Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers
A critical step in tissue and wound repair is the removal of eschar—accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activi...
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| Veröffentlicht in: | Analytical biochemistry 1989-10, Vol.182 (1), p.20-24 |
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| creator | Boswell, Glenda S. Dimitrijevich, S.Dan Gracy, Robert W. |
| description | A critical step in tissue and wound repair is the removal of eschar—accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered
in vivo. We have, therefore, developed an
in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([
3H]thymidine, [
14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations. |
| doi_str_mv | 10.1016/0003-2697(89)90711-2 |
| format | Article |
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in vivo. We have, therefore, developed an
in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([
3H]thymidine, [
14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/0003-2697(89)90711-2</identifier><identifier>PMID: 2532485</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>550201 - Biochemistry- Tracer Techniques ; AMINO ACIDS ; Analytical, structural and metabolic biochemistry ; AZINES ; BASIC BIOLOGICAL SCIENCES ; Biological and medical sciences ; BIOLOGICAL RECOVERY ; CARBON 14 COMPOUNDS ; Carbon Radioisotopes ; CARBOXYLIC ACIDS ; COAGULANTS ; Deoxyribonucleases - analysis ; DNA-ASE ; DOUBLE LABELLING ; DRUGS ; ENZYME ACTIVITY ; ENZYMES ; Enzymes and enzyme inhibitors ; ESTERASES ; FIBRIN ; Fibrinolysin - analysis ; fibroblasts ; Fibroblasts - metabolism ; Fundamental and applied biological sciences. Psychology ; General aspects, investigation methods ; HEALING ; HEMATOLOGIC AGENTS ; HEMOSTATICS ; HETEROCYCLIC COMPOUNDS ; Humans ; HYDROGEN COMPOUNDS ; HYDROLASES ; INJURIES ; ISOTOPE APPLICATIONS ; Kinetics ; LABELLED COMPOUNDS ; LABELLING ; LEUCINE ; Microchemistry - methods ; NUCLEOSIDES ; NUCLEOTIDES ; ORGANIC ACIDS ; ORGANIC COMPOUNDS ; ORGANIC NITROGEN COMPOUNDS ; PHOSPHODIESTERASES ; plasmin ; PROTEINS ; PYRIMIDINES ; RADIOASSAY ; RECOVERY ; RIBOSIDES ; SCLEROPROTEINS ; Skin - cytology ; THYMIDINE ; TRACER TECHNIQUES ; Tritium ; TRITIUM COMPOUNDS ; WOUNDS</subject><ispartof>Analytical biochemistry, 1989-10, Vol.182 (1), p.20-24</ispartof><rights>1989</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c444t-474a608327ea108f4a99eb66c03f0a365b3979b5846eb9814241b3c0b85315e83</citedby><cites>FETCH-LOGICAL-c444t-474a608327ea108f4a99eb66c03f0a365b3979b5846eb9814241b3c0b85315e83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0003-2697(89)90711-2$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,315,781,785,886,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6637716$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2532485$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/7105806$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Boswell, Glenda S.</creatorcontrib><creatorcontrib>Dimitrijevich, S.Dan</creatorcontrib><creatorcontrib>Gracy, Robert W.</creatorcontrib><title>Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A critical step in tissue and wound repair is the removal of eschar—accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered
in vivo. We have, therefore, developed an
in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([
3H]thymidine, [
