heat shock on amino acid transport and cell volume in 3T3 cells
- Author
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Petronini, P. G., Caccamo, A. E., Alfieri, R. R., Bonelli, M. A., and Borghetti, A. F.
- Published
- 2001
- Full Text
- View/download PDF
3. Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer
- Author
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Bazzola, L, primary, Foroni, C, additional, Andreis, D, additional, Zanoni, V, additional, R Cappelletti, M, additional, Allevi, G, additional, Aguggini, S, additional, Strina, C, additional, Milani, M, additional, Venturini, S, additional, Ferrozzi, F, additional, Giardini, R, additional, Bertoni, R, additional, Turley, H, additional, Gatter, K, additional, Petronini, P G, additional, Fox, S B, additional, Harris, A L, additional, Martinotti, M, additional, Berruti, A, additional, Bottini, A, additional, Reynolds, A R, additional, and Generali, D, additional
- Published
- 2014
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4. SHIGA TOXIN 1, AS DNA REPAIR INHIBITOR, POTENTIATES THE EFFECT OF MAFOSFAMIDE ON RAJI CELLS
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BRIGOTTI, MAURIZIO, ARFILLI, VALENTINA, CARNICELLI, DOMENICA, ROCCHI, LAURA, Bonelli M., Alfieri R. R., Ricci F., Tazzari P. L., Pagliaro P., Paolillo M., Petronini P. G., Sestili P., Brigotti M., Arfilli V., Carnicelli D., Rocchi L., Bonelli M., Alfieri R.R., Ricci F., Tazzari P.L., Pagliaro P., Paolillo M., Petronini P.G., and Sestili P.
- Subjects
SHIGA TOXIN 1 ,DNA REPAIR INHIBITORS ,B CELL LYMPHOMA ,MAFOSFAMIDE - Published
- 2009
5. RUOLO DEL DANNO AL DNA NELLA RISPOSTA CITOTOSSICA INDOTTA DA TOSSINE RIBOSOMIALI IN CELLULE ENDOTELIALI UMANE IN COLTURA
- Author
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Sestili, Piero, Martinelli, C., Carnicelli, D., Alfieri, R., Petronini, P. G., and Brigotti, M.
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Ribotoxins ,DNA damage ,Cytotoxicity - Published
- 2006
6. Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells
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Brigotti, M., Alfieri, R., Sestili, Piero, Bonelli, M., Petronini, P. G., Guidarelli, Andrea, Barbieri, L., Stirpe, F., and Sperti, S.
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ribosome-inactivating proteins ,alkaline-halo assay ,AP sites ,cytotoxins - Published
- 2002
7. Adaptive cellular response to osmotic stress in pig articular chondrocytes
- Author
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Borghetti, P., DELLA SALDA, Leonardo, DE ANGELIS, E., Maltarello, M. C., Petronini, P. G., Cabassi, E., Marcato, P. S., Maraldi, N. M., and Borghetti, A. F.
- Published
- 1995
8. Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer.
- Author
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Bazzola, L, Foroni, C, Andreis, D, Zanoni, V, R Cappelletti, M, Allevi, G, Aguggini, S, Strina, C, Milani, M, Venturini, S, Ferrozzi, F, Giardini, R, Bertoni, R, Turley, H, Gatter, K, Petronini, P G, Fox, S B, Harris, A L, Martinotti, M, and Berruti, A
- Subjects
DRUG tolerance ,LETROZOLE ,CYCLOPHOSPHAMIDE ,BREAST cancer treatment ,ESTROGEN ,CD31 antigen ,PHARMACOKINETICS - Abstract
Purpose:To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC).Methods:Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers.Results:Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by
18 FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P<0.00001) and in 9 out of 13 patients at the definitive surgery compared with baseline (P<0.03). There was also a significant suppression of CD31 and VEGF-A expression in response to treatment (P=0.01 and P=0.007, respectively).Conclusions:The LCS combination is feasible and tolerable. The tumour response and target biomarker modulation indicate that the combination is clinically and biologically active. [ABSTRACT FROM AUTHOR]- Published
- 2015
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9. Osmotically inducible uptake of betaine via amino acid transport system A in SV-3T3 cells
- Author
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Petronini, P G, primary, De Angelis, E, additional, Borghetti, A F, additional, and Wheeler, K P, additional
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- 1994
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10. The Effect of Verapamil and Diltiazem on Alveolar Type II Cells during Warm and Cold Metabolic Ischaemia
- Author
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Spaggiari, L, primary, Rusca, M, additional, Alfieri, R, additional, Petronini, P G, additional, Carbognani, P, additional, Cattelani, L, additional, Borghetti, A F, additional, and Bobbio, P, additional
- Published
- 1994
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11. Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress
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Petronini, P G, primary, De Angelis, E M, additional, Borghetti, A F, additional, and Wheeler, K P, additional
- Published
- 1993
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12. Decreased Expression of Heat Shock Protein 70 mRNA and Protein in WI-38 Human Fibroblasts Aging in Vitro
- Author
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CAMPANINI, C., primary, PETRONINI, P. G., additional, ALFIERI, R., additional, and BORGHETTI, A. F., additional
- Published
- 1992
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13. Modulation by betaine of cellular responses to osmotic stress
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Petronini, P G, primary, De Angelis, E M, additional, Borghetti, P, additional, Borghetti, A F, additional, and Wheeler, K P, additional
- Published
- 1992
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14. Different HSP70 expression and cell survival during adaptive responses of 3T3 and transformed 3T3 cells to osmotic stress.
- Author
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Petronini, PG, Alfieri, R, De Angelis, E, Campanini, C, Borghetti, AF, Wheeler, KP, Petronini, P G, Borghetti, A F, and Wheeler, K P
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- 1993
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15. Primer tRNA^T^r^p of RSV-transformed or RAV-1-infected cells up-regulates the antiribosomal activity of gelonin
- Author
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Brigotti, M., Petronini, P. G., Borghetti, A. F., Guidotti, G. G., Sperti, S., and Montanaro, L.
