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1
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Circulating and non-circulating proteins and nucleic acids as biomarkers and therapeutic molecules in ovarian cancer. Genes Dis 2022. [DOI: 10.1016/j.gendis.2022.07.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
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2
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Tao Z, Zhu H, Zhang J, Huang Z, Xiang Z, Hong T. Recent advances of eosinophils and its correlated diseases. Front Public Health 2022; 10:954721. [PMID: 35958837 PMCID: PMC9357997 DOI: 10.3389/fpubh.2022.954721] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Accepted: 07/04/2022] [Indexed: 11/22/2022] Open
Abstract
Eosinophils are differentiated by bone marrow multipotent progenitor cells and are further released into peripheral blood after maturation. Human eosinophils can exhibit unique multi-leaf nuclear morphology, which are filled with cytoplasmic granules that contain cytotoxicity and immune regulatory proteins. In recent years, many studies focused on the origin, differentiation and development process of eosinophils. It has been discovered that the eosinophils have the regulatory functions of innate and adaptive immunity, and can also function in several diseases, including asthma, chronic obstructive pulmonary diseases, acute respiratory distress syndrome, malignant tumors and so on. Hence, the role and effects of eosinophils in various diseases are emphasized. In this review, we comprehensively summarized the development and differentiation process of eosinophils, the research progress of their related cytokines, diseases and current clinical treatment options, and discussed the potential drug target, aiming to provide a theoretical and practical basis for the clinical prevention and treatment of eosinophil-related diseases, especially respiratory diseases. To conclude, the guiding significance of future disease treatment is proposed based on the recent updated understandings into the cell functions of eosinophils.
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Affiliation(s)
- Zhang Tao
- Department of Pulmonary Diseases, Yancheng Traditional Chinese Medicine Hospital, Yancheng, China
| | - Hua Zhu
- Department of Gastroenterology, Yancheng Third People's Hospital, Yancheng, China
- School of Medicine, Affiliated Yancheng Hospital, Southeast University, Yancheng, China
| | - Jiateng Zhang
- Zhejiang University School of Medicine, Hangzhou, China
- Chu Kochen Honors College of Zhejiang University, Hangzhou, China
| | - Zhiming Huang
- Zhejiang University School of Medicine, Hangzhou, China
- Chu Kochen Honors College of Zhejiang University, Hangzhou, China
| | - Ze Xiang
- Zhejiang University School of Medicine, Hangzhou, China
- Chu Kochen Honors College of Zhejiang University, Hangzhou, China
| | - Tu Hong
- Zhejiang University School of Medicine, Hangzhou, China
- Chu Kochen Honors College of Zhejiang University, Hangzhou, China
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Grozdanovic MM, Doyle CB, Liu L, Maybruck BT, Kwatia MA, Thiyagarajan N, Acharya KR, Ackerman SJ. Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis. J Allergy Clin Immunol 2020; 146:377-389.e10. [PMID: 31982451 DOI: 10.1016/j.jaci.2020.01.013] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2019] [Revised: 10/28/2019] [Accepted: 01/03/2020] [Indexed: 01/10/2023]
Abstract
BACKGROUND The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. OBJECTIVE We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. METHODS Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. RESULTS CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CONCLUSIONS CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.
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Affiliation(s)
- Milica M Grozdanovic
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Ill
| | - Christine B Doyle
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Ill
| | - Li Liu
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Ill
| | - Brian T Maybruck
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Ill
| | - Mark A Kwatia
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Ill
| | - Nethaji Thiyagarajan
- Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom
| | - K Ravi Acharya
- Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom
| | - Steven J Ackerman
- Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Ill.
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Zhao HM, Qin WQ, Wang PJ, Wen ZM. Eosinopenia is a predictive factor for the severity of acute ischemic stroke. Neural Regen Res 2019; 14:1772-1779. [PMID: 31169195 PMCID: PMC6585555 DOI: 10.4103/1673-5374.258411] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Previous data have revealed an association between eosinopenia and mortality of acute ischemic stroke. However, the relationship of eosinopenia with infarct volume, infection rate, and poor outcome of acute ischemic stroke is still unknown. The retrospective study included 421 patients (273 males, 65%; mean age, 68.0 ± 13.0 years) with first acute ischemic stroke who were hospitalized in the Second Affiliated Hospital of Soochow University, China, from January 2017 to February 2018. Laboratory data, neuroimaging results, and modified Rankin Scale scores were collected. Patients were divided into four groups according to their eosinophil percentage level (< 0.4%, 0.4–1.1%, 1.1–2.3%, ≥ 2.3%). Spearman’s correlation analysis showed that the percentage of eosinophils was negatively correlated with infarct volume (rs = −0.514, P < 0.001). Receiver operating characteristic analysis demonstrated that eosinopenia predicted a large infarct volume more accurately than neutrophilia; the area under curve was 0.906 and 0.876, respectively; a large infarct was considered as that with a diameter larger than 3 cm and involving more than two major arterial blood supply areas. Logistic regression analysis revealed that eosinophil percentage was an independent risk factor for acute ischemic stroke (P = 0.002). Moreover, eosinophil percentage was significantly associated with large infarct volume, high infection rate (pulmonary and urinary tract infections), and poor outcome (modified Rankin Scale score > 3) after adjusting for potential confounding factors (P-trend < 0.001). These findings suggest that eosinopenia has the potential to predict the severity of acute ischemic stroke. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University, China (approval number: K10) on November 10, 2015.
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Affiliation(s)
- Hui-Min Zhao
- Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
| | - Wen-Qian Qin
- Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
| | - Pei-Ji Wang
- Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
| | - Zhong-Min Wen
- Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
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5
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Wezler X, Dübel S, Schirrmann T. Antibody fusion proteins with human ribonucleases 1 to 8. Hum Antibodies 2018; 26:177-192. [PMID: 29689715 DOI: 10.3233/hab-180337] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity.
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Affiliation(s)
- Xenia Wezler
- Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, 38106 Braunschweig, Germany
| | - Stefan Dübel
- Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, 38106 Braunschweig, Germany
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6
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Akbari B, Farajnia S, Ahdi Khosroshahi S, Safari F, Yousefi M, Dariushnejad H, Rahbarnia L. Immunotoxins in cancer therapy: Review and update. Int Rev Immunol 2017; 36:207-219. [DOI: 10.1080/08830185.2017.1284211] [Citation(s) in RCA: 60] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Affiliation(s)
- Bahman Akbari
- Department of Medical Laboratory Sciences, School of Paramedicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Safar Farajnia
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Fatemeh Safari
- Department of Medical Laboratory Sciences, School of Paramedicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Mohammadreza Yousefi
- Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Hassan Dariushnejad
- Department of Medical Laboratory Sciences, School of Paramedicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Leila Rahbarnia
- Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
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Abstract
First described by Paul Ehrlich in 1879, who noted its characteristic staining by acidophilic dyes, for many years, the eosinophil was considered to be an end-effector cell associated with helminth infections and a cause of tissue damage. Over the past 30 years, research has helped to elucidate the complexity of the eosinophil's function and establish its role in host defense and immunity. Eosinophils express an array of ligand receptors which play a role in cell growth, adhesion, chemotaxis, degranulation, and cell-to-cell interactions. They play a role in activation of complement via both classical and alternative pathways. Eosinophils synthesize, store and secrete cytokines, chemokines, and growth factors. They can process antigen, stimulate T cells, and promote humoral responses by interacting with B cells. Eosinophils can function as antigen presenting cells and can regulate processes associated with both T1 and T2 immunity. Although long known to play a role in defense against helminth organisms, the interactions of eosinophils with these parasites are now recognized to be much more complex. In addition, their interaction with other pathogens continues to be investigated. In this paper, we review the eosinophil's unique biology and structure, including its characteristic granules and the effects of its proteins, our developing understanding of its role in innate and adaptive immunity and importance in immunomodulation, and the part it plays in defense against parasitic, viral, fungal and bacterial infections. Rather than our worst enemy, the eosinophil may, in fact, be one of the most essential components in host defense and immunity.
