A Human Interleukin 3 Analog with Increased Biological and Binding Activities
Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first α-helix, [Ala21,Leu22]IL-3 and [...
Gespeichert in:
| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1992-12, Vol.89 (24), p.11842-11846 |
|---|---|
| Hauptverfasser: | , , , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 11846 |
|---|---|
| container_issue | 24 |
| container_start_page | 11842 |
| container_title | Proceedings of the National Academy of Sciences - PNAS |
| container_volume | 89 |
| creator | Lopez, A. F. Shannon, M. F. Barry, S. Phillips, J. A. Cambareri, B. Dottore, M. Simmons, P. Vadas, M. A. |
| description | Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first α-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted α-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the α chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists. |
| doi_str_mv | 10.1073/pnas.89.24.11842 |
| format | Article |
| fullrecord | <record><control><sourceid>jstor_proqu</sourceid><recordid>TN_cdi_proquest_miscellaneous_16385465</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><jstor_id>2360812</jstor_id><sourcerecordid>2360812</sourcerecordid><originalsourceid>FETCH-LOGICAL-c554t-80cf5f0a2b7d62b4f6e28304b6226cf2f8e3810c498d537ca509414b19215ce63</originalsourceid><addsrcrecordid>eNp9kc1vEzEQxS0EKiFw5wBihRDismH8uV6JS1oBrVTEBc6W12unDhtvansL_Pd1SAiFA5Ily_N-zzOah9BTDAsMDX27DTotZLsgbIGxZOQemmFocS1YC_fRDIA0dSmzh-hRSmsAaLmEE3SCmeAM5Ax9Wlbn00aH6iJkGwc7ffOhotUy6GFcVd99viqKiVYn21enfixVb_RQ6bB7ht6HVbU02d_47G16jB44PST75HDP0dcP77-cndeXnz9enC0va8M5y7UE47gDTbqmF6RjTlgiKbBOECKMI05aKjEY1sqe08ZoDi3DrMMtwdxYQefo3f7f7dRtbG9syFEPahv9RsefatRe_a0Ef6VW443iIDgt9tcHexyvJ5uy2vhk7DDoYMcpKSyo5GVDBXz5D7gep1h2kxQBTASm5cwR7CETx5Sidcc5MKhdSmqXkpKtIkz9SqlYnt-d_49hH0vRXx10ncq2XdTB-HTEGJcNlk3B3hywXYPf6p1Gyk3DkO2PXNAX_0cL8WxPrFMe4xEhVIDEhN4CeH-7kQ</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>201261361</pqid></control><display><type>article</type><title>A Human Interleukin 3 Analog with Increased Biological and Binding Activities</title><source>Jstor Complete Legacy</source><source>MEDLINE</source><source>PubMed Central</source><source>Alma/SFX Local Collection</source><source>Free Full-Text Journals in Chemistry</source><creator>Lopez, A. F. ; Shannon, M. F. ; Barry, S. ; Phillips, J. A. ; Cambareri, B. ; Dottore, M. ; Simmons, P. ; Vadas, M. A.</creator><creatorcontrib>Lopez, A. F. ; Shannon, M. F. ; Barry, S. ; Phillips, J. A. ; Cambareri, B. ; Dottore, M. ; Simmons, P. ; Vadas, M. A.</creatorcontrib><description>Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first α-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted α-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the α chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.89.24.11842</identifier><identifier>PMID: 1465408</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Amino Acid Sequence ; Amino acids ; Antineoplastic drugs. Cytokines ; Basophils ; Binding, Competitive ; Biochemistry ; Biological and medical sciences ; Biotechnology ; Blood ; Bone marrow cells ; Cell Adhesion - drug effects ; Cell Division - drug effects ; Cell growth ; Cell lines ; Cellular biology ; Endothelial cells ; Fundamental and applied biological sciences. Psychology ; Health. Pharmaceutical industry ; Histamine release ; Humans ; In Vitro Techniques ; Industrial applications and implications. Economical aspects ; Interleukin-3 - analogs & derivatives ; Interleukin-3 - chemistry ; Interleukin-3 - pharmacology ; Interleukins ; Molecular Sequence Data ; Monocytes ; Monocytes - cytology ; Mutagenesis, Site-Directed ; Production of active biomolecules ; Receptors ; Receptors, Interleukin-3 - metabolism ; Structure-Activity Relationship</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1992-12, Vol.89 (24), p.11842-11846</ispartof><rights>Copyright 1992 The National Academy of Sciences of the United States of America</rights><rights>1993 INIST-CNRS</rights><rights>Copyright National Academy of Sciences Dec 15, 1992</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c554t-80cf5f0a2b7d62b4f6e28304b6226cf2f8e3810c498d537ca509414b19215ce63</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/89/24.