AMERICAN
`ASSOCIATION FOR THE'
`ADVANCEMENT OF
`SCIENCE
`
`•
`CIENCE
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`24- OCTOBER 1986
`VOL. 234 n PAGES 401-516
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`Ex. 1091 - Page 1
`
`(cid:9)
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`

`AMERICAN
`ASSOCIATION FOle•THETti,,
`ADVANCEMENT 011
`SCIENCE
`
`ARY
`
`OFFIC
`
`MAR 2.i
`
`IENCE
`
`24 OCTOBER 1986
`VOL. 234 • PAGES 401-516
`
`$2.50
`
`Ex. 1091 - Page 2
`
`

`

`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`SCIENCE ISSN 0036-8075
`
`24 OCTOBER 1986
`VOLUME 234
`NUMBER 4775
`
`407 (cid:9)
`
`This Week in Science
`
`Editorial
`Letters
`
`409 (cid:9)
`
`411 (cid:9)
`
`Spanking, Reason, and the Environment
`
`International Peace Week: C. C. PRICE • Water Diversion in the Soviet Union:
`P. P. MICKLIN • Galileo and the Catholic Church: 0. GINGERICH • Vitamins,
`Fiber, and Cancer: B. KALLmAN • Hazardous Waste Disposal: S. L. DANIELS •
`Mystery Cloud: Additional Observations: D. L. MCKENNA and D. A. WALKER
`
`News & Comment
`
`Research News
`
`Articles
`
`415 (cid:9)
`
`Sex and Needles, Not Insects and Pigs, Spread AIDS in Florida Town
`French R&D: a la Reagan with Dash of De Gaulle
`417 (cid:9)
`418 (cid:9) Woburn Case May Spark Explosion of Lawsuits
`Briefing: Science Agencies Fare Well in Budget Battles • Education Secretary
`420 (cid:9)
`Uses Harvard Podium to Take Host to Task • Hanford Plant Closed Over
`Safety Violation • Hoechst Tests Lead EPA to Ban Herbicide • Panel
`Questions Shuttle Flight Rate
`
`423 (cid:9)
`
`424 (cid:9)
`
`427 (cid:9)
`
`New Class of Animal Virus Found in Virulent Form of Human Hepatitis
`
`An Optical Measurement of Berry's Phase
`
`Shaping New Tools for Paleoceanographers
`
`433 (cid:9)
`
`Crustal Structure of Yunnan Province, People's Republic of China, from Seismic
`Refraction Profiles: R.-J. KAN, H.-X. Hu, R.-S. ZENG, W. D. MOONEY,
`T. V. MCEVILLY
`The trans Golgi Network: Sorting at the Exit Site of the Golgi Complex:
`G. GRIFFITHS and K. SIMONS
`443 (cid:9) What Has Happened to Productivity Growth?: M. N. BAILY
`
`438 (cid:9)
`
`Research Articles
`
`451 (cid:9)
`
`Saturation Mutagenesis of the Yeast his3 Regulatory Site: Requirements for
`Transcriptional Induction and for Binding by GCN4 Activator Protein:
`D. E. HILL, I. A. HOPE, J. P. MACKE, K. STRUHL
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`SCIENCE, VOL. 234
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`404
`
`Ex. 1091 - Page 3
`
`