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8
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Pulido D, Moussaoui M, Nogués MV, Torrent M, Boix E. Towards the rational design of antimicrobial proteins. FEBS J 2013; 280:5841-52. [DOI: 10.1111/febs.12506] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Revised: 08/19/2013] [Accepted: 08/23/2013] [Indexed: 12/15/2022]
Affiliation(s)
- David Pulido
- Department of Biochemistry and Molecular Biology; Universitat Autònoma de Barcelona; Cerdanyola del Vallès Spain
| | - Mohammed Moussaoui
- Department of Biochemistry and Molecular Biology; Universitat Autònoma de Barcelona; Cerdanyola del Vallès Spain
| | - M. Victòria Nogués
- Department of Biochemistry and Molecular Biology; Universitat Autònoma de Barcelona; Cerdanyola del Vallès Spain
| | - Marc Torrent
- Department of Biochemistry and Molecular Biology; Universitat Autònoma de Barcelona; Cerdanyola del Vallès Spain
- Medical Research Council Laboratory of Molecular Biology; Francis Crick Avenue; Cambridge CB2 0QH UK
| | - Ester Boix
- Department of Biochemistry and Molecular Biology; Universitat Autònoma de Barcelona; Cerdanyola del Vallès Spain
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9
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Kita H. Eosinophils: multifunctional and distinctive properties. Int Arch Allergy Immunol 2013; 161 Suppl 2:3-9. [PMID: 23711847 DOI: 10.1159/000350662] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The eosinophil is a granulocyte prominent in allergic diseases and inflammatory responses against helminthic parasites. The eosinophil was named by Paul Ehrlich in 1879, and derives from the intense staining of its granules with the acidic dye eosin. It has been the subject of extensive investigation ever since. It is strongly associated with human diseases involving mucosal surfaces, such as allergic asthma, atopic dermatitis and gastrointestinal disorders. Eosinophils are likely involved in tissue homeostasis, modulation of adaptive immune responses, innate immunity to certain microbes and pathological changes in allergic disorders. Thus, the eosinophil is considered a multifunctional leukocyte that contributes to a wide variety of physiological and pathological processes, depending on its location and activation status. Further studies will be necessary to better understand the biology of this extraordinary leukocyte and to reveal the importance of the cell in human health and disease.
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Affiliation(s)
- Hirohito Kita
- Division of Allergic Diseases, Departments of Internal Medicine and Immunology, Mayo Clinic, Rochester, MN 55905, USA.
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10
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Provost V, Larose MC, Langlois A, Rola-Pleszczynski M, Flamand N, Laviolette M. CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2. J Leukoc Biol 2013; 94:213-22. [PMID: 23532518 DOI: 10.1189/jlb.0212074] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5-oxo-eicosatetraenoate-induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.
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11
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Zeng Q, Wen H, Wen Q, Chen X, Wang Y, Xuan W, Liang J, Wan S. Cucumber mosaic virus as drug delivery vehicle for doxorubicin. Biomaterials 2013; 34:4632-42. [PMID: 23528229 DOI: 10.1016/j.biomaterials.2013.03.017] [Citation(s) in RCA: 105] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2013] [Accepted: 03/07/2013] [Indexed: 02/07/2023]
Abstract
Taking advantage of the unique structure feature of cucumber mosaic virus (CMV), we have anchored folic acid (FA) as targeting moiety on the rigid CMV capsid and loaded significant amount of doxorubicin (Dox) into the interior cavity of CMV through the formation of Dox-RNA conjugate to provide a nanosized control delivery system for cancer therapy. The FA-CMV-Dox assemblies were characterized using transmission electron microscopy and size exclusion chromatography, which disclose that they have comparable size and morphology to the native CMV particles. The Dox-loaded viral particles exhibit sustained in vitro Dox release profile over 5 days at physiological pH but can be liberated from the conjugates with the presence of elevated level of RNase. The in vitro effects of folate receptor (FR)-targeted CMV-Dox nanoconjugates on cellular internalization and cell proliferation were evaluated by live-cell imaging, MTT and TUNEL assay, respectively, in mouse cardiomyocytes and FR over expression OVCAR-3 tumor cells. The in vivo efficacy was also investigated in the OVCAR-3 BALB/c nude mouse xenograft model through histological alterations and TUNEL assessment. The FA-CMV-Dox particles significantly decrease the accumulation of Dox in the nuclei of mouse myocardial cells and improve the uptake of Dox in the ovarian cancer, leading to less cardiotoxicity and enhanced antitumor effect. We believe that CMV offers a new way to fabricate nanosized drug delivery vehicles.
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MESH Headings
- Animals
- Antibiotics, Antineoplastic/administration & dosage
- Antibiotics, Antineoplastic/adverse effects
- Antibiotics, Antineoplastic/pharmacology
- Antibiotics, Antineoplastic/therapeutic use
- Apoptosis/drug effects
- Cell Line, Tumor
- Cells, Cultured
- Cucumovirus/chemistry
- Cucumovirus/metabolism
- Delayed-Action Preparations/chemistry
- Delayed-Action Preparations/metabolism
- Doxorubicin/administration & dosage
- Doxorubicin/adverse effects
- Doxorubicin/pharmacology
- Doxorubicin/therapeutic use
- Drug Delivery Systems
- Female
- Folic Acid/chemistry
- Folic Acid/metabolism
- Humans
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Models, Molecular
- Myocytes, Cardiac/pathology
- Ovarian Neoplasms/drug therapy
- Ovarian Neoplasms/pathology
- Ovary/drug effects
- Ovary/pathology
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Affiliation(s)
- Qingbing Zeng
- Biomaterial Research Center, School of Pharmaceutical Sciences, Southern Medical University, 1023 Southern Shatai Street, Guangzhou, GD 510515, China.
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In silico prediction and in vitro characterization of multifunctional human RNase3. BIOMED RESEARCH INTERNATIONAL 2013; 2013:170398. [PMID: 23484086 PMCID: PMC3581242 DOI: 10.1155/2013/170398] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/31/2012] [Accepted: 12/02/2012] [Indexed: 12/18/2022]
Abstract
Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members.
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13
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Pawankar R, Mori S, Ozu C, Kimura S. Overview on the pathomechanisms of allergic rhinitis. Asia Pac Allergy 2011; 1:157-67. [PMID: 22053313 PMCID: PMC3206239 DOI: 10.5415/apallergy.2011.1.3.157] [Citation(s) in RCA: 180] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2011] [Accepted: 09/25/2011] [Indexed: 12/21/2022] Open
Abstract
Allergic rhinitis a chronic inflammatory disease of the upper airways that has a major impact on the quality of life of patients and is a socio-economic burden. Understanding the underlying immune mechanisms is central to developing better and more targeted therapies. The inflammatory response in the nasal mucosa includes an immediate IgE-mediated mast cell response as well as a latephase response characterized by recruitment of eosinophils, basophils, and T cells expressing Th2 cytokines including interleukin (IL)-4, a switch factor for IgE synthesis, and IL-5, an eosinophil growth factor and on-going allergic inflammation. Recent advances have suggested new pathways like local synthesis of IgE, the IgE-IgE receptor mast cell cascade in on-going allergic inflammation and the epithelial expression of cytokines that regulate Th2 cytokine responses (i.e., thymic stromal lymphopoietin, IL-25, and IL-33). In this review, we briefly review the conventional pathways in the pathophysiology of allergic rhinitis and then elaborate on the recent advances in the pathophysiology of allergic rhinitis. An improved understanding of the immune mechanisms of allergic rhinitis can provide a better insight on novel therapeutic targets.