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- 1998
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16. The effect of betaine on SV3T3 cells exposed to hypertonicity
- Author
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Silvotti, Lucia, Petronini, Pier G, Borghetti, Angelo F, and Wheeler, Kenneth P
- Published
- 1990
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17. Heat shock response in human fibroblasts during ageing
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Petronini, Pier G, Campanini, Cinzia, Marmiroli, Nelson, and Borghetti, Angelo F
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- 1990
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18. Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer
- Author
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Helen Turley, Letizia Bazzola, Sergio Venturini, Ramona Bertoni, Mario Martinotti, Vanessa Zanoni, Andrew R. Reynolds, Roberto Giardini, Francesco Ferrozzi, Daniele Andreis, Adrian L. Harris, Alberto Bottini, Carla Strina, Alfredo Berruti, Piergiorgio Petronini, Daniele Generali, G Allevi, Sergio Aguggini, Kevin C. Gatter, Manuela Milani, Chiara Foroni, Mariarosa Cappelletti, Stephen B. Fox, Bazzola, L., Foroni, C., Andreis, D., Zanoni, V., Cappelletti, M. R., Allevi, G., Aguggini, S., Strina, C., Milani, M., Venturini, S., Ferrozzi, F., Giardini, R., Bertoni, R., Turley, H., Gatter, K., Petronini, P. G., Fox, S. B., Harris, A. L., Martinotti, M., Berruti, A., Bottini, A., Reynolds, A. R., and Generali, Daniele
- Subjects
Oncology ,Cancer Research ,breast cancer ,sorafenib ,letrozole ,Cyclophosphamide ,Colorectal cancer ,ANTINEOPLASTIC AGENTS ,Pharmacology ,urologic and male genital diseases ,ADMINISTRATION, METRONOMIC, AGED, ANTINEOPLASTIC AGENTS, ANTINEOPLASTIC COMBINED CHEMOTHERAPY PROTOCOLS, BREAST NEOPLASMS, CYCLOPHOSPHAMIDE, FEMALE, HUMANS, MIDDLE AGED, NIACINAMIDE, NITRILES, PHENYLUREA COMPOUNDS, RANDOMIZED CONTROLLED TRIALS AS TOPIC, TRIAZOLES, TUMOR MARKERS, BIOLOGICAL ,Antineoplastic Agent ,Prostate cancer ,TUMOR MARKERS ,METRONOMIC ,BIOLOGICAL ,TRIAZOLES ,CYCLOPHOSPHAMIDE ,skin and connective tissue diseases ,Randomized Controlled Trials as Topic ,Tumor ,Letrozole ,Medicine (all) ,endocrine resistance ,neoadjuvant ,primary hormone therapy ,Administration, Metronomic ,Aged ,Antineoplastic Agents ,Antineoplastic Combined Chemotherapy Protocols ,Biomarkers, Tumor ,Breast Neoplasms ,Female ,Humans ,Middle Aged ,Niacinamide ,Nitriles ,Phenylurea Compounds ,Triazoles ,HUMANS ,Sorafenib ,female genital diseases and pregnancy complications ,NIACINAMIDE ,FEMALE ,PHENYLUREA COMPOUNDS ,Settore SECS-S/01 - STATISTICA ,Liver cancer ,Nitrile ,Breast Neoplasm ,AGED ,medicine.drug ,Human ,Phenylurea Compound ,medicine.medical_specialty ,RANDOMIZED CONTROLLED TRIALS AS TOPIC ,ADMINISTRATION ,Breast cancer ,Internal medicine ,medicine ,ANTINEOPLASTIC COMBINED CHEMOTHERAPY PROTOCOLS ,neoplasms ,Antineoplastic Combined Chemotherapy Protocol ,NITRILES ,business.industry ,medicine.disease ,digestive system diseases ,MIDDLE AGED ,BREAST NEOPLASMS ,Clinical Study ,Triazole ,Skin cancer ,business ,Biomarkers - Abstract
Purpose: To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC). Methods: Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers. Results: Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by 18FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P
- Published
- 2015
19. PD-L1 overexpression induces STAT signaling and promotes the secretion of pro-angiogenic cytokines in non-small cell lung cancer (NSCLC).
- Author
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Cavazzoni A, Digiacomo G, Volta F, Alfieri R, Giovannetti E, Gnetti L, Bellini L, Galetti M, Fumarola C, Xu G, Bonelli M, La Monica S, Verzè M, Leonetti A, Eltayeb K, D'Agnelli S, Moron Dalla Tor L, Minari R, Petronini PG, and Tiseo M
- Subjects
- Humans, B7-H1 Antigen, Leukocytes, Mononuclear pathology, Signal Transduction, Tumor Microenvironment, Carcinoma, Non-Small-Cell Lung drug therapy, Lung Neoplasms drug therapy
- Abstract
Background: Monoclonal antibodies (ICI) targeting the immune checkpoint PD-1/PD-L1 alone or in combination with chemotherapy have demonstrated relevant benefits and established new standards of care in first-line treatment for advanced non-oncogene addicted non-small cell lung cancer (NSCLC). However, a relevant percentage of NSCLC patients, even with high PD-L1 expression, did not respond to ICI, highlighting the presence of intracellular resistance mechanisms that could be dependent on high PD-L1 levels. The intracellular signaling induced by PD-L1 in tumor cells and their correlation with angiogenic signaling pathways are not yet fully elucidated., Methods: The intrinsic role of PD-L1 was initially checked in two PD-L1 overexpressing NSCLC cells by transcriptome profile and kinase array. The correlation of PD-L1 with VEGF, PECAM-1, and angiogenesis was evaluated in a cohort of advanced NSCLC patients. The secreted cytokines involved in tumor angiogenesis were assessed by Luminex assay and their effect on Huvec migration by a non-contact co-culture system., Results: PD-L1 overexpressing cells modulated pathways involved in tumor inflammation and JAK-STAT signaling. In NSCLC patients, PD-L1 expression was correlated with high tumor intra-vasculature. When challenged with PBMC, PD-L1 overexpressing cells produced higher levels of pro-angiogenic factors compared to parental cells, as a consequence of STAT signaling activation. This increased production of cytokines involved in tumor angiogenesis largely stimulated Huvec migration. Finally, the addition of the anti-antiangiogenic agent nintedanib significantly reduced the spread of Huvec cells when exposed to high levels of pro-angiogenic factors., Conclusions: In this study, we reported that high PD-L1 modulates STAT signaling in the presence of PBMC and induces pro-angiogenic factor secretion. This could enforce the role of PD-L1 as a crucial regulator of the tumor microenvironment stimulating tumor progression, both as an inhibitor of T-cell activity and as a promoter of tumor angiogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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20. Soluble PD-L1 and Circulating CD8+PD-1+ and NK Cells Enclose a Prognostic and Predictive Immune Effector Score in Immunotherapy Treated NSCLC patients.
- Author
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Mazzaschi G, Minari R, Zecca A, Cavazzoni A, Ferri V, Mori C, Squadrilli A, Bordi P, Buti S, Bersanelli M, Leonetti A, Cosenza A, Ferri L, Rapacchi E, Missale G, Petronini PG, Quaini F, and Tiseo M
- Subjects
- B7-H1 Antigen, CD8-Positive T-Lymphocytes, Humans, Immunotherapy, Killer Cells, Natural, Prognosis, Programmed Cell Death 1 Receptor, Carcinoma, Non-Small-Cell Lung therapy, Lung Neoplasms therapy
- Abstract
Introduction: Upfront criteria to foresee immune checkpoint inhibitors (ICIs) efficacy are far from being identified. Thus, we integrated blood descriptors of pro-inflammatory/immunosuppressive or effective anti-tumor response to non-invasively define predictive immune profiles in ICI-treated advanced non-small cell lung cancer (NSCLC)., Methods: Peripheral blood (PB) was prospectively collected at baseline from 109 consecutive NSCLC patients undergoing ICIs as first or more line treatment. Soluble PD-L1 (sPD-L1) (immunoassay), CD8+PD-1+ and NK (FACS) cells were assessed and interlaced to generate an Immune effector Score (I
eff S). Lung Immune Prognostic Index (LIPI) was computed by LDH levels and derived Neutrophil-to-Lymphocyte Ratio (dNLR). All these parameters were correlated with survival outcome and treatment response., Results: High sPD-L1 and low CD8+PD-1+ and NK number had negative impact on PFS (P < 0.001), OS (P < 0.01) and ICI-response (P < 0.05). Thus, sPD-L1high , CD8+PD-1+low and NKlow were considered as risk factors encompassing Ieff S, whose prognostic power outperformed that of individual features and slightly exceeded that of LIPI. Accordingly, the absence of these risk factors portrayed a favorable Ieff S characterizing patients with significantly (P < 0.001) prolonged PFS (median NR vs 2.3 months) and OS (median NR vs 4.1) and greater benefit from ICIs (P < 0.01). We then combined each risk parameter composing Ieff S and LIPI (LDHhigh , dNLRhigh ), thus defining three distinct prognostic classes. A remarkable impact of Ieff S-LIPI integration was documented on survival outcome (PFS, HR = 4.61; 95%CI = 2.32-9.18; P < 0.001; OS, HR=4.03; 95%CI=1.91-8.67; P < 0.001) and ICI-response (AUC=0.90, 95%CI=0.81-0.97, P < 0.001)., Conclusion: Composite risk models based on blood parameters featuring the tumor-host interaction might provide accurate prognostic scores able to predict ICI benefit in NSCLC patients., (Copyright © 2020. Published by Elsevier B.V.)- Published
- 2020
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21. Epidermal growth factor receptor irreversible inhibitors: chemical exploration of the cysteine-trap portion.