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/2360812$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/2360812$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27903,27904,53769,53771,57995,58228</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4587187$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1465408$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lopez, A. F.</creatorcontrib><creatorcontrib>Shannon, M. F.</creatorcontrib><creatorcontrib>Barry, S.</creatorcontrib><creatorcontrib>Phillips, J. A.</creatorcontrib><creatorcontrib>Cambareri, B.</creatorcontrib><creatorcontrib>Dottore, M.</creatorcontrib><creatorcontrib>Simmons, P.</creatorcontrib><creatorcontrib>Vadas, M. A.</creatorcontrib><title>A Human Interleukin 3 Analog with Increased Biological and Binding Activities</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first α-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted α-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the α chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Antineoplastic drugs. Cytokines</subject><subject>Basophils</subject><subject>Binding, Competitive</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Blood</subject><subject>Bone marrow cells</subject><subject>Cell Adhesion - drug effects</subject><subject>Cell Division - drug effects</subject><subject>Cell growth</subject><subject>Cell lines</subject><subject>Cellular biology</subject><subject>Endothelial cells</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Health. Pharmaceutical industry</subject><subject>Histamine release</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Industrial applications and implications. Economical aspects</subject><subject>Interleukin-3 - analogs & derivatives</subject><subject>Interleukin-3 - chemistry</subject><subject>Interleukin-3 - pharmacology</subject><subject>Interleukins</subject><subject>Molecular Sequence Data</subject><subject>Monocytes</subject><subject>Monocytes - cytology</subject><subject>Mutagenesis, Site-Directed</subject><subject>Production of active biomolecules</subject><subject>Receptors</subject><subject>Receptors, Interleukin-3 - metabolism</subject><subject>Structure-Activity Relationship</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc1vEzEQxS0EKiFw5wBihRDismH8uV6JS1oBrVTEBc6W12unDhtvansL_Pd1SAiFA5Ily_N-zzOah9BTDAsMDX27DTotZLsgbIGxZOQemmFocS1YC_fRDIA0dSmzh-hRSmsAaLmEE3SCmeAM5Ax9Wlbn00aH6iJkGwc7ffOhotUy6GFcVd99viqKiVYn21enfixVb_RQ6bB7ht6HVbU02d_47G16jB44PST75HDP0dcP77-cndeXnz9enC0va8M5y7UE47gDTbqmF6RjTlgiKbBOECKMI05aKjEY1sqe08ZoDi3DrMMtwdxYQefo3f7f7dRtbG9syFEPahv9RsefatRe_a0Ef6VW443iIDgt9tcHexyvJ5uy2vhk7DDoYMcpKSyo5GVDBXz5D7gep1h2kxQBTASm5cwR7CETx5Sidcc5MKhdSmqXkpKtIkz9SqlYnt-d_49hH0vRXx10ncq2XdTB-HTEGJcNlk3B3hywXYPf6p1Gyk3DkO2PXNAX_0cL8WxPrFMe4xEhVIDEhN4CeH-7kQ</recordid><startdate>19921215</startdate><enddate>19921215</enddate><creator>Lopez, A. F.</creator><creator>Shannon, M. F.</creator><creator>Barry, S.</creator><creator>Phillips, J. A.</creator><creator>Cambareri, B.</creator><creator>Dottore, M.</creator><creator>Simmons, P.</creator><creator>Vadas, M. A.</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>19921215</creationdate><title>A Human Interleukin 3 Analog with Increased Biological and Binding Activities</title><author>Lopez, A. F. ; Shannon, M. F. ; Barry, S. ; Phillips, J. A. ; Cambareri, B. ; Dottore, M. ; Simmons, P. ; Vadas, M. A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c554t-80cf5f0a2b7d62b4f6e28304b6226cf2f8e3810c498d537ca509414b19215ce63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Antineoplastic drugs. Cytokines</topic><topic>Basophils</topic><topic>Binding, Competitive</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Blood</topic><topic>Bone