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Abstract
Eosinophils are leukocytes resident in mucosal tissues. During T-helper 2 (Th2)-type inflammation, eosinophils are recruited from bone marrow and blood to the sites of immune response. While eosinophils have been considered end-stage cells involved in host protection against parasite infection and immunopathology in hypersensitivity disease, recent studies changed this perspective. Eosinophils are now considered multifunctional leukocytes involved in tissue homeostasis, modulation of adaptive immune responses, and innate immunity to certain microbes. Eosinophils are capable of producing immunoregulatory cytokines and are actively involved in regulation of Th2-type immune responses. However, such new information does not preclude earlier observations showing that eosinophils, in particular human eosinophils, are also effector cells with proinflammatory and destructive capabilities. Eosinophils with activation phenotypes are observed in biological specimens from patients with disease, and deposition of eosinophil products is readily seen in the affected tissues from these patients. Therefore, it would be reasonable to consider the eosinophil a multifaceted leukocyte that contributes to various physiological and pathological processes depending on their location and activation status. This review summarizes the emerging concept of the multifaceted immunobiology of eosinophils and discusses the roles of eosinophils in health and disease and the challenges and perspectives in the field.
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Affiliation(s)
- Hirohito Kita
- Division of Allergic Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.
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15
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Molfino NA, Gossage D, Kolbeck R, Parker JM, Geba GP. Molecular and clinical rationale for therapeutic targeting of interleukin-5 and its receptor. Clin Exp Allergy 2011; 42:712-37. [PMID: 22092535 DOI: 10.1111/j.1365-2222.2011.03854.x] [Citation(s) in RCA: 161] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2010] [Revised: 07/26/2011] [Accepted: 07/28/2011] [Indexed: 12/17/2022]
Abstract
Interleukin-5 is a Th2 homodimeric cytokine involved in the differentiation, maturation, migration, development, survival, trafficking and effector function of blood and local tissue eosinophils, in addition to basophils and mast cells. The IL-5 receptor (IL-5R) consists of an IL-5-specific α subunit that interacts in conformationally dynamic ways with the receptor's βc subunit, an aggregate of domains it shares with binding sites of IL-3 and granulocyte-macrophage colony-stimulating factor. IL-5 and IL-5R drive allergic and inflammatory immune responses characterizing numerous diseases, such as asthma, atopic dermatitis, chronic obstructive pulmonary disease, eosinophilic gastrointestinal diseases, hyper-eosinophilic syndrome, Churg-Strauss syndrome and eosinophilic nasal polyposis. Although corticosteroid therapy is the primary treatment for these diseases, a substantial number of patients exhibit incomplete responses and suffer side-effects. Two monoclonal antibodies have been designed to neutralize IL-5 (mepolizumab and reslizumab). Both antibodies have demonstrated the ability to reduce blood and tissue eosinophil counts. One additional monoclonal antibody, benralizumab (MEDI-563), has been developed to target IL-5R and attenuate eosinophilia through antibody-dependent cellular cytotoxicity. All three monoclonal antibodies are being clinically evaluated. Antisense oligonucleotide technology targeting the common βc IL-5R subunit is also being used therapeutically to inhibit IL-5-mediated effects (TPI ASM8). Small interfering RNA technology has also been used therapeutically to inhibit the expression of IL-5 in animal models. This review summarizes the structural interactions between IL-5 and IL-5R and the functional consequences of such interactions, and describes the pre-clinical and clinical evidence supporting IL-5R as a therapeutic target.
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Affiliation(s)
- N A Molfino
- MedImmune, LLC, Gaithersburg, MD 20878, USA.
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16
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The eight human "canonical" ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins. FEBS Lett 2010; 584:2194-200. [PMID: 20388512 DOI: 10.1016/j.febslet.2010.04.018] [Citation(s) in RCA: 121] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2010] [Revised: 04/07/2010] [Accepted: 04/07/2010] [Indexed: 01/25/2023]
Abstract
Human ribonucleases (RNases) are members of a large superfamily of rapidly evolving homologous proteins. Upon completion of the human genome, eight catalytically active RNases (numbered 1-8) were identified. These structurally distinct RNases, characterized by their various catalytic differences on different RNA substrates, constitute a gene family that appears to be the sole vertebrate-specific enzyme family. Apart from digestion of dietary RNA, a wide variety of biological actions, including neurotoxicity, angiogenesis, immunosuppressivity, and anti-pathogen activity, have been recently reported for almost all members of the family. Recent evolutionary studies suggest that RNases started off in vertebrates as host defence or angiogenic proteins.
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17
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Mathew M, Verma RS. Humanized immunotoxins: a new generation of immunotoxins for targeted cancer therapy. Cancer Sci 2009; 100:1359-65. [PMID: 19459847 PMCID: PMC11158948 DOI: 10.1111/j.1349-7006.2009.01192.x] [Citation(s) in RCA: 120] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Chemotherapy, radiation, and surgery are the conventional treatment modalities for cancer. The success achieved with these approaches has been limited due to several factors like chemoresistance to drugs, non-specificity leading to peripheral toxicity, and non-resectable tumors. To combat these problems, the concept of targeted therapy using immunotoxins was developed. Immunotoxins are chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety and can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments directed against these antigens are fused with bacterial or plant toxins and are made use of as immunotoxins. Pseudomonas exotoxin, anthrax toxin, and diphtheria toxin are the commonly used bacterial toxins. Ricin, saporin, gelonin, and poke weed antiviral protein are the plant toxins utilized in immunotoxin constructs. Several such fusion proteins are in clinical trials, and denileukin difitox is a FDA-approved fusion protein. In spite of the promise shown by bacterial- and plant toxin-based chimeric proteins, their clinical application is hampered by several factors like immunogenicity of the toxin moiety and non-specific toxicity leading to vascular leak syndrome. In order to overcome these problems, a novel generation of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein or RNase has been developed. This review summarizes the advances in this new class of fusion protein and the future directions to be explored.
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Affiliation(s)
- Mrudula Mathew
- Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India
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Harder J, Gläser R, Schröder JM. Human antimicrobial proteins effectors of innate immunity. ACTA ACUST UNITED AC 2008; 13:317-38. [PMID: 18182460 DOI: 10.1177/0968051907088275] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
We live in a world populated by an enormous number of micro-organisms. This necessitates the existence of highly effective mechanisms to control microbial growth. Through many research efforts, a chemical defense system based on the production of antimicrobial proteins (AMPs) has been identified. AMPs are endogenous, small proteins exhibiting antimicrobial activity against a wide variety of micro-organisms. The wide distribution of these molecules in the plant and animal kingdom reflects their biological significance. Various human AMPs show a potent effect on pathogenic micro-organisms including antibiotic-resistant bacteria. Thus, there is great interest in understanding the role of AMPs within innate immunity and evaluating their use and/or specific induction to fend off infections. In this review, we provide an overview of the characteristics of human AMPs and discuss examples where AMPs may be involved in the pathogenesis of infectious and inflammatory diseases.
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Affiliation(s)
- Jürgen Harder
- Clinical Research Unit, Department of Dermatology, University Hospital Schleswig-Holstein, Kiel, Germany.
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19
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Fan TC, Chang HT, Chen IW, Wang HY, Chang MDT. A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein. Traffic 2007; 8:1778-1795. [PMID: 17944807 DOI: 10.1111/j.1600-0854.2007.00650.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Eosinophil cationic protein (ECP), a human RNAseA superfamily member, highly implicated in asthma pathology, is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In this study, we show that cell surface-bound heparan sulfate proteoglycans serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas-2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan-defective cells compared with their wild-type counterparts. Furthermore, pharmacological treatment combined with siRNA knockdown identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and ADP-ribosylating factor 6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and phosphatidylinositol-3-kinase activities, and is compatible with the characteristics of raft-dependent macropinocytosis. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.