- Author
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Carmi C, Lodola A, Rivara S, Vacondio F, Cavazzoni A, Alfieri RR, Ardizzoni A, Petronini PG, and Mor M
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- Humans, Protein Kinase Inhibitors chemistry, Receptor Protein-Tyrosine Kinases antagonists & inhibitors, Receptor Protein-Tyrosine Kinases metabolism, Structure-Activity Relationship, Cysteine chemistry, ErbB Receptors antagonists & inhibitors, Protein Kinase Inhibitors pharmacology
- Abstract
Covalent EGFR irreversible inhibitors showed promising potential for the treatment of gefitinib-resistant tumors and for imaging purposes. They contain a cysteine-reactive portion forming a covalent bond with the protein. Irreversible kinase inhibitors have been advanced to clinical studies, mostly characterized by an acrylamide or butynamide warhead. However, the clinical usefulness of these compounds has been hampered by resistances, toxicity and pharmacokinetic problems. Investigation on the structure-activity and structure-reactivity relationships may provide useful information for compounds with improved selectivity and pharmacokinetic properties. This review focuses on the exploration of the cysteine-trap portions able to irreversibly inhibit EGFR and other erbB receptors.
- Published
- 2011
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22. Effect of inducible FHIT and p53 expression in the Calu-1 lung cancer cell line.
- Author
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Cavazzoni A, Galetti M, Fumarola C, Alfieri RR, Roz L, Andriani F, Carbognani P, Rusca M, Sozzi G, and Petronini PG
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- Acid Anhydride Hydrolases genetics, Acid Anhydride Hydrolases physiology, Apoptosis, Blotting, Northern, Blotting, Western, Cell Line, Tumor, Cell Proliferation, Cyclin-Dependent Kinase Inhibitor p21 genetics, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Gene Expression Regulation, Neoplastic, Genetic Vectors genetics, Humans, Neoplasm Proteins genetics, Neoplasm Proteins physiology, Proto-Oncogene Proteins c-mdm2 genetics, Proto-Oncogene Proteins c-mdm2 metabolism, Time Factors, Transfection, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 physiology, Acid Anhydride Hydrolases metabolism, Neoplasm Proteins metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
Loss of FHIT expression and p53 mutations are critical events in the early stages of lung carcinogenesis. The restoration of Fhit function in FHIT-negative cancer cells has been reported to cause tumour suppression by inhibition of cell proliferation and/or activation of apoptotic pathways. However, the studies designed to elucidate the biological role of Fhit and its potential interaction with p53 have produced conflicting results. We investigated here the effects of the simultaneous restoration of FHIT and p53 in Calu-1 cells by using a hormone-inducible gene expression system. We demonstrate that the restoration of FHIT expression reinforces the anti-proliferative effect associated with the simultaneous replacement of p53. Indeed, a more pronounced inhibition of cell proliferation associated with an earlier and higher induction of p21(waf1) mRNA and protein expression was observed in Fhit/p53-expressing cells compared with cells expressing p53 alone. This effect was not due to Fhit-mediated up-regulation of p53 expression; in fact p53 protein was expressed at the same level in both FHIT-positive and FHIT-negative cell clones. Consistent with this result, Fhit did not affect the expression of MDM2, a protein known to interact directly with p53 and target p53 for proteolytic degradation, thus down-regulating its activity.
- Published
- 2007
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23. Evaluation of endogenous pig retrovirus expression and of tumorigenicity in nude mice.
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Ferrari M, Corradi A, Petronini PG, Tosini A, Toniolo A, Robotti C, and Moratti R
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- Animals, Antiviral Agents pharmacology, Mice, Mice, Nude, Nelfinavir pharmacology, Polymerase Chain Reaction, Retroviridae drug effects, Retroviridae genetics, Retroviridae Infections transmission, Reverse Transcriptase Polymerase Chain Reaction, Swine, Swine Diseases virology, Tumor Virus Infections, Retroviridae physiology, Retroviridae Infections veterinary
- Published
- 2004
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24. Heat-induced proteasomic degradation of HSF1 in serum-starved human fibroblasts aging in vitro.
- Author
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Bonelli MA, Alfieri RR, Poli M, Petronini PG, and Borghetti AF
- Subjects
- Cells, Cultured, Culture Media, Serum-Free, Cysteine Proteinase Inhibitors pharmacology, Female, HSP70 Heat-Shock Proteins metabolism, Heat Shock Transcription Factors, Hot Temperature, Humans, Leupeptins pharmacology, Proteasome Endopeptidase Complex, Protein Binding, Transcription Factors, Cellular Senescence, Cysteine Endopeptidases metabolism, DNA-Binding Proteins metabolism, Fibroblasts physiology, HSP70 Heat-Shock Proteins genetics, Multienzyme Complexes metabolism
- Abstract
The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation., (Copyright 2001 Academic Press.)
- Published
- 2001
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25. Osmotic regulation of ATA2 mRNA expression and amino acid transport System A activity.
- Author
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Alfieri RR, Petronini PG, Bonelli MA, Caccamo AE, Cavazzoni A, Borghetti AF, and Wheeler KP
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- Animals, Biological Transport physiology, Carrier Proteins genetics, Cells, Cultured, Cycloheximide pharmacology, Dactinomycin pharmacology, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Hypertonic Solutions pharmacology, Membrane Proteins genetics, Nucleic Acid Synthesis Inhibitors pharmacology, Protein Synthesis Inhibitors pharmacology, Swine, Transfection, Water-Electrolyte Balance drug effects, beta-Alanine pharmacokinetics, Amino Acid Transport System A, Amino Acids metabolism, Carrier Proteins metabolism, Endothelium, Vascular metabolism, Membrane Proteins metabolism, RNA, Messenger metabolism, Water-Electrolyte Balance physiology, beta-Alanine analogs & derivatives
- Abstract
When porcine endothelial cells were exposed to hypertonicity, both the level of ATA2 (amino acid transporter 2) mRNA and activity of amino acid transport System A increased transiently, peaking after about 6 and 9 h, respectively. Cycloheximide, like actinomycin D, prevented both responses, showing that an earlier step also involves protein synthesis. Withdrawal of hypertonicity after 6 h increased the rate of down regulation. These findings confirm that ATA2 is a major isoform of System A and show that changes in the expression of ATA2 mRNA precede both the induction and subsequent down regulation of transport activity., (Copyright 2001 Academic Press.)