marrow cells</topic><topic>Cell Adhesion - drug effects</topic><topic>Cell Division - drug effects</topic><topic>Cell growth</topic><topic>Cell lines</topic><topic>Cellular biology</topic><topic>Endothelial cells</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Health. Pharmaceutical industry</topic><topic>Histamine release</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Industrial applications and implications. Economical aspects</topic><topic>Interleukin-3 - analogs & derivatives</topic><topic>Interleukin-3 - chemistry</topic><topic>Interleukin-3 - pharmacology</topic><topic>Interleukins</topic><topic>Molecular Sequence Data</topic><topic>Monocytes</topic><topic>Monocytes - cytology</topic><topic>Mutagenesis, Site-Directed</topic><topic>Production of active biomolecules</topic><topic>Receptors</topic><topic>Receptors, Interleukin-3 - metabolism</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lopez, A. F.</creatorcontrib><creatorcontrib>Shannon, M. F.</creatorcontrib><creatorcontrib>Barry, S.</creatorcontrib><creatorcontrib>Phillips, J. A.</creatorcontrib><creatorcontrib>Cambareri, B.</creatorcontrib><creatorcontrib>Dottore, M.</creatorcontrib><creatorcontrib>Simmons, P.</creatorcontrib><creatorcontrib>Vadas, M. A.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lopez, A. F.</au><au>Shannon, M. F.</au><au>Barry, S.</au><au>Phillips, J. A.</au><au>Cambareri, B.</au><au>Dottore, M.</au><au>Simmons, P.</au><au>Vadas, M. A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Human Interleukin 3 Analog with Increased Biological and Binding Activities</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1992-12-15</date><risdate>1992</risdate><volume>89</volume><issue>24</issue><spage>11842</spage><epage>11846</epage><pages>11842-11846</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first α-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted α-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the α chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>1465408</pmid><doi>10.1073/pnas.89.24.11842</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0027-8424 |
| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1992-12, Vol.89 (24), p.11842-11846 |
| issn | 0027-8424 1091-6490 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_16385465 |
| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence Amino acids Antineoplastic drugs. Cytokines Basophils Binding, Competitive Biochemistry Biological and medical sciences Biotechnology Blood Bone marrow cells Cell Adhesion - drug effects Cell Division - drug effects Cell growth Cell lines Cellular biology Endothelial cells Fundamental and applied biological sciences. Psychology Health. Pharmaceutical industry Histamine release Humans In Vitro Techniques Industrial applications and implications. Economical aspects Interleukin-3 - analogs & derivatives Interleukin-3 - chemistry Interleukin-3 - pharmacology Interleukins Molecular Sequence Data Monocytes Monocytes - cytology Mutagenesis, Site-Directed Production of active biomolecules Receptors Receptors, Interleukin-3 - metabolism Structure-Activity Relationship |
| title | A Human Interleukin 3 Analog with Increased Biological and Binding Activities |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-27T11%3A11%3A23IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-jstor_proqu&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20Human%20Interleukin%203%20Analog%20with%20Increased%20Biological%20and%20Binding%20Activities&rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20-%20PNAS&rft.au=Lopez,%20A.%20F.&rft.date=1992-12-15&rft.volume=89&rft.issue=24&rft.spage=11842&rft.epage=11846&rft.pages=11842-11846&rft.issn=0027-8424&rft.eissn=1091-6490&rft.coden=PNASA6&rft_id=info:doi/10.1073/pnas.89.24.11842&rft_dat=%3Cjstor_proqu%3E2360812%3C/jstor_proqu%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=201261361&rft_id=info:pmid/1465408&rft_jstor_id=2360812&rfr_iscdi=true |