14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.</description><subject>550201 - Biochemistry- Tracer Techniques</subject><subject>AMINO ACIDS</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>AZINES</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>Biological and medical sciences</subject><subject>BIOLOGICAL RECOVERY</subject><subject>CARBON 14 COMPOUNDS</subject><subject>Carbon Radioisotopes</subject><subject>CARBOXYLIC ACIDS</subject><subject>COAGULANTS</subject><subject>Deoxyribonucleases - analysis</subject><subject>DNA-ASE</subject><subject>DOUBLE LABELLING</subject><subject>DRUGS</subject><subject>ENZYME ACTIVITY</subject><subject>ENZYMES</subject><subject>Enzymes and enzyme inhibitors</subject><subject>ESTERASES</subject><subject>FIBRIN</subject><subject>Fibrinolysin - analysis</subject><subject>fibroblasts</subject><subject>Fibroblasts - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>General aspects, investigation methods</subject><subject>HEALING</subject><subject>HEMATOLOGIC AGENTS</subject><subject>HEMOSTATICS</subject><subject>HETEROCYCLIC COMPOUNDS</subject><subject>Humans</subject><subject>HYDROGEN COMPOUNDS</subject><subject>HYDROLASES</subject><subject>INJURIES</subject><subject>ISOTOPE APPLICATIONS</subject><subject>Kinetics</subject><subject>LABELLED COMPOUNDS</subject><subject>LABELLING</subject><subject>LEUCINE</subject><subject>Microchemistry - methods</subject><subject>NUCLEOSIDES</subject><subject>NUCLEOTIDES</subject><subject>ORGANIC ACIDS</subject><subject>ORGANIC COMPOUNDS</subject><subject>ORGANIC NITROGEN COMPOUNDS</subject><subject>PHOSPHODIESTERASES</subject><subject>plasmin</subject><subject>PROTEINS</subject><subject>PYRIMIDINES</subject><subject>RADIOASSAY</subject><subject>RECOVERY</subject><subject>RIBOSIDES</subject><subject>SCLEROPROTEINS</subject><subject>Skin - cytology</subject><subject>THYMIDINE</subject><subject>TRACER TECHNIQUES</subject><subject>Tritium</subject><subject>TRITIUM COMPOUNDS</subject><subject>WOUNDS</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU2LFDEQhoMo6-zqP1AIIqKH1konnY-LILt-waIH9WpI0tVupDsZk25h_73dzjBHPdWhnirqqZeQRwxeMmDyFQDwppVGPdfmhQHFWNPeITsGRjbAwdwluxNyn5zX-hOAMdHJM3LWdrwVutuR71_itIyzS5iXSl2t7pYOudD96OoUE3Wpp1efXEW61Jh-0OL6mEfnccSe3iyTS3SIvmS_8nOlOdEphpKDKyViqQ_IvcGNFR8e6wX59u7t18sPzfXn9x8v31w3QQgxN0IJJ0HzVqFjoAfhjEEvZQA-gOOy89wo4zstJHqjmWgF8zyA1x1nHWp-QZ4c9uY6R1tDnDHchJwShtkqBp0GuULPDtC-5F8L1tlOsQYcx4O9VUZAKzn8F2QdVwZas4LiAK7KtRYc7L7EyZVby8BuIdktAbslYLWxf0Oy7Tr2-Lh_8RP2p6FjKmv_6bHvanDjUFwKsZ4wKblSbPN5fcBw_ezv9d2bOKaAfSybd5_jv-_4A-HVrEg</recordid><startdate>19891001</startdate><enddate>19891001</enddate><creator>Boswell, Glenda S.</creator><creator>Dimitrijevich, S.Dan</creator><creator>Gracy, Robert W.</creator><general>Elsevier Inc</general><general>Elsevier</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>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M81</scope><scope>P64</scope><scope>7X8</scope><scope>OTOTI</scope></search><sort><creationdate>19891001</creationdate><title>Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers</title><author>Boswell, Glenda S. ; Dimitrijevich, S.Dan ; Gracy, Robert W.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c444t-474a608327ea108f4a99eb66c03f0a365b3979b5846eb9814241b3c0b85315e83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>550201 - Biochemistry- Tracer Techniques</topic><topic>AMINO ACIDS</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>AZINES</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>Biological and medical sciences</topic><topic>BIOLOGICAL RECOVERY</topic><topic>CARBON 14 COMPOUNDS</topic><topic>Carbon Radioisotopes</topic><topic>CARBOXYLIC ACIDS</topic><topic>COAGULANTS</topic><topic>Deoxyribonucleases - analysis</topic><topic>DNA-ASE</topic><topic>DOUBLE LABELLING</topic><topic>DRUGS</topic><topic>ENZYME ACTIVITY</topic><topic>ENZYMES</topic><topic>Enzymes and enzyme inhibitors</topic><topic>ESTERASES</topic><topic>FIBRIN</topic><topic>Fibrinolysin - analysis</topic><topic>fibroblasts</topic><topic>Fibroblasts - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>General aspects, investigation methods</topic><topic>HEALING</topic><topic>HEMATOLOGIC AGENTS</topic><topic>HEMOSTATICS</topic><topic>HETEROCYCLIC COMPOUNDS</topic><topic>Humans</topic><topic>HYDROGEN COMPOUNDS</topic><topic>HYDROLASES</topic><topic>INJURIES</topic><topic>ISOTOPE APPLICATIONS</topic><topic>Kinetics</topic><topic>LABELLED COMPOUNDS</topic><topic>LABELLING</topic><topic>LEUCINE</topic><topic>Microchemistry - methods</topic><topic>NUCLEOSIDES</topic><topic>NUCLEOTIDES</topic><topic>ORGANIC ACIDS</topic><topic>ORGANIC COMPOUNDS</topic><topic>ORGANIC NITROGEN COMPOUNDS</topic><topic>PHOSPHODIESTERASES</topic><topic>plasmin</topic><topic>PROTEINS</topic><topic>PYRIMIDINES</topic><topic>RADIOASSAY</topic><topic>RECOVERY</topic><topic>RIBOSIDES</topic><topic>SCLEROPROTEINS</topic><topic>Skin - cytology</topic><topic>THYMIDINE</topic><topic>TRACER TECHNIQUES</topic><topic>Tritium</topic><topic>TRITIUM COMPOUNDS</topic><topic>WOUNDS</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Boswell, Glenda S.</creatorcontrib><creatorcontrib>Dimitrijevich, S.Dan</creatorcontrib><creatorcontrib>Gracy, Robert W.</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Boswell, Glenda S.</au><au>Dimitrijevich, S.Dan</au><au>Gracy, Robert W.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1989-10-01</date><risdate>1989</risdate><volume>182</volume><issue>1</issue><spage>20</spage><epage>24</epage><pages>20-24</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>A critical step in tissue and wound repair is the removal of eschar—accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered
in vivo. We have, therefore, developed an
in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([
3H]thymidine, [
14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>2532485</pmid><doi>10.1016/0003-2697(89)90711-2</doi><tpages>5</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Analytical biochemistry, 1989-10, Vol.182 (1), p.20-24 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_osti_scitechconnect_7105806 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | 550201 - Biochemistry- Tracer Techniques AMINO ACIDS Analytical, structural and metabolic biochemistry AZINES BASIC BIOLOGICAL SCIENCES Biological and medical sciences BIOLOGICAL RECOVERY CARBON 14 COMPOUNDS Carbon Radioisotopes CARBOXYLIC ACIDS COAGULANTS Deoxyribonucleases - analysis DNA-ASE DOUBLE LABELLING DRUGS ENZYME ACTIVITY ENZYMES Enzymes and enzyme inhibitors ESTERASES FIBRIN Fibrinolysin - analysis fibroblasts Fibroblasts - metabolism Fundamental and applied biological sciences. Psychology General aspects, investigation methods HEALING HEMATOLOGIC AGENTS HEMOSTATICS HETEROCYCLIC COMPOUNDS Humans HYDROGEN COMPOUNDS HYDROLASES INJURIES ISOTOPE APPLICATIONS Kinetics LABELLED COMPOUNDS LABELLING LEUCINE Microchemistry - methods NUCLEOSIDES NUCLEOTIDES ORGANIC ACIDS ORGANIC COMPOUNDS ORGANIC NITROGEN COMPOUNDS PHOSPHODIESTERASES plasmin PROTEINS PYRIMIDINES RADIOASSAY RECOVERY RIBOSIDES SCLEROPROTEINS Skin - cytology THYMIDINE TRACER TECHNIQUES Tritium TRITIUM COMPOUNDS WOUNDS |
| title | Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers |
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