`

`Mature sporangium of Rhinosporidium seeberi discharging endospores
`COVER (cid:9)
`through a single pore. The organisms were present in a large polyp located in the
`external nares of a dog. Mature sporangia are approximately 100 to 400
`micrometers in diameter and have relatively thin walls. They contain endospores
`that may be in various stages of maturation. This section is 1 micrometer in
`thickness and stained with toluidine blue. See page 474. [Donald J. Meuten,
`School of Veterinary Science, North Carolina State University, Raleigh, NC
`27606]
`
`459
`461 (cid:9)
`
`464 (cid:9)
`
`467 (cid:9)
`
`470 (cid:9)
`
`474 (cid:9)
`
`476 (cid:9)
`
`479 (cid:9)
`
`483 (cid:9)
`
`490 (cid:9)
`
`494 (cid:9)
`
`10% •
`8,01w 0 (cid:9)
`
`s: R. M. HABERLE
`Interannual Variability of Global D (cid:9)
`Gap Junctional Conductance and Permeability Are Linearly Related:
`V. VERSELIS, R. L. WHITE, D. C. SPRAY, M. V. L. BENNETT
`Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens:
`A. M. LLOYD, A. R. BARNASON, S. G. ROGERS, M. C. BYRNE, R. T. FRALEY,
`R. B. HORSCH
`Participation of c-myc Protein in DNA Synthesis of Human Cells:
`G. P. STUDZINSKI, Z. S. BRELVI, S. C. FELDMAN, R. A. WATT
`Shock and Tissue Injury Induced by Recombinant Human Cachectin:
`K. J. TRACEY, B. BEUTLER, S. F. LOVVRY, J. MERRYWEATHER, S. WOLPE,
`I. W. MILSARK, R. J. HARIRI, T. J. FAHEY III, A. ZENTELLA et al.
`Cultivation of Rhinosporidium seeberi in Vitro: Interaction with Epithelial Cells:
`M. G. LEVY, D. J. MEUTEN, E. B. BREITSCHWERDT
`Human Monoclonals from Antigen-Specific Selection of B Lymphocytes and
`Transformation by EBV: P. CASALI, G. INGHIRAMI, M. NAKAMURA,
`T. F. DAVIES, A. L. NOTKINS
`Synchronized Rearrangement of T-Cell y and 13 Chain Genes in Fetal Thymocyte
`Development: W. BoRN, G. RATHBUN, P. TUCKER, P. MARRACK, J. KAPPLER
`
`Science and Security: The Future of Arms Control: Program • Advance Registration
`Form
`
`The Positive Sum Strategy, reviewed by R. C. LEVIN • Circulation, Respiration,
`and Metabolism, S. C. WOOD • Cell Motility, D. R. BURGESS • Books Received
`
`Expert System for Experimental Design • Microprocessor-Monitored Photometer
`• Kit for RNA Synthesis • Digital Oscilloscope Peripheral • Pyrolytic Analyzers
`• Literature
`
`Editorial Board
`David Baltimore
`William F. Brinkman
`Ansley J. Coale
`Joseph L. Goldstein
`James D. Idol, Jr.
`Leon Knopoff
`Seymour Lipset
`Walter Massey
`Oliver E. Nelson
`Allen Newell
`Ruth Patrick
`David V. Ragone
`Vera C. Rubin
`Howard E. Simmons
`Solomon H. Snyder
`Robert M. Solow
`
`Board of Reviewing
`Editors
`Oais Al-Awqati
`James P. Allison
`Luis W. Alvarez
`Don L Anderson
`C. Paul Bianchi
`Elizabeth H. Blackburn
`Floyd E. Bloom
`Charles R. Cantor
`James H. Clark
`Bruce F. Eldridge
`Stanley Falkow
`Theodore H. Geballe
`Roger I. M. Glass
`
`Stephen P. Goff
`Robert B. Goldberg
`Patricia S. Goldman-Rakic
`Corey S. Goodman
`Richard M. Held
`Gloria Heppner
`Eric F. Johnson
`Konrad B. Krauskopf
`Karl L Magleby
`Joseph B. Martin
`John C. McGiff
`Alton Meister
`Mortimer Mishkin
`Peter Olson
`Gordon H. Orians
`John S. Pearse
`Yeshayau Pocker
`Jean Paul Revel
`
`Frederic M. Richards
`James E. Rothman
`Thomas C. Schelling
`Ronald H. Schwartz
`Stephen M. Schwartz
`Otto T. Solbrig
`Robert T. N. Tjian
`Virginia Trimble
`Geerat J. Vermeij
`Martin G. Weigert
`Harold Weintraub
`Irving L. Weissman
`George M. Whitesides
`Owen N. Witte
`William B. Wood
`Harriet Zuckerman
`
`TABLE OF CONTENTS +05
`
`, (cid:9)
`
`e.
`
`Reports
`
`AAAS Meetings
`
`Book Reviews
`
`Products & Materials
`
`Board of Directors
`Gerard Piel
`Retiring President,
`Chairman
`Lawrence Bogorad
`President
`Sheila E. Widnall
`President-elect
`
`Robert McC. Adams
`Robert W. Berliner
`Floyd E. Bloom
`Mary E. Clutter
`Mildred S. Dresselhaus
`Donald N. Langenberg
`Dorothy Nelkin
`Linda S. Wilson
`William T. Golden
`Treasurer
`William D. Carey
`Executive Officer
`
`24 OCTOBER 1986
`
`Ex. 1091 - Page 4
`
`