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Affiliation(s)
- Tan-Chi Fan
- Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China
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20
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Sikriwal D, Seth D, Dey P, Batra JK. Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis. Mol Cell Biochem 2007; 303:175-81. [PMID: 17483910 DOI: 10.1007/s11010-007-9471-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2007] [Accepted: 03/30/2007] [Indexed: 11/25/2022]
Abstract
Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P(-1). Although RNase A also contains a P(-1) subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P(-1) subsite residues Arg(36), Asn(39), and Gln(40) of EDN in its catalytic activity. The individual mutation of Arg(36), Asn (39), and Gln(40) resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P(-1) subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates.
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Affiliation(s)
- Deepa Sikriwal
- Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
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21
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Rosenberg HF, Phipps S, Foster PS. Eosinophil trafficking in allergy and asthma. J Allergy Clin Immunol 2007; 119:1303-10; quiz 1311-2. [PMID: 17481712 DOI: 10.1016/j.jaci.2007.03.048] [Citation(s) in RCA: 303] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2007] [Revised: 03/27/2007] [Accepted: 03/28/2007] [Indexed: 02/08/2023]
Abstract
Blood eosinophilia and tissue eosinophilia are characteristic features of allergic inflammation and asthma, conditions associated with prominent production of T(H)2 cytokines IL-4, IL-5, and IL-13. In this review, we will consider recent advances in our understanding of the molecular mechanisms that promote expansion and differentiation of eosinophil progenitors in bone marrow, eosinophil recruitment in response to chemokine receptor 3 agonists eosinophil transit mediated by specific ligand-receptor interactions, and prolonged survival of eosinophils in peripheral tissues. Novel rational therapies including antiselectin and antichemokine receptor modalities designed to block eosinophil development and trafficking are discussed, together with the implications of recent clinical studies that have evaluated the efficacy of humanized anti-IL-5 mAb therapy.
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Affiliation(s)
- Helene F Rosenberg
- Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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22
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23
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Rudolph B, Podschun R, Sahly H, Schubert S, Schröder JM, Harder J. Identification of RNase 8 as a novel human antimicrobial protein. Antimicrob Agents Chemother 2006; 50:3194-6. [PMID: 16940129 PMCID: PMC1563533 DOI: 10.1128/aac.00246-06] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The antimicrobial activity of the human RNase A superfamily member RNase 8 was evaluated. Recombinant RNase 8 exhibited broad-spectrum microbicidal activity against potential pathogenic microorganisms (including multidrug-resistant strains) at micro- to nanomolar concentrations. Thus, RNase 8 was identified as a novel antimicrobial protein and may contribute to host defense.
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Affiliation(s)
- Bente Rudolph
- Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany
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24
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Swartz JM, Dyer KD, Cheever AW, Ramalingam T, Pesnicak L, Domachowske JB, Lee JJ, Lee NA, Foster PS, Wynn TA, Rosenberg HF. Schistosoma mansoni infection in eosinophil lineage-ablated mice. Blood 2006; 108:2420-7. [PMID: 16772607 PMCID: PMC1895572 DOI: 10.1182/blood-2006-04-015933] [Citation(s) in RCA: 144] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2005] [Accepted: 05/29/2006] [Indexed: 12/30/2022] Open
Abstract
We explore the controversial issue of the role of eosinophils in host defense against helminthic parasites using the established Schistosoma mansoni infection model in 2 novel mouse models of eosinophil lineage ablation (DeltadblGATA and TgPHIL). No eosinophils were detected in bone marrow of infected DeltadblGATA or TgPHIL mice, despite the fact that serum IL-5 levels in these infected mice exceeded those in infected wild type by approximately 4-fold. Liver granulomata from infected DeltadblGATA and TgPHIL mice were likewise depleted of eosinophils compared with those from their respective wild types. No eosinophil-dependent differences in granuloma number, size, or fibrosis were detected at weeks 8 or 12 of infection, and differential accumulation of mast cells was observed among the DeltadblGATA mice only at week 12. Likewise, serum levels of liver transaminases, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) increased in all mice in response to S mansoni infection, with no eosinophil-dependent differences in hepatocellular damage observed. Finally, eosinophil ablation had no effect on worm burden or on egg deposition. Overall, our data indicate that eosinophil ablation has no impact on traditional measures of disease in the S mansoni infection model in mice. However, eosinophils may have unexplored immunomodulatory contributions to this disease process.
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Affiliation(s)
- Jonathan M Swartz
- Laboratory of Allergic Diseases, NIAID, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1883, USA
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25
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Abstract
The Ribonuclease A superfamily includes an extensive network of distinct and divergent gene lineages. Although all ribonucleases of this superfamily share invariant structural and catalytic elements and some degree of enzymatic activity, the primary sequences have diverged significantly, ostensibly to promote novel function. We will review the literature on the evolution and biology of the RNase A ribonuclease lineages that have been characterized specifically as involved in host defense including: (1) RNases 2 and RNases 3, also known as the eosinophil ribonucleases, which are rapidly-evolving cationic proteins released from eosinophilic leukocytes, (2) RNase 7, an anti-pathogen ribonuclease identified in human skin, and (3) RNase 5, also known as angiogenin, another rapidly-evolving ribonuclease known to promote blood vessel growth with recently-discovered antibacterial activity. Interestingly, some of the characterized anti-pathogen activities do not depend on ribonuclease activity per se. We discuss the ways in which the anti-pathogen activities characterized in vitro might translate into experimental confirmation in vivo. We will also consider the possibility that other ribonucleases, such as the dimeric bovine seminal ribonuclease and the frog oocyte ribonucleases, may have host defense functions and therapeutic value that remain to be explored. (190 words).
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Affiliation(s)
- Kimberly D Dyer
- Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Ramos AL, Discipio RG, Ferreira AM. Eosinophil cationic protein damages protoscoleces in vitro and is present in the hydatid cyst. Parasite Immunol 2006; 28:347-55. [PMID: 16879306 DOI: 10.1111/j.1365-3024.2006.00842.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Eosinophils are locally recruited during the establishment and chronic phases of cystic hydatidosis. This study provides evidence that eosinophil cationic protein (ECP), one of the major components of eosinophil granules, can damage Echinococcus granulosus protoscoleces (PSC). The toxicity of ECP was investigated in vitro by following parasite viability in the presence of this protein. ECP was found to damage PSC at micromolar concentrations; the effect was blocked by specific antibodies and heparin, and was more severe than the one caused by similar concentrations of RNase A, suggesting that the cationic nature of ECP, and not its ribonuclease activity, is involved in toxicity. This observation may highlight the capacity of eosinophils to control secondary hydatidosis, derived from PSC leakage from a primary cyst. To further assess the relevance of the previous result during infection, the presence of eosinophil proteins was investigated in human hydatid cysts. ECP was found to be strongly associated with the laminated layer of the cyst wall, and present at micromolar concentrations in the hydatid fluid. Overall, these results demonstrate that eosinophils degranulate in vivo at the host-parasite interface, and that the released ECP reaches concentrations that could be harmful for the parasite.