- Published
- 2001
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26. Induction of BGT-1 and amino acid system A transport activities in endothelial cells exposed to hyperosmolarity.
- Author
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Petronini PG, Alfieri RR, Losio MN, Caccamo AE, Cavazzoni A, Bonelli MA, Borghetti AF, and Wheeler KP
- Subjects
- Amino Acid Transport Systems, Animals, Betaine metabolism, Carrier Proteins genetics, Cations, Cell Size, Cells, Cultured, Dogs, GABA Plasma Membrane Transport Proteins, Kinetics, Ninhydrin analysis, Osmolar Concentration, Potassium metabolism, Pulmonary Artery, RNA, Messenger analysis, Sodium metabolism, Sodium Chloride administration & dosage, Sucrose administration & dosage, Swine, gamma-Aminobutyric Acid metabolism, Carrier Proteins biosynthesis, Carrier Proteins metabolism, Cell Line, Endothelium, Vascular metabolism, Hypertonic Solutions
- Abstract
We studied the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. In 0.5 osmol/kgH(2)O medium, initial cell shrinkage was followed by a regulatory volume increase (RVI), complete after 1 h, concomitant with an increase in cellular K(+) content. Then the activity of amino acid transport System A increased, accompanied by an accumulation of ninhydrin-positive solutes (NPS), reaching a peak at approximately 6 h. The subsequent decline in System A activity was paralleled by an induction of the betaine-GABA transporter (BGT-1), detected as increases of BGT-1 mRNA and of transport activity, which peaked at approximately 24 h. Inhibitors of transcription or translation prevented induction of both transport activities. The increased expression of BGT-1, which involved activation of "tonicity-responsive enhancer," was inhibited by 5 mM extracellular betaine. Cellular K(+) concentration gradually declined after the accumulation of NPS and during the induction of BGT-1. This very effective adaptation to hypertonicity suggests it has a physiological role.
- Published
- 2000
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27. Attenuated expression of 70-kDa heat shock protein in WI-38 human fibroblasts during aging in vitro.
- Author
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Bonelli MA, Alfieri RR, Petronini PG, Brigotti M, Campanini C, and Borghetti AF
- Subjects
- Base Sequence, Biomarkers, Cell Line, Cell Nucleus metabolism, Cellular Senescence physiology, Culture Media, Serum-Free, Cytoplasm metabolism, DNA Primers genetics, Fibroblasts cytology, Fibroblasts metabolism, Gene Expression, Heat-Shock Response, Humans, In Vitro Techniques, RNA, Messenger genetics, RNA, Messenger metabolism, beta-Galactosidase metabolism, Cellular Senescence genetics, HSP70 Heat-Shock Proteins genetics
- Abstract
We examined the effects of cellular aging on the expression of the heat shock-inducible HSP70 gene in WI-38 diploid human fibroblasts serially passaged in vitro. The senescence of the cells was established by evaluating population doubling level, cell density at confluency, and cell morphology along with the detection of senescence-associated beta-galactosidase activity (histochemically detectable at pH 6), a reliable marker of aging in low-density cultures. A marked decrease in the synthesis and accumulation of the inducible HSP70 protein was observed in serum-fed late passage cells exposed to a severe heat shock (30 min at 45 degrees C) in comparison to early passage cells. However, the degree of HSF-DNA binding, monitored by gel retardation assay was similar in both early and late passage cells. Similarly, Northern blotting analysis indicated that comparable amounts of inducible HSP70 mRNA were present in the total RNA fraction, in the total polyadenylated RNA fraction, or in the nuclear polyadenylated RNA fraction extracted from both early and late passage cells. In contrast, much less inducible HSP70 mRNA was detected in the total cytoplasmic RNA fraction or in the polyadenylated cytoplasmic RNA fraction of late passage cells. Thus age-related differences in heat-induced HSP70 synthesis and accumulation observed in serum-fed WI-38 cells appeared to result from an impairment in the posttranscriptional processing of the HSP70 mRNA at a level following the polyadenylation step and preceding translocation from the nucleus to the cytoplasm. When HF were serum deprived for 20 h before heat shock, the induction of HSP70 mRNA was less than 30% reduced in early passage cells in comparison to serum-fed cells; however, the level of HSP70 mRNA was markedly (over 80%) decreased in serum-deprived late passage cells. This result indicated that the presence of serum has a strong influence on heat shock-induced HSP70 gene expression in human fibroblasts aging in vitro., (Copyright 1999 Academic Press.)
- Published
- 1999
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28. Induction of betaine-gamma-aminobutyric acid transport activity in porcine chondrocytes exposed to hypertonicity.
- Author
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de Angelis E, Petronini PG, Borghetti P, Borghetti AF, and Wheeler KP
- Subjects
- Amino Acids metabolism, Animals, Biological Transport, Blotting, Northern, Cell Survival, Cells, Cultured, Chondrocytes drug effects, Cycloheximide pharmacology, Dactinomycin pharmacology, Hypertonic Solutions, Phenotype, Protein Synthesis Inhibitors pharmacology, RNA, Messenger biosynthesis, Swine, Betaine metabolism, Chondrocytes metabolism, Gastrointestinal Agents metabolism, gamma-Aminobutyric Acid metabolism
- Abstract
1. We measured the rates of uptake of selected amino acids and betaine by primary cultures of chondrocytes from porcine articular cartilage after the cells had been incubated in 'isotonic' (0.3 osmol l-1) or hypertonic (0.5 osmol l-1) media. 2. Na+-dependent uptake of methylaminoisobutyric acid increased rapidly when the cells were exposed to hypertonic conditions, reached a peak after 6-9 h, and then gradually decreased so that after 24 h it was only slightly above the control value. Conversely, (Na+ + Cl-)-dependent influx of gamma-aminobutyric acid (GABA) remained low for the first 9 h of hypertonic incubation, but then increased markedly to reach a plateau value after 24-30 h. Betaine influx also increased in cells incubated in hypertonic medium, being mainly Na+ dependent after 6 h, but (Na+ + Cl-)-dependent after 24 h. 3. This pattern indicates that exposure of the chondrocytes to hypertonicity induces first amino acid transport system A and then, as this decreases again, betaine-GABA transport activity. 4. Induction of betaine-GABA transport activity did not require continuous exposure of chondrocytes to hypertonicity; but the magnitude of the increase measured at the end of a 24 h incubation period was proportional to the length of time the cells had been exposed to hypertonicity during the 24 h. 5. Isolation and culture of chondrocytes in 0.4 osmol l-1 medium, instead of 0.3 osmol l-1, significantly increased their betaine-GABA transport activity, but not their system A activity. 6. Induction of betaine-GABA transport activity was prevented by addition of either actinomycin D or cycloheximide to the medium, but no mRNA for the betaine-GABA transporter known as BGT-1 was detected by Northern blot analysis of extracts of chondrocytes.
- Published
- 1999
- Full Text
- View/download PDF
29. The effect of free-radical-scavenger system "N-Acetylcysteine/Glutathione" for hypothermic prolonged lung cells preservation.
