1992 Fiscal Year Final Research Report Summary
Structure and Functions of an IL-1 inhibitor found at inflammatory site in rabbit.
| Project/Area Number |
03454172
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Experimental pathology
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
YOSHINAGA Masaru Kumamoto University School of Medicine, Department of Pathology Professor, 医学部, 教授 (90040196)
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| Project Period (FY) |
1991 – 1992
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| Keywords | IL-1 / IL-1 receptor antagonist / Inflammation / Inflammatory exudate cells / neutrophils / Anti-inflammatory agent |
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| Research Abstract |
A rabbit interleukin-1 (IL-1) inhibitor in inflammatory peritoneal exudate cells was purified to apparent homogeneity. This inhibitor was extracted from exudate cells of the 24-hr stage of peritoneal inflammation and purified using isoelectrofocusing (IEF), gel filtration, followed in this order by high-performance liquid chromatography steps with hydroxylapatite and anionic ion exchanger. The purified factor showed a single band on silver-stained SDS-PAGE. This molecule of MW 19 kD and pI 5.5 inhibited the binding of both IL-1alpha and beta to receptors on a thymoma cell line, EL-4 and a B-cell line, 70Z/3. We determined its primary structure by a combination of peptide chemistry and molecular cloning. The inhibitor was synthesized as a precursor composed of 177 amino acids and was processed to a mature molecule of 143 amino acids. The N-terminal amino acid of the mature inhibitor was N-acetyl-methionine residue. The deduced amino acid sequence of the inhibitor showed a 77% homology t
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o the human IL-1 receptor antagonist (IL-1ra) and essentially the same mode of action as seen with human IL-1ra. We consider the this inhibitor is a rabbit counterpart of human IL-1ra, although there are differences with respect to the molecular structure; the N-terminus of the mature rabbit IL-1ra at a position of nine amino acids downstream from that of human IL-1ra.
Further, we made a recombinant rabbit IL-1ra (rrIL-1ra) expressed in E. coli. Using the rrIL-1ra, we also made a sheep anti-serum and developed an ELISA system for the rabbit IL-1ra. The rrIL-1ra was purified using DEAE-cellulose and Sephadex G-75, in a conventional chromatography system, followed by a linear NaCl gradient-elution of DEAE-cellulose in an FPLC system. Its specific biologic activity was almost the same as the authentic natural product. Using the above described tools, we examined the generation of IL-1ra during the course of LPS-arthritis in rabbits. Production of IL-1ra peaked at 9 hr (196.7 ng/joint) and the amount was 180-200 fold molar excess of IL-1 found at the lesion, and a large amount of IL-1ra was sustained for one week. The LPS-induced arthritis was strongly suppressed by intra-articular injection of 10 mug of IL-1ra when the severity of arthritis was monitored by the infiltration of leukocytes and destruction of articular cartilage. Then, we concluded that IL-1ra play an regulatory role in inflammation and may become a potent anti-inflammatory agent. Less
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[Publications] Goto F,Goto K,Miyata T,Ohkawara S.,Takao T,Mori S,Furukawa S,Maeda T,Iwanaga S,Shimonishi Y and Yoshinaga M: "Interlcukin-1 rcceplor antagonist in inflammatory cells of rabbits. Production,purification and detemination of promary structure." Immunology. 77. 235-244 (1992)
Description
「研究成果報告書概要(和文)」より
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[Publications] Goto F, Goto K, Miyata T, Ohkawara S., Takao T, Mori S, Furukawa S, Maeda T, Iwanaga S, Shimonishi Y and Yoshinaga M: "Interleukin-1 receptor antagonist in inflammatory exudate cells of rabbits. Production, purification and determination of primary structure." Immunology. 77. 235-244 (1992)
Description
「研究成果報告書概要(欧文)」より
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