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Affiliation(s)
- A L Ramos
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química. Instituto de Higiene, Avda A. Navarro 3051, p2, Montevideo CP.11600, Uruguay
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27
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Ye B, Skates S, Mok SC, Horick NK, Rosenberg HF, Vitonis A, Edwards D, Sluss P, Han WK, Berkowitz RS, Cramer DW. Proteomic-based discovery and characterization of glycosylated eosinophil-derived neurotoxin and COOH-terminal osteopontin fragments for ovarian cancer in urine. Clin Cancer Res 2006; 12:432-41. [PMID: 16428483 DOI: 10.1158/1078-0432.ccr-05-0461] [Citation(s) in RCA: 110] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
PURPOSE The objective was to identify and characterize low molecular weight proteins/peptides in urine and their posttranslational modifications that might be used as a screening tool for ovarian cancer. EXPERIMENTAL DESIGN Urine samples collected preoperatively from postmenopausal women with ovarian cancer and benign conditions and from nonsurgical controls were analyzed by surface-enhanced laser desorption/ionization mass spectrometry and two-dimensional gel electrophoresis. Selected proteins from mass profiles were purified by chromatography and followed by liquid chromatography-tandem mass spectrometry sequence analysis. Specific antibodies were generated for further characterization, including immunoprecipitation and glycosylation. Quantitative and semiquantitative ELISAs were developed for preliminary validation in patients of 128 ovarian cancer, 52 benign conditions, 44 other cancers, and 188 healthy controls. RESULTS A protein (m/z approximately 17,400) with higher peak intensities in cancer patients than in benign conditions and controls was identified and subsequently defined as eosinophil-derived neurotoxin (EDN). A glycosylated form of EDN was specifically elevated in ovarian cancer patients. A cluster of COOH-terminal osteopontin was identified from two-dimensional gels of urine from cancer patients. Modified forms EDN and osteopontin fragments were elevated in early-stage ovarian cancers and a combination of both resulted to 93% specificity and 72% sensitivity. CONCLUSIONS Specific elevated posttranslationally modified urinary EDN and osteopontin COOH-terminal fragments in ovarian cancer might lead to potential noninvasive screening tests for early diagnosis. Urine with less complexity than serum and relatively high thermodynamic stability of peptides or metabolites is a promising study medium for discovery of the novel biomarkers which may present in many non-urinary tract neoplastic diseases.
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MESH Headings
- Adenocarcinoma, Clear Cell/pathology
- Adenocarcinoma, Clear Cell/surgery
- Adenocarcinoma, Clear Cell/urine
- Adenocarcinoma, Mucinous/pathology
- Adenocarcinoma, Mucinous/surgery
- Adenocarcinoma, Mucinous/urine
- Amino Acid Sequence
- Biomarkers, Tumor/urine
- Carcinoma, Endometrioid/pathology
- Carcinoma, Endometrioid/surgery
- Carcinoma, Endometrioid/urine
- Case-Control Studies
- Cystadenocarcinoma, Serous/pathology
- Cystadenocarcinoma, Serous/surgery
- Cystadenocarcinoma, Serous/urine
- Electrophoresis, Gel, Two-Dimensional
- Enzyme-Linked Immunosorbent Assay
- Eosinophil-Derived Neurotoxin/urine
- Female
- Glycosylation
- Humans
- Molecular Sequence Data
- Neoplasm Invasiveness
- Neoplasms, Glandular and Epithelial/pathology
- Neoplasms, Glandular and Epithelial/surgery
- Neoplasms, Glandular and Epithelial/urine
- Osteopontin
- Ovarian Neoplasms/pathology
- Ovarian Neoplasms/surgery
- Ovarian Neoplasms/urine
- Prognosis
- Proteome
- Sialoglycoproteins/urine
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Affiliation(s)
- Bin Ye
- Laboratory of Gynecologic Oncology, Department of Obstetrics and Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Dana-Farber Cancer Center, 221 Longwood Avenue, LMRC-601B, Boston, MA 02115, USA.
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Abstract
Eosinophils are one of the cells that play a critical role in the pathogenesis of allergic diseases. The increase in the number of eosinophils in such diseases is regulated by interleukin-5 (IL-5). The author have prepared recombinant rat IL-5 using a baculovirus expression system and examined its biological activities in rat eosinophils. It was demonstrated that recombinant rat IL-5 prolongs the survival of mature eosinophils and differentiates immature eosinophils into mature eosinophils, suggesting that rat IL-5 is a factor for eosinophilia in rats. Recombinant rat eosinophil-associated ribonuclease (Ear)-1 and Ear-2 were also prepared. Eosinophil granule proteins are thought to cause tissue damage due to their cytotoxic activity, but using recombinant rat Ear-1 and Ear-2, it was found that rat Ear-1 and Ear-2 have strong RNase A activity and bactericidal activity, suggesting that these proteins play critical roles in host defense. Finally, the important role of acetylation of histones was clarified in the differentiation of HL-60 clone 15 cells into eosinophils using the histone deacetylase inhibitors sodium n-butyrate, apicidin, and trichostatin A. These findings would be useful for further investigations of the role of eosinophils in allergic inflammation.
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Affiliation(s)
- Kenji Ishihara
- Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki Aoba, Sendai, Japan.
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29
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Shichijo K, Makiyama K, Wen CY, Matsuu M, Nakayama T, Nakashima M, Ihara M, Sekine I. Antibody to eosinophil cationic protein suppresses dextran sulfate sodium-induced colitis in rats. World J Gastroenterol 2005; 11:4505-10. [PMID: 16052679 PMCID: PMC4398699 DOI: 10.3748/wjg.v11.i29.4505] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To produce an antibody against rat eosinophil cationic protein (ECP) and to examine the effects of the antibody in rats with dextran sulfate sodium (DSS)-induced colitis.
METHODS: An antibody was raised against rat ECP. Rats were treated with 3% DSS in drinking water for 7 d and received the antibody or normal serum. The colons were examined histologically and correlated with clinical symptoms. Immunohistochemistry and Western blot analysis were estimated as a grade of inflammation.
RESULTS: The ECP antibody stained the activated eosinophils around the injured crypts in the colonic mucosa. Antibody treatment reduced the severity of colonic ulceration and acute clinical symptoms (diarrhea and/or blood-stained stool). Body weight gain was significantly greater and the colon length was significantly longer in anti-ECP-treated rats than in normal serum-treated rats. Expression of ECP in activated eosinophils was associated with the presence of erosions and inflammation. The number of Ki-67-positive cells in the regenerated surface epithelium increased in anti-ECP-treated rats compared with normal serum-treated rats. Western blot analysis revealed reduced expression of macrophage migration inhibitory factor (MIF) in anti-ECP-treated rats.
CONCLUSION: Our results indicate that treatment with ECP antibody, improved DSS-induced colitis in rats, possibly by increasing the regenerative activity of the colonic epithelium and downregulation of the immune response, and suggest that anti-ECP may promote intestinal wound healing in patients with ulcerative colitis (UC).
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Affiliation(s)
- Kazuko Shichijo
- Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
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Cho S, Beintema JJ, Zhang J. The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories. Genomics 2005; 85:208-20. [PMID: 15676279 DOI: 10.1016/j.ygeno.2004.10.008] [Citation(s) in RCA: 133] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2004] [Accepted: 10/13/2004] [Indexed: 12/22/2022]
Abstract
The RNase A superfamily has been important in biochemical, structural, and evolutionary studies and is believed to be the sole vertebrate-specific enzyme family. To understand the origin and diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of human, mouse, rat, and chicken. We report a previously unnoticed gene cluster in mouse chromosome 10 and a number of new genes, including mammalian RNases 11-13, which are close relatives of the recently identified RNases 9 and 10. Gene expression data imply male-reproductive functions for RNases 9-13, although their sequences suggest the lack of ribonucleolytic activities. In contrast to the presence of 13-20 functional genes in mammals, chicken has only 3 RNase genes, which are evolutionarily close to mammalian RNase 5, like other nonmammalian RNases. This and other evidence suggests that the RNase A superfamily originated from an RNase 5-like gene and expanded in mammals. Together with the fact that multiple lineages of the superfamily, including RNases 2, 3, 5, and 7, have antipathogenic activities, we suggest that the superfamily started off as a host-defense mechanism in vertebrates. Consistent with this hypothesis, all members of the superfamily exhibit high rates of amino acid substitution as is commonly observed in immunity genes.