- Author
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Spaggiari L, Alfieri R, Rusca M, Carbognani P, Cattelani L, Bobbio A, Petronini PG, Borghetti FA, and Bobbio P
- Subjects
- Cells, Cultured, Cold Temperature, Humans, Lung Transplantation physiology, Organ Preservation Solutions pharmacology, Acetylcysteine pharmacology, Free Radical Scavengers metabolism, Glutathione pharmacology, Lung cytology, Lung metabolism, Organ Preservation methods
- Published
- 1998
30. Expression of human CD44v6 in non-small-cell lung cancer.
- Author
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Carbognani P, Spaggiari L, Romani A, Solli P, Corradi A, Cantoni AM, Petronini PG, Borghetti AF, Rusca M, and Bobbio P
- Subjects
- Adenocarcinoma genetics, Adenocarcinoma immunology, Adenocarcinoma pathology, Aged, Alternative Splicing, Carcinoma, Large Cell genetics, Carcinoma, Large Cell immunology, Carcinoma, Large Cell pathology, Carcinoma, Non-Small-Cell Lung pathology, Carcinoma, Squamous Cell genetics, Carcinoma, Squamous Cell immunology, Carcinoma, Squamous Cell pathology, Female, Gene Expression, Humans, Lung Neoplasms pathology, Male, Middle Aged, Prognosis, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung immunology, Hyaluronan Receptors genetics, Hyaluronan Receptors metabolism, Lung Neoplasms genetics, Lung Neoplasms immunology
- Abstract
Introduction: The CD44 is a membrane glycoprotein that functions as lymph node homing receptor in lymphocyte activation and is involved in homo- and heterotypic cell adhesion. In several tumor cell lines the expression of splice variants (CD44v6 and CD44v7) are correlated with the metastatic potential and confer an advantage in the early steps of the metastatic cascade. In our study we examined 35 cases of non-small-cell lung cancers (NSCLC) in order to detect the presence of CD44v6 and to compare its expression with the histologic type, degree of differentiation, stage of the tumor and survival of the patients., Methods: CD44v6 expression in frozen tissue sections of 35 patients with NSCLC who underwent pneumonectomy or lobectomy was analyzed with the VFF-7 monoclonal antibody that detected the CD44v6 variant. The data on survival were analyzed by the actuarial method and compared by the log rank test., Results: The expression of CD44v6 occurred in all the 20 cases of epidermoid carcinomas tested and in 2 out of the 3 cases of undifferentiated large cell carcinoma and was absent in all the 12 adenocarcinomas. No relationship was found between the presence of this marker and the grading or the stage of the pathology. The 3-year survival rate was 73% for CD44v6-positive and 65% for CD44v6-negative cancer and the comparison was not statistically significant., Conclusion: These results suggest that in lung cancer the expression of CD44v6 is not a useful prognostic factor.
- Published
- 1998
- Full Text
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31. An original application of plasma expanders: heart-lung preservation.
- Author
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Spaggiari L, Rusca M, Carbognani P, Alfieri R, Urbani S, Petronini PG, Cattelani L, Borghetti AF, and Bobbio P
- Subjects
- Adenosine, Allopurinol, Endothelium, Vascular cytology, Glutathione, Humans, In Vitro Techniques, Insulin, Organ Preservation, Pulmonary Artery cytology, Raffinose, Heart, Lung, Organ Preservation Solutions, Plasma Substitutes, Polygeline
- Abstract
The lack of an ideal heart-lung preservation solution is one of the principal factor that limits the wide spread of transplantation. The aim of this work was to investigate the efficacy of Haemaccel (HM) on isolated human pulmonary artery endothelial cells comparing its effects with those of University of Wisconsin (UWS). Subcultures of HPAEC were inoculated at the density of 5,000 cells per cm2 in 9 cm2 well-plates. Cells were incubated with HM and UWS for 6 hrs at 10 degrees C. Cellular viability was analysed by the total protein content (cytotoxicity index) and by the rate of protein synthesis (metabolic index). The results showed that HM and UWS did no show a significant differences in the toxicity when compared with the control; on the contrary, HM seems to determine a less inhibitory effect on cellular metabolism permitting a more rapid cellular metabolic recovery than UWS. Thus, HM appears to be more suitable for the preservation of isolated HPAEC than UWS.
- Published
- 1996
32. Activation of heat-shock transcription factor 1 by hypertonic shock in 3T3 cells.
- Author
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Alfieri R, Petronini PG, Urbani S, and Borghetti AF
- Subjects
- 3T3 Cells, Animals, Heat Shock Transcription Factors, Mice, Osmotic Pressure, Potassium metabolism, Sodium metabolism, Transcription Factors, DNA-Binding Proteins metabolism
- Abstract
The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 degrees C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.
- Published
- 1996
- Full Text
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33. Cell susceptibility to apoptosis by glutamine deprivation and rescue: survival and apoptotic death in cultured lymphoma-leukemia cell lines.
- Author
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Petronini PG, Urbani S, Alfieri R, Borghetti AF, and Guidotti GG
- Subjects
- Cell Survival drug effects, Culture Media metabolism, Energy Metabolism, Glucose deficiency, Humans, Leukemia metabolism, Leukemia pathology, Lymphoma metabolism, Lymphoma pathology, Signal Transduction, Tumor Cells, Cultured, Apoptosis drug effects, Apoptosis physiology, Glutamine deficiency, Glutamine pharmacology
- Abstract
Human leukemia/lymphoma cells maintained in culture medium without provision of fresh nutrients lose viability and die by a process resembling apoptosis within a few days. Upon incubation in an FCS-supplemented RPMI 1640 medium containing 2 mM L-glutamine CEM, Namalwa, HL-60 and U937 cells, seeded at initial densities of 0.2 to 1 x 10(6) cells/ml, ceased growing within 3-5 days and progressively entered an apoptotic pathway, as assessed by nucleosomal DNA fragmentation and morphology. Both the major energy-source nutrients in the medium, glucose and glutamine, became rapidly exhausted during the incubation. Further studies were performed using CEM cells. Incubation in glutamine-free or glucose-free medium renewed every 24 h showed that glutamine deprivation is associated with cell death by apoptosis independent of energetic failure, whereas glucose deprivation is followed by rapid loss of mitochondrial function with sharp drop of intracellular ATP and cell death by necrosis. A 12-24 h incubation in glutamine-depleted medium was required to direct the cells toward the apoptotic pathway. Growth arrest followed by apoptotic death was detected in CEM cells when medium glutamine concentration remained below 0.3-0.4 mM for at least 24 h, but a reinstatement of medium glutamine to 2 mM within this period rescued the cells from growth arrest and death.
- Published
- 1996
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34. Adaptive cellular response to osmotic stress in pig articular chondrocytes.