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Affiliation(s)
- Soochin Cho
- Department of Ecology and Evolutionary Biology, University of Michigan, 3003 Natural Science Building, 830 North University Avenue, Ann Arbor, MI 48109, USA
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31
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Ishihara K, Hong J, Zee O, Ohuchi K. [The role of eosinophils in allergic inflammation]. Nihon Yakurigaku Zasshi 2005; 125:265-70. [PMID: 15997162 DOI: 10.1254/fpj.125.265] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
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32
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Nitto T, Dyer KD, Mejia RA, Byström J, Wynn TA, Rosenberg HF. Characterization of the divergent eosinophil ribonuclease, mEar 6, and its expression in response to Schistosoma mansoni infection in vivo. Genes Immun 2005; 5:668-74. [PMID: 15526002 DOI: 10.1038/sj.gene.6364143] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The eosinophil-associated ribonucleases (Ears) are rapidly evolving proteins found in multigene clusters that are unique to each rodent species. Of the 15 independent genes in the Mus musculus cluster, only mEars 1 and 2 are expressed at significant levels at homeostasis. Here we characterize the expression of mEar 6 in the liver and spleen in mice in response to infection with the helminthic parasite, Schistosoma mansoni. Interestingly, expression of mEar 6 is not directly related to the elevated levels of serum IL-5 or tissue eosinophilia characteristic of this disease, as no mEar 6 transcripts were detected in the liver or the spleen from uninfected IL-5-transgenic mice. The coding sequence of mEar 6 has diverged under positive selection pressure (K(a)/K(s) > 1.0) and has a unique unpaired cysteine near the carboxy-terminus of the protein. The high catalytic efficiency of recombinant mEar 6 (k(cat)/K(m) = 0.9 x 10(6)/M/s) is similar to that of the cluster's closest human ortholog, eosinophil-derived neurotoxin (EDN/RNase 2). In summary, we have identified mEar 6 as one of only two RNase A superfamily ribonucleases known to be expressed specifically in response to pathophysiologic stress in vivo.
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Affiliation(s)
- T Nitto
- Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, room 11N104, 9000 Rockville Pike, Bethesda, MD 20892, USA.
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Wu CM, Chang HT, Chang MT. Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells. Biochem J 2005; 382:841-8. [PMID: 15233624 PMCID: PMC1133959 DOI: 10.1042/bj20040894] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2004] [Revised: 06/30/2004] [Accepted: 07/02/2004] [Indexed: 11/17/2022]
Abstract
ECP (eosinophil cationic protein) is a major component of eosinophil granule proteins, and is used as a clinical biomarker for asthma and allergic inflammatory disease. ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known. In the present study, mECP-eGFP-6H, a recombinant fusion protein containing mature ECP (mECP), enhanced green fluorescence protein (eGFP) and a His(6) tag (6H), has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP. We found that mECP-eGFP-6H entered into GH3 neuroendocrine cells and inhibited the growth of the cells with an IC(50) of 0.8 microM. By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein-protein interaction between mECP and CPE (carboxypeptidase E), a well characterized metalloprotease. Further in vivo yeast two-hybrid screening has also revealed that residues 318-387 located in a region of unknown function in mature CPE are indispensable for association with mECP. In addition, the uptake of mECP-eGFP-6H is suppressed by dominant-negative expression of the recycling defect mutant pre-pro-HA-CPE(S471A,E472A) in GH3 cells, suggesting that the entry of mECP-eGFP-6H is associated with the recycling of CPE in GH3 cells. Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cells, and such an endocytotic process allows the cytotoxic ECP to inhibit growth of the target cells.
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Affiliation(s)
- Chia-Mao Wu
- Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China
| | - Hao-Teng Chang
- Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China
| | - Margaret Dah-Tsyr Chang
- Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China
- To whom correspondence should be addressed (email )
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34
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Harder J, Schröder JM. Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins. J Leukoc Biol 2005; 77:476-86. [PMID: 15629886 DOI: 10.1189/jlb.0704409] [Citation(s) in RCA: 171] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Patients with psoriasis, a chronic, hyperproliferative and noninfectious skin disease, suffer surprisingly fewer cutaneous infections than would be expected. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides psoriatic skin with resistance against infection. We subsequently began a systematic analysis of in vitro antimicrobially active proteins in psoriatic-scale extracts. A biochemical approach with rigorous purification and characterization combined with antimicrobial testing identified a number of mostly new human antibiotic peptides and proteins. In this review, we will focus on the most prominent antimicrobial proteins in psoriatic-scale extracts, which we identified as the S100-protein psoriasin, human beta-defensin 2 (hBD-2), RNase 7, lysozyme, and human neutrophil defensin 1-3. Apart from these cutaneous, antimicrobial proteins, only a few others, including hBD-3, have been characterized. A great number of minor antimicrobial proteins await further structural characterization.
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Affiliation(s)
- Jürgen Harder
- Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 7, D-24105 Kiel, Germany.
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35
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Swartz JM, Byström J, Dyer KD, Nitto T, Wynn TA, Rosenberg HF. Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes. J Leukoc Biol 2004; 76:812-9. [PMID: 15277569 DOI: 10.1189/jlb.0304182] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Plasminogen activator inhibitor-2 (PAI-2) as a potential eosinophil protein was inferred from our gene microarray study of mouse eosinophilopoiesis. Here, we detect 47 kDa intracellular and approximately 60 kDa secretory forms of PAI-2 in purified human eosinophil extracts. PAI-2 is present at variable concentrations in eosinophil lysates, ranging from 30 to 444 ng/10(6) cells, with a mean of 182 ng/10(6) cells from 10 normal donors, which is the highest per-cell concentration among all leukocyte subtypes evaluated. Enzymatic assay confirmed that eosinophil-derived PAI-2 is biologically active and inhibits activation of its preferred substrate, urokinase. Immunohistochemical and immunogold staining demonstrated PAI-2 localization in eosinophil-specific granules. Immunoreactive PAI-2 was detected in extracellular deposits in and around the eosinophil-enriched granuloma tissue encapsulating the parasitic egg in livers of wild-type mice infected with the helminthic parasite Schistosoma mansoni. Among the possibilities, we consider a role for eosinophil-derived PAI-2 in inflammation and remodeling associated with parasitic infection as well as allergic airways disease, respiratory virus infection, and host responses to tumors and metastasis in vivo.
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Affiliation(s)
- Jonathan M Swartz
- Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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36
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Dyer KD, Nitto T, Moreau JM, McDevitt AL, Rosenberg HF. Identification of a purine-rich intronic enhancer element in the mouse eosinophil-associated ribonuclease 2 (mEar 2) gene. Mamm Genome 2004; 15:126-34. [PMID: 15058383 DOI: 10.1007/s00335-003-2304-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
The Mus musculus eosinophil-associated ribonuclease (mEar) gene cluster includes multiple distinct coding sequences that are highly divergent orthologs of the human eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3). We present a transcriptional analysis of the gene encoding mEar 2, the only member of this cluster with a well-defined expression profile. In this work, we demonstrate that the presence of non-coding exon 1 and the intron in tandem with a 361-bp 5' promoter of mEar 2 results in enhanced reporter gene expression, as much as 6-to 10-fold over the activity observed with the 5' promoter alone. We have identified a conserved purine-rich element in the intron of the mEar 2 gene that is necessary for maximum transcription and that interacts specifically with NFAT-binding proteins in nuclear extracts derived from the mouse LA4 epithelial cell line. Similar intronic enhancers have been described as regulating transcription of the human EDN gene, suggesting an overall conservation of an important regulatory strategy.