- Author
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Borghetti P, Della Salda L, De Angelis E, Maltarello MC, Petronini PG, Cabassi E, Marcato PS, Maraldi NM, and Borghetti AF
- Subjects
- Amino Acids metabolism, Animals, Biological Transport physiology, Cartilage, Articular ultrastructure, Cell Division physiology, Cells, Cultured, Histocytochemistry, Microscopy, Electron, Microscopy, Electron, Scanning, Osmolar Concentration, Saline Solution, Hypertonic, Stress, Mechanical, Swine, Adaptation, Physiological, Cartilage, Articular cytology, Protein Biosynthesis
- Abstract
The authors studied the effects of a wide range of medium osmolarities (from 0.28 osM (physiological osmolarity of plasma and synovial fluid) to 0.58 osM) by altering Na+ concentration in high density cultures of pig articular chondrocytes in order to analyze the behaviour of some functional and structural parameters during cell adaptation to these imposed changes in the ionic environment. Biochemical and morphological results indicated that, even if isolated from the tissue matrix and cultured in vitro, chondrocytes maintained active osmoregulation systems which are present in living conditions. They showed a similar biochemical and morphological behavior when cultured at 0.28 osM and 0.38 osM but they were able, with regard to protein synthesis, aminoacid transport and proliferation rates, to respond quickly and to adapt to 0.48 osM medium as well. On the contrary, the treatment at the highest osmolarity (0.58 osM) early altered these biochemical parameters and was detrimental or even gave rise to lethal damage during long-term treatment. Furthermore, while chondrocytes cultured in 0.28-0.38 osM medium maintained phenotypic characteristics in culture, the higher osmolarities (0.48-0.58 osM) caused morphological changes in cell populations resulting in loss of phenotypic cell stability as demonstrated by their taking on a fibroblast-like shape as well as a lack of ability to assembly matrix proteoglycans.
- Published
- 1995
- Full Text
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35. Effect of an alkaline shift on induction of the heat shock response in human fibroblasts.
- Author
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Petronini PG, Alfieri R, Campanini C, and Borghetti AF
- Subjects
- Base Sequence, DNA-Binding Proteins metabolism, Female, Fibroblasts, Gene Expression Regulation, Heat Shock Transcription Factors, Hot Temperature, Humans, Hydrogen-Ion Concentration, In Vitro Techniques, Molecular Sequence Data, Oligodeoxyribonucleotides chemistry, Time Factors, Transcription Factors, Transcriptional Activation, HSP70 Heat-Shock Proteins genetics
- Abstract
The simultaneous exposure of WI-38 human fibroblasts (HF) to a heat shock (45 degrees C, 30 min) and an alkaline shift (> or = pH 8.0) in the incubation medium increased and extended the expression of heat shock proteins (hsps). Hsp70 was the most prominent inducible hsp synthesized during the recovery phase after the double shock, and the increase in synthesis depended on the degree of alkalinization during the heat shock. The accumulation of inducible hsp70, which was shown by Western blotting to occur in the late part of the recovery period, was more pronounced in the cells exposed to alkaline medium during the heat shock. Northern blotting did not reveal any increase in hsp70 mRNA, although time course studies following the double shock indicated a more prolonged presence of mRNA. Hsp70 gene activation was evaluated by a gel retardation assay using a 32P-labelled DNA oligonucleotide containing the heat shock consensus element (HSE) and a heat shock-induced specific binding protein (heat shock transcription factor, HSTF) from the cell extract. Heat shock activated HSTF-DNA binding and induced hsp70 mRNA expression as well as the synthesis and accumulation of hsp70. Alkaline shift, which by itself did not induce hsps expression, activated HSTF DNA-binding. However, in combination with heat shock, alkaline shift enhanced and prolonged HSTF-HSE complex association and hsp expression at both mRNA and protein levels. Since the alkaline shift-induced activation of hsp gene does not allow full transcription, these results provide further support for the multistep nature of the heat shock transcriptional response.
- Published
- 1995
- Full Text
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36. Some questions about Eurocollins solution used for lung preservation.
- Author
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Spaggiari L, Carbognani P, Rusca M, Alfieri R, Petronini PG, Cattelani L, Salcuni P, Borghetti AF, and Bobbio P
- Subjects
- Animals, Cell Survival, Cells, Cultured, Epithelial Cells, Epithelium metabolism, Glucose metabolism, Isotonic Solutions, Lung Transplantation, Methionine metabolism, Osmolar Concentration, Protein Biosynthesis, Pulmonary Alveoli cytology, Pulmonary Alveoli metabolism, Rats, Rats, Wistar, Ringer's Lactate, Sulfur Radioisotopes, Time Factors, Hypertonic Solutions, Lung, Organ Preservation
- Abstract
Currently, Eurocollins' (EC) solution (high-potassium concentration) is the most widely clinically used pulmonary perfusate. However, recently, experimental studies have reported an increase of the lung ischemic period using low-potassium solutions. The purpose of our study, is to investigate the influence of the EC ionic composition and the effect of hyperosmolarity due to the glucose concentration on isolated alveolar type II epithelial cells. Pneumocytes type II were isolated from pathogen free Wistar rats using the modified Dobbs' method. Cells were incubated for 6 hours at 4 degrees C in EC, Collins (CL) and Ringer Lactate (RL) solutions. After that, cellular viability was evaluated by analysis of the protein synthesis assay by measuring the 35 S methionine uptake during an incorporation period of one hour at 37 degrees C (picomol 35 S met/mg proteins/h). Mean +/- standard deviation and Student "t"-test were used for data presentation and results comparison. Cellular viability at time 0 (control) before cellular incubation was 3.93 +/- 0.38. After 6 hours at 4 degrees C the results were respectively as follows: EC = 2.16 +/- 0.13; CL = 2.63 +/- 0; RL = 3.21 +/- 0.04. Our results suggest that the low-potassium extracellular type solution (RL) shows a protection on isolated type II epithelial cells statistically significant (p < 0.05) if compared with EC solution. Moreover CL solution, that has the same ionic composition EC but without glucose, presents a less cytotoxic effects on incubated cells than EC, confirming a deleterious influence of solution hyperosmolarity.
- Published
- 1994
37. The influence of high and low doses of diltiazem on isolated alveolar type II cells during normothermic and hypothermic ischemia: cytoprotection or cytotoxicity?
- Author
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Spaggiari L, Carbognani P, Rusca M, Alfieri R, Petronini PG, Urbani S, Cattelani L, Dal Corso HM, Bobbio A, and Dell'Abate P
- Subjects
- Animals, Calcium Channel Blockers toxicity, Cell Separation, Cell Survival drug effects, Cells, Cultured, Diltiazem toxicity, Pulmonary Alveoli blood supply, Rats, Rats, Wistar, Temperature, Time Factors, Calcium Channel Blockers administration & dosage, Diltiazem administration & dosage, Hypothermia pathology, Ischemia pathology, Pulmonary Alveoli cytology, Pulmonary Alveoli drug effects
- Abstract
The alteration of calcium homeostasis is of outstanding importance for the cytotoxic reactions that place after ischemia, for this reason calcium channel blockers have been used with the purpose to protect the lung during transplantation. This work analyses the effect of Diltiazem at two different doses (10 mg/l and 50 mg/l) on Wistar rat alveolar type II cells, incubated for 8 hours at 37 degrees C and at 4 degrees in an electrolytic solution. Total protein content and the rate of protein synthesis derived from 35S Methionine uptake were used to evaluate cells viability. The data showed that Diltiazem did not improve cellular viability after warm and cold metabolic ischemia either using 10 mg/l or 50 mg/l, while at 4 degrees C a significantly cytotoxic effect (p < 0.05) was observed. At this temperature toxicity was independent on the dose used.
- Published
- 1994
38. [Analysis of type II pneumocyte protection by pneumoplegic solutions. An experimental in vitro study of lung preservation for transplantation].
- Author
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Spaggiari L, Alfieri R, Rusca M, Carbognani P, Petronini PG, Cattelani L, Dell'Abate P, Bobbio A, Solli PG, and Endrizzi C
- Subjects
- Albumins, Animals, Cell Survival, Cells, Cultured, Gluconates, Hypertonic Solutions, Isotonic Solutions, Male, Rats, Rats, Wistar, Ringer's Lactate, Lung cytology, Lung Transplantation, Organ Preservation methods, Solutions
- Abstract
With the aim of studying the physiopathological mechanism of lung preservation, we have valued the Pneumocytes Type II viability after six hours of incubation at 4 degrees C in extracellular (Ringer Lactate) and intracellular (Collins, Euro-Collins and Belzer) solutions. The cells have been cut off from adult rat's lung using the modified Dobbs' method. Alveolar Type II viability have been valued using two methods: the total protein content in each culture and the metabolic function of the cells using the rate of protein synthesis by means of 35 S methionine uptake assay. The results have demonstrated a significant difference (p < 0.05) using extracellular solution instead of intracellular. Besides, we have observed a better Pneumocytes Type II survival during Belzer's solution incubation comparing with Collins and Euro-Collins (p < 0.05). Pneumocytes type II require for a better preservation a specific solution that must be similar to extracellular fluid and added with substances able to minimize noxious events during preservation.
- Published
- 1993
39. Modulation of cell growth and host protein synthesis during HIV infection in vitro.
- Author
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Di Rienzo AM, Petronini PG, Guetard D, Favilla R, Borghetti AF, Montagnier L, and Piedimonte G
- Subjects
- CD4-Positive T-Lymphocytes microbiology, Cell Division, Cell Line, Humans, Proteins metabolism, Virus Replication, CD4-Positive T-Lymphocytes cytology, HIV-1 physiology, Protein Biosynthesis
- Abstract
During HIV infection of CEM cells cultured in vitro, significant differences in growth rate and protein turnover were observed with different viral preparations. There was a significant inhibition of proliferation after infection with crude HIV supernatants. On the other hand, infection with purified HIV particles obtained by filtration, differential centrifugation, and isopycnic sedimentation led to a progressively increasing stimulation of cell growth. This early stimulation was prevented by neutralizing the virus with soluble CD4 molecules. Study of cell growth in the presence of a purified membrane preparation indicated that membrane fragments contaminating the crude HIV supernatant were responsible for the observed growth inhibition. Interestingly, the stimulation of proliferation was also observed with heat-inactivated virus or after inhibition of viral replication with ZDV. In the presence of purified HIV virions, the rate of general protein synthesis was not inhibited, as is usually observed with crude viral supernatants. However, a marked reduction in protein content and increased protein degradation was found in cultures infected with either crude or purified HIV preparations.
- Published
- 1992
40. Differential adaptive response to hyperosmolarity of 3T3 and transformed SV3T3 cells.
- Author
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Silvotti L, Petronini PG, Mazzini A, Piedimonte G, and Borghetti AF
- Subjects
- Amino Acids metabolism, Animals, Biological Transport, Cell Division, Cell Line, Cytoplasm metabolism, Electrophoresis, Gel, Two-Dimensional, Mice, Potassium metabolism, Proline metabolism, Protein Biosynthesis, Sodium metabolism, Cell Transformation, Neoplastic metabolism, Heat-Shock Proteins metabolism, Osmolar Concentration
- Abstract
Both 3T3 and simian virus 40-transformed 3T3 (SV3T3) cells were used to investigate differences in population kinetics, protein synthesis, monovalent ion levels, and amino acid accumulations between normal and transformed cells exposed to hyperosmolarity at 0.5 Osm. Under similar culture conditions, SV3T3 cells were found to be more sensitive in their proliferative response than normal cells to the hyperosmolar treatment. In the normal 3T3 cells, the increase in transport of amino acids was less sustained and was associated with higher levels of accumulated amino acids. The equilibrium distribution of intracellular monovalent cations and the rate of protein synthesis also returned faster to baseline values in the normal cells than in the transformed cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed the induction of a 69-kDa polypeptide in the 3T3 cells but not in the SV3T3 cells after exposure to hyperosmolarity. On electrofocusing and relative mass analysis, this polypeptide closely migrated with the 70-kDa heat shock protein (hsp) family, although it was unrelated immunologically to the inducible 72-kDa hsp.
- Published
- 1991
- Full Text
- View/download PDF
41. Attenuated induction of heat shock proteins in human fibroblasts during ageing in vitro.
- Author
-
Borghetti AF, Campanini C, Alfieri R, and Petronini PG
- Subjects
- Blotting, Northern, Cells, Cultured, Fibroblasts metabolism, Humans, Methionine metabolism, RNA, Messenger analysis, Cellular Senescence, Fibroblasts cytology, Heat-Shock Proteins biosynthesis
- Published
- 1991
42. Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide.
- Author
-
Petronini PG, Tramacere M, Wheeler KP, and Borghetti AF
- Subjects
- Animals, Biological Transport, Chick Embryo, Cycloheximide pharmacology, Dactinomycin pharmacology, Fibroblasts drug effects, In Vitro Techniques, Kinetics, Osmolar Concentration, Fibroblasts metabolism, Proline metabolism, Sodium Chloride metabolism, Sucrose metabolism
- Abstract
The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.
- Published
- 1990
- Full Text
- View/download PDF
43. Protease activation during HIV infection in a CD4-positive cell line.
- Author
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Piedimonte G, Petronini PG, Guetard D, Favier V, Borghetti AF, and Montagnier L
- Subjects
- CD4 Antigens immunology, Cell Survival, Child, Preschool, Cytopathogenic Effect, Viral, Enzyme Activation drug effects, HIV Protease, HIV-1 immunology, HIV-1 physiology, Humans, Kinetics, Leukocyte Count, Protease Inhibitors pharmacology, Protein Denaturation, Tumor Cells, Cultured, Endopeptidases biosynthesis, Gene Products, pol biosynthesis, HIV Infections enzymology, HIV-1 enzymology
- Abstract
The mechanism of cytopathic effects associated with HIV infection in a continuous line of CD4-positive lymphocytes (CEM cells, clone 13) has been studied. Here we report the following observations: (1) HIV infection killed a variable but always significant number of cells without a strict relationship with the syncytia formation; (2) an important decrease in the proliferation rate occurred soon after infection; (3) a marked inhibition of protein synthesis took place within the first few hours of infection and clearly before the beginning of viral protein expression. In addition, when three-day-old cultures were incubated in serum-free medium, a larger degradation of proteins was observed in infected cells in comparison to controls. An increase in protein degradation activity was observed also in vitro with extracts obtained from HIV-infected cells and incubated in the presence of endogenous- or exogenous-labeled substrates. Extracts from cells infected with heat-inactivated HIV did not show a similar degradative activity. The possible induction or activation of latent proteases during the development of the HIV infection is discussed.