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Affiliation(s)
- Kimberly D Dyer
- Eosinophil Pathophysiology Section, LHD, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
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37
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Yang D, Rosenberg HF, Chen Q, Dyer KD, Kurosaka K, Oppenheim JJ. Eosinophil-derived neurotoxin (EDN), an antimicrobial protein with chemotactic activities for dendritic cells. Blood 2003; 102:3396-403. [PMID: 12855582 DOI: 10.1182/blood-2003-01-0151] [Citation(s) in RCA: 120] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Recent publications have highlighted the chemotactic activities of antimicrobial proteins derived from the granules of neutrophils and basophils. Eosinophil granules also contain antimicrobial proteins. One of them is eosinophil-derived neurotoxin (EDN), a protein belonging to the ribonuclease A (RNase A) superfamily, which has recently been found to have antiviral activity in vitro. We found that EDN was selectively chemotactic for dendritic cells (DCs). The DC chemotactic activity of EDN was inhibited by either pretreatment of DCs with pertussis toxin or by simultaneous addition of placental RNase inhibitor to inhibit the activity of EDN. EDN was not chemotactic for leukocytes other than DCs. Mouse eosinophil-associated RNase 2 (mEAR2), one of a cluster of divergent orthologs of human EDN, was also chemotactic for human as well as mouse DCs. Sequence and mutational analysis demonstrated the importance of the N-terminal region of mEAR2 in mediating its chemotactic effect on DCs. EDN also induced the activation of p42/44 mitogen-activated protein kinase (MAPK) in DCs. Furthermore, injection of mEAR2 into the air pouches of mice resulted in the recruitment of DCs into the air pouches. Thus, EDN and its mouse ortholog, mEAR2, are eosinophil granule-derived antimicrobial RNases that function as chemoattractants for DCs in vitro and in vivo.
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Affiliation(s)
- De Yang
- Basic Research Program, SAIC-Frederick, Laboratory of Molecular Regulation, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg 560, Rm 31-19, Frederick, MD 21702-1201, USA.
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38
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Ishihara K, Asai K, Nakajima M, Mue S, Ohuchi K. Preparation of recombinant rat eosinophil-associated ribonuclease-1 and -2 and analysis of their biological activities. BIOCHIMICA ET BIOPHYSICA ACTA 2003; 1638:164-72. [PMID: 12853122 DOI: 10.1016/s0925-4439(03)00077-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.
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Affiliation(s)
- Kenji Ishihara
- Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan
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39
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Kumar K, Jenkins JL, Jardine AM, Shapiro R. Inhibition of mammalian ribonucleases by endogenous adenosine dinucleotides. Biochem Biophys Res Commun 2003; 300:81-6. [PMID: 12480524 DOI: 10.1016/s0006-291x(02)02800-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The most potent low molecular weight inhibitors of pancreatic RNase superfamily enzymes reported to date are synthetic derivatives of adenosine 5(')-pyrophosphate. Here we have investigated the effects of six natural nucleotides that also incorporate this moiety (NADP(+), NADPH, ATP, Ap(3)A, Ap(4)A, and Ap(5)A) on the activities of RNase A and two of its homologues, eosinophil-derived neurotoxin and angiogenin. With eosinophil-derived neurotoxin and angiogenin, Ap(5)A is comparable to the tightest binding inhibitors identified previously (K(i) values at pH 5.9 are 370 nM and 100 microM, respectively); it ranks among the strongest small antagonists of RNase A as well (K(i)=230 nM). The K(i) for NADPH with angiogenin is similar to that of Ap(5)A. These findings suggest that Ap(5)A and NADPH may serve as useful new leads for inhibitor design. Examination of inhibition under physiological conditions indicates that NADPH, ATP, and Ap(5)A may suppress intracellular RNase activity significantly in vivo.
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Affiliation(s)
- Kapil Kumar
- Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, One Kendall Square, Building 600, Third Floor, Cambridge, MA 02139, USA
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40
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Niini T, Vettenranta K, Hollmén J, Larramendy ML, Aalto Y, Wikman H, Nagy B, Seppänen JK, Ferrer Salvador A, Mannila H, Saarinen-Pihkala UM, Knuutila S. Expression of myeloid-specific genes in childhood acute lymphoblastic leukemia - a cDNA array study. Leukemia 2002; 16:2213-21. [PMID: 12399964 DOI: 10.1038/sj.leu.2402685] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2002] [Accepted: 05/31/2002] [Indexed: 11/09/2022]
Abstract
Several specific cytogenetic changes are known to be associated with childhood acute lymphoblastic leukemia (ALL), and many of them are important prognostic factors for the disease. Little is known, however, about the changes in gene expression in ALL. Recently, the development of cDNA array technology has enabled the study of expression of hundreds to thousands of genes in a single experiment. We used the cDNA array method to study the gene expression profiles of 17 children with precursor-B ALL. Normal B cells from adenoids were used as reference material. We discuss the 25 genes that were most over-expressed compared to the reference. These included four genes that are normally expressed only in the myeloid lineages of the hematopoietic cells: RNASE2, GCSFR, PRTN3 and CLC. We also detected over-expression of S100A12, expressed in nerve cells but also in myeloid cells. In addition to the myeloid-specific genes, other over-expressed genes included AML1, LCP2 and FGF6. In conclusion, our study revealed novel information about gene expression in childhood ALL. The data obtained may contribute to further studies of the pathogenesis and prognosis of childhood ALL.
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Affiliation(s)
- T Niini
- Department of Pathology, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Finland
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41
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Harder J, Schroder JM. RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin. J Biol Chem 2002; 277:46779-84. [PMID: 12244054 DOI: 10.1074/jbc.m207587200] [Citation(s) in RCA: 318] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We analyzed healthy human skin for the presence of endogenous antimicrobial proteins that might explain the unusually high resistance of human skin against infections. A novel 14.5-kDa antimicrobial ribonuclease, termed RNase 7, was isolated from skin-derived stratum corneum. RNase 7 exhibited potent ribonuclease activity and thus may contribute to the well known ribonuclease activity of human skin. RNase 7 revealed broad spectrum antimicrobial activity against many pathogenic microorganisms and remarkably potent activity (lethal dose of 90% < 30 nm) against a vancomycin-resistant Enterococcus faecium. Molecular cloning from skin-derived primary keratinocytes and purification of RNase 7 from supernatants of cultured primary keratinocytes indicate that keratinocytes represent the major cellular source in skin and that RNase 7 is secreted. RNase 7 mRNA expression was detected in various epithelial tissues including skin, respiratory tract, genitourinary tract, and at a low level, in the gut. In addition to a constitutive expression, RNase 7 mRNA was induced in cultured primary keratinocytes by interleukin-1beta, interferon-gamma, and bacterial challenge. This is the first report demonstrating RNases as a novel class of epithelial inducible antimicrobial proteins, which may play an important role in the innate immune defense system of human epithelia.
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Affiliation(s)
- Jurgen Harder
- Clinical Research Unit, Department of Dermatology, University Hospital Kiel, Schittenhelmstrasse 7, D-24105 Kiel, Germany
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42
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Tang PC, Huang HC, Wang SC, Jeng JC, Liao YD. Regulation of ribonuclease expression by estradiol in Rana catesbeiana (Bullfrog). Nucleic Acids Res 2002; 30:3286-93. [PMID: 12136111 PMCID: PMC135762 DOI: 10.1093/nar/gkf442] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.
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Affiliation(s)
- Pin-Chi Tang
- Institute of Biomedical Sciences, Academia Sinica, 128, Yen-Chiu-Yuan Road, Sec. 2, Taipei 115, Taiwan
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43
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Maeda T, Kitazoe M, Tada H, de Llorens R, Salomon DS, Ueda M, Yamada H, Seno M. Growth inhibition of mammalian cells by eosinophil cationic protein. EUROPEAN JOURNAL OF BIOCHEMISTRY 2002; 269:307-16. [PMID: 11784325 DOI: 10.1046/j.0014-2956.2001.02653.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC(50) values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were estimated to be approximately 1-5 microm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.
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Affiliation(s)
- Takashi Maeda
- Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology,Okayama University, Japan
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44
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Sugihara R, Kumamoto T, Ito T, Ueyama H, Toyoshima I, Tsuda T. Human muscle protein degradation in vitro by eosinophil cationic protein (ECP). Muscle Nerve 2001; 24:1627-34. [PMID: 11745972 DOI: 10.1002/mus.1198] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
To clarify the role of tissue eosinophils in and around inflammatory foci, we purified eosinophil cationic protein (ECP) and examined its effect on muscle protein degradation in vitro. Eosinophil cationic protein was purified from the buffy coat of blood from healthy volunteers. Myofibrillar, soluble sarcoplasmic, and membrane-associated cytoskeletal proteins were fractionated from latissimus dorsi muscle obtained by orthopedic procedures done on a patient with no neurologic abnormalities. After incubation of these fractions with purified ECP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed. Eosinophil cationic protein degraded the myofibrillar proteins, especially the myosin heavy chain (MHC) and alpha-actinin. It also degraded membrane-associated cytoskeletal proteins dystrophin and spectrin, whereas soluble sarcoplasmic proteins did not undergo proteolysis. Quantitative analysis of the MHC degradation showed that the ECP reaction was dose-dependent and that the optimal pH was 7.0. Protein degradation was not inhibited by heparin or the protease inhibitors leupeptin, E-64, and pepstatin A. Our results suggest that ECP functions in the degradation of myofibrillar and membrane-associated cytoskeletal proteins, indicating that tissue eosinophils have a specific role in muscle fiber degradation in some myopathies associated with numerous tissue eosinophils, such as eosinophilic myositis, eosinophilic myalgia syndrome, and eosinophilic endocardial disease.
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Affiliation(s)
- R Sugihara
- Third Department of Internal Medicine, Oita Medical University, Hasama 1-1, Oita 879-5593, Japan
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45
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Rosenberg HF, Domachowske JB. Eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens. J Leukoc Biol 2001. [DOI: 10.1189/jlb.70.5.691] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Affiliation(s)
- Helene F. Rosenberg
- Eosinophil Biology Unit, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; and
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46
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Bousquet J, Van Cauwenberge P, Khaltaev N. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol 2001; 108:S147-334. [PMID: 11707753 DOI: 10.1067/mai.2001.118891] [Citation(s) in RCA: 2121] [Impact Index Per Article: 88.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- J Bousquet
- Department of Allergy and Respiratory Diseases, University Hospital and INSERM, Montpellier, France
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47
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Holloway DE, Hares MC, Shapiro R, Subramanian V, Acharya KR. High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins. Protein Expr Purif 2001; 22:307-17. [PMID: 11437607 DOI: 10.1006/prep.2001.1434] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Angiogenin (Ang) is a small basic protein which belongs to the pancreatic ribonuclease superfamily. It potently induces the formation of new blood vessels and has emerged as a promising anticancer target. Mice possess genes encoding one ortholog (mAng) and three homologs of Ang, designated angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3), and angiogenin-4 (mAng-4). Structural and functional study of these homologs has been hampered by the low yield of protein from the existing heterologous expression system. In the experiments described, we used a pET expression vector to express these proteins in the cytoplasm of Escherichia coli BL21-CodonPlus(DE3)-RIL cells, whereupon substantial amounts of each accumulated in the form of insoluble aggregates. The proteins were renatured using an arginine-assisted procedure and subsequently purified by cation-exchange chromatography and reversed-phase HPLC; each purified protein was shown to be enzymatically active toward tRNA. The yields of pure mAngrp and mAng-3 were 7.6 and 12 mg/liter culture, respectively, representing substantial increases over previously reported experiments. This is also the first report of the expression and purification of mAng-4, obtained here in a yield of 30 mg/liter culture. The ready availability of milligram quantities of these proteins will enable further functional studies and high-resolution structural analyses to be conducted.
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Affiliation(s)
- D E Holloway
- Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom
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48
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Leonidas DD, Boix E, Prill R, Suzuki M, Turton R, Minson K, Swaminathan GJ, Youle RJ, Acharya KR. Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN). High resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors. J Biol Chem 2001; 276:15009-17. [PMID: 11154698 DOI: 10.1074/jbc.m010585200] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic RNase A family. Although its physiological function is still unclear, it has been shown that EDN is a neurotoxin capable of inducing the Gordon phenomenon in rabbits. EDN is also a potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now determined high resolution (1.8 A) crystal structures for EDN in complex with adenosine-3',5'-diphosphate (3',5'-ADP), adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate refined at 1.6 A. The inhibition constant of these mononucleotides for EDN has been determined. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They also provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN.
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Affiliation(s)
- D D Leonidas
- Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
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49
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McDevitt AL, Deming MS, Rosenberg HF, Dyer KD. Gene structure and enzymatic activity of mouse eosinophil-associated ribonuclease 2. Gene 2001; 267:23-30. [PMID: 11311552 DOI: 10.1016/s0378-1119(01)00392-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Mouse eosinophil-associated ribonuclease-2 (mEAR-2) is one of a cluster of genes identified in the genome of the mouse Mus musculus that are highly divergent orthologs of the primate ribonucleases, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). Northern analysis revealed expression of genes hybridizing to mEAR-2 in mouse lung, liver and spleen tissues. We obtained full-length cDNA by hybridization screening of mouse eosinophil and lung cDNA libraries and by rapid amplification of cDNA ends (RACE) from liver, spleen and lung RNA. Using these methods we have isolated the 195 base pair (bp) 3' untranslated region (UTR) that includes a typical polyadenylation signal preceding a poly A tail and the 5' UTR which includes 63-71 bp and three distinct transcriptional start sites. Using unidirectional PCR we isolated a 361-bp 5' promoter region and delineated the intronic / exonic boundaries which include a non-coding exon 1, a single intron, and a coding exon 2, a structure that is typical of genes of the RNase A superfamily. Consensus sites for PU.1 and EoTF, both active as intronic enhancer elements of the gene encoding EDN, are also present in the intron of the gene encoding mEAR-2. The catalytic activity of recombinant baculovirus-derived mEAR-2 is similar to that of rhEDN from this source, with catalytic constants k(cat)/K(m)=5.6x10(6) M(-1) s(-1) and 10.5x10(6) M(-1) s(-1), respectively, against a standard yeast tRNA substrate. Sequence analysis of the non-coding regions and enzymatic characterization of the gene product provide further evidence indicating that mEAR-2 is a structural and functional ortholog of primate EDNs and ECPs.
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Affiliation(s)
- A L McDevitt
- Eosinophil Biology Unit, Laboratory of Host Defenses 10/11N104, NIAID / National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1886, USA
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50
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Liao YD, Huang HC, Leu YJ, Wei CW, Tang PC, Wang SC. Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog). Nucleic Acids Res 2000; 28:4097-104. [PMID: 11058105 PMCID: PMC113159 DOI: 10.1093/nar/28.21.4097] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2000] [Revised: 09/20/2000] [Accepted: 09/20/2000] [Indexed: 11/14/2022] Open
Abstract
Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.
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Affiliation(s)
- Y D Liao
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
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