- Published
- 1990
- Full Text
- View/download PDF
44. Cell density and L-leucine transport in simian-virus-40-transformed 3T3 cells.
- Author
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Piedimonte G, Petronini PG, and Borghetti AF
- Subjects
- Amino Acids metabolism, Animals, Biological Transport, Cells, Cultured, Kinetics, Mice, Cell Transformation, Viral, Leucine metabolism, Simian virus 40 metabolism
- Published
- 1980
- Full Text
- View/download PDF
45. Density-dependent regulation of amino acid transport in a Burkitt lymphoma cell line.
- Author
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Piedimonte G, Baginski I, Silvotti L, Petronini PG, and Borghetti AF
- Subjects
- Biological Transport, Burkitt Lymphoma pathology, Cell Count, Humans, Tumor Cells, Cultured, Amino Acids metabolism, Burkitt Lymphoma metabolism
- Abstract
Rate of proliferation and amino acid transport were assessed in the Burkitt's lymphoma-derived Namalwa cells by measurements of growth rate and proline and serine uptake. Cell density of the cultures was varied by modifying the number of cells initially seeded and growing for different periods of time. Under these experimental conditions the growth rate was not correlated with cell density. In contrast, the activity of amino acid transport through Systems A and ASC, as assessed by the uptake of proline and serine, respectively, decreased as a function of cell density. This marked decrease of transport activity cannot be explained by large alterations of cell morphology since it was observed at a cell density range where minimal change of cell volume and surface area occurred. When a constant number of cells suspended in an identical volume of medium sedimented on different settling areas, a marked effect on amino acid transport activity occurred. These results indicate that cell to cell contacts may be involved in the density-dependent regulation of transport.
- Published
- 1989
- Full Text
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46. Osmoregulation of amino acid transport activity in cultured fibroblasts.
- Author
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Tramacere M, Petronini PG, Severini A, and Borghetti AF
- Subjects
- Animals, Biological Transport drug effects, Cations, Cells, Cultured, Chickens, Cycloheximide pharmacology, Dactinomycin pharmacology, Kinetics, Sodium physiology, Amino Acids metabolism, Water-Electrolyte Balance
- Abstract
The effect of exposure of chick embryo cells to increasing concentrations of Na+ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na+. Changes were measurable as early as 1 h after altering Na+ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na+ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with L-proline and L-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na+. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na+ treatment occurred through a mechanism affecting Vmax rather than Km.
- Published
- 1984
- Full Text
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47. Induction of stress proteins by hyperosmolarity in normal and transformed cells.
- Author
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Borghetti AF, Petronini PG, Piedimonte G, Silvotti L, and Tramacere M
- Subjects
- Animals, Chick Embryo, Electrophoresis, Polyacrylamide Gel, Fibroblasts metabolism, Molecular Weight, Simian virus 40, Cell Transformation, Viral, Heat-Shock Proteins biosynthesis, Osmolar Concentration
- Abstract
The synthesis of at least three proteins, with molecular weights of approximately 87, 70, and 53 kd, was enhanced following the exposure of chick embryo fibroblasts to hyperosmolar shock of 30 min at 0.6 osM. Two of these proteins, the 87 and 70 kd, comigrated on one-dimensional gel electrophoresis with the stress proteins induced by heat shock after 30 min at 44 degrees C. In 3T3 cells, the hyperosmolar shock enhanced the expression of two proteins of 88 and 52 kd, whereas the heat shock increased the synthesis of several new polypeptides including the 88 and 52 kd mw. In SV40-transformed 3T3 cells the synthesis of two proteins of 72 and 69 kd was enhanced by heat shock, but no change of the protein pattern was recorded after the hyperosmolar shock.
- Published
- 1986
- Full Text
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48. The regulation by cell density of amino acid transport system L in SV40 3T3 cells.
- Author
-
Petronini PG, Piedimonte G, and Borghetti AF
- Subjects
- Amino Acids pharmacology, Animals, Biological Transport drug effects, Cells, Cultured, Kinetics, Mice, Mice, Inbred BALB C, Cell Transformation, Viral, Leucine metabolism, Phenylalanine metabolism, Simian virus 40 genetics
- Abstract
The rate of transport of phenylalanine by System L has been measured in SV40 3T3 cells at various cell densities. When the activity of the L system was determined before any cell depletion of intracellular amino acids, a density-dependent increase in transport paralleled the decrease in cell density. This regulation was lost after cell depletion but reappeared after reloading the cells with pertinent substrates of System L. The phenylalanine transport activity modulated by cell density appeared to be related to the internal level of amino acids capable of exchange up to a definite concentration, beyond which transport activity by System L did not parallel a further increase of internal substrate level. Analysis of the relationship between influx and substrate concentration suggested that two saturable components contribute to entry of phenylalanine and leucine in depleted and in reloaded cells: a low-affinity and a high-affinity component. Both kinetic parameters of the high-affinity component appeared to be modulated by the loading treatment, but only V changed markedly. Activation energies for the high-affinity component of the amino acid transport reaction were calculated from an Arrhenius plot in reloaded cells, and were found to be different for low- and high-density cultures. This result is consistent with the interpretation that cell density modulated the rates at which the amino acid-carrier complex can move within the cell membrane.
- Published
- 1982
- Full Text
- View/download PDF
49. Hyperosmolarity-induced stress proteins in chick embryo fibroblasts.
- Author
-
Petronini PG, Tramacere M, Mazzini A, Piedimonte G, Silvotti L, and Borghetti AF
- Subjects
- Animals, Biological Transport drug effects, Chick Embryo, Dactinomycin pharmacology, Fibroblasts metabolism, Glucose pharmacology, Osmolar Concentration, Potassium metabolism, Proteins isolation & purification, Sodium administration & dosage, Sodium metabolism, Sucrose pharmacology, Culture Media pharmacology, Fibroblasts drug effects, Hypertonic Solutions pharmacology, Protein Biosynthesis
- Abstract
The effects of a short exposure of chick embryo fibroblasts to a hyperosmolar medium on monovalent cation content, rate of protein synthesis, and polypeptide pattern expression were studied. The hyperosmolar shock gave an immediate and pronounced inhibition of the protein-synthesis rate temporally related to a marked alteration of the intracellular Na+ content. Following the return of the cells to an osmolar environment, the internal Na+ content quickly resumed its previous level, while the recovery of the protein-synthesis rate was more gradual. During the recovery period, there was enhanced expression of at least 12 proteins. The 4 major induced proteins exhibited apparent molecular weights of 96, 87, 70, and 48 kDa. A reduction in the synthesis of five protein bands including three large polypeptides of 220, 160, and 140 kDa was also observed. A comparison with the 3 major proteins induced by a 44 degrees C heat shock indicated an apparent similarity with only two of the hyperosmolarity-inducible polypeptides. Moreover, evidence has been also obtained of the close similarity between the 96 and 75 kDa glucose-regulated proteins and the 96 and 75 kDa proteins inducible by a hyperosmolar shock or by a continuous hyperosmolar treatment, respectively. The kinetics of the stress-proteins appearance indicated nonsimultaneous induction. The presence of actinomycin D during the exposure of the cells to the stress and the recovery period suggested that the expression of some hyperosmolarity-enhanced proteins is regulated at the transcriptional level.
- Published
- 1987
- Full Text
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50. Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts.
- Author
-
Tramacere M, Petronini PG, and Borghetti AF
- Subjects
- Animals, Biological Transport, Cells, Cultured, Chick Embryo, Fibroblasts metabolism, Osmolar Concentration, Phenylalanine metabolism, Amino Acids metabolism
- Abstract
The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.
- Published
- 1984
- Full Text
- View/download PDF
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