`

`SCIENCE
`
`;i***•%•-
`-ft•t ft•-•••• •
`. (cid:9)
`••16.1" •
`Filber.1000
`[14111, 6. fa• (cid:9)
`4140
`4/0 , , ••••
`
`•
`
`ssk
`
`AAAS Meetings
`
`Book Reviews
`
`Mature sporangium of Rhinosporidium seeberi discharging endospores
`COVER (cid:9)
`through a single pore. The organisms were present in a large polyp located in the
`external nares of a dog. Mature sporangia are approximately 100 to 400
`micrometers in diameter and have relatively thin walls. They contain endospores
`that may be in various stages of maturation. This section is 1 micrometer in
`thickness and stained with toluidine blue. See page 474. [Donald J. Meuten,
`School of Veterinary Science, North Carolina§tate University, Raleigh, NC
`..„
`v,3Cr CO7,
`27606] (cid:9)
`•;;Ls
`OCT 2 SB5
`
`cv
`
`s: R. M. HABERLE
`Interannual Variability of Global I (cid:9)
`Gap Junctional Conductance and Permeability Are Linearly Related:
`V. VERSELIS, R. L. WHITE, D. C. SPRAY, M. V. L. BENNETT
`Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens:
`A. M. LLOYD, A. R. BARNASON, S. G. ROGERS, M. C. BYRNE, R. T. FRALEY,
`R. B. HORSCH
`Participation of c-myc Protein in DNA Synthesis of Human Cells:
`G. P. STUDZINSKI, Z S. BRELVI, S. C FELDMAN, R. A. WATT
`Shock and Tissue Injury Induced by Recombinant Human Cachectin:
`K. J. TRACEY, B. BEUTLER, S. F. LOWRY, J. MERRYWEATHER, S. WOLPE,
`I. W. MILSARK, R. J. HARIRI, T. J. FAHEY III, A. ZENTELLA et al.
`Cultivation of Rhinosporidium seeberi in Vitro: Interaction with Epithelial Cells:
`M. G. LEVY, D. J. MEUTEN, E B. BREITSCHWERDT
`Human Monoclonals from Antigen-Specific Selection of B Lymphocytes and
`Transformation by EBV: P. CASALI, G. INGHIRAMI, M. NAKAMURA,
`T. F. DAVIES, A. L. NorioNs
`Synchronized Rearrangement of T-Cell y and 0 Chain Genes in Fetal Thymocyte
`Development:. W. BORN, G. RATHBUN, P. TUCKER, P. MARRACIC, J. KAFFLER
`
`Science and Security: The Future of Arms Control: Program • Advance Registration
`Form
`
`459 (cid:9)
`461 (cid:9)
`
`464 (cid:9)
`
`467 (cid:9)
`
`470 (cid:9)
`
`474 (cid:9)
`
`476 (cid:9)
`
`479 (cid:9)
`
`483 (cid:9)
`
`490. The Positive Sum Strategy, reviewed by R. C. LEVIN • Circulation, Respiration,
`and Metabolism, S. C. WOOD • Cell Motility, D. R. BURGESS • Books Received
`
`Products & Materials
`
`494 (cid:9)
`
`Expert System for Experimental Design • Microprocessor-Monitored Photometer
`• Kit for RNA Synthesis • Digital Oscilloscope Peripheral • Pyrolytic Analyzers
`• Literature
`
`Board of Directors (cid:9)
`Gerard Piel
`Retiring President,
`Chairman
`Lawrence Bogorad (cid:9)
`President (cid:9)
`Sheila E. Widnall
`President-elect
`
`Robert McC. Adams
`Robert W. Berliner
`Floyd E. Bloom
`Mary E. Clutter
`Mildred S. Dresselhaus
`Donald N. Langenberg
`Dorothy Nelkin
`Linda S. Wilson
`William T. Golden
`Treasurer
`William D. Carey
`Executive Officer
`
`Editorial Board
`David Baltimore
`William F. Brinkman
`Ansley J. Coale
`Joseph L Goldstein
`James D. Idol, Jr.
`Leon Knopoll
`Seymour Lipset
`Walter Massey
`Oliver E. Nelson
`Allen Newell
`Ruth Patrick
`David V. Ragone
`Vera C. Rubin
`Howard E. Simmons
`Solomon H. Snyder
`Robert M. Solow
`
`Board of Reviewing (cid:9)
`Editors (cid:9)
`Oais Al-Awqati
`• James P. Allison
`Luis W. Alvarez
`Don L Anderson
`C. Paul Bianchi
`Elizabeth H. Blackburn
`Floyd E. Bloom
`Charles R. Cantor
`James H. Clark
`Bruce F. Eldridge
`Stanley Falkow
`Theodore H. Geballe
`Roger I. M. Glass
`
`Stephen P. Goff
`Robert B. Goldberg
`Patricia S. Goldman-Rakic
`Corey S. Goodman
`Richard M. Held
`Gloria Heppner
`Erie F. Johnson
`Konrad B. Krauskopf
`Karl L Magleby
`Joseph B. Martin
`John C. McGill
`Mon Meister
`Mortimer Mishkin
`Peter Olson
`Gordon H. Orians
`John S. Pearse
`Yeshayau Pocker
`Jean Paul Revel
`
`Frederic M. Richards
`James E. Rothman
`Thomas C. Schelling '
`Ronald H. Schwartz
`Stephen M. Schwartz
`Otto T. Solbrig
`Robert T. N. Tjian
`Virginia Trimble
`Geerat J. Vermeij
`Martin G. Weigert
`Harold Weintraub
`Irving L. Weissman
`George M. Whitesides
`Owen N. Witte .
`William B. Wood
`Harriet Zuckerman
`
`24 OCTOBER 1986
`
`TABLE OF CONTENTS 405
`
`Ex. 1091 - Page 5
`
`(cid:9)
`(cid:9)
`

`

`SCIENCE
`
`•
`
`Mature sporangium of Rhinosporidium seeberi discharging endospores
`COVER (cid:9)
`through a single pore. The organisms were present in a large polyp located in the
`external tares of a dog. Mature sporangia are approximately 100 to 400
`micrometers in diameter and have relatively thin walls. They contain endospores
`that may be in various stages of maturation. This section is 1 micrometer in
`thickness and stained with toluidine blue. See page 474. [Donald J. Meuten,
`School of Veterinary Science, North Carolina §tate University, Raleigh, NC
`msVCFCc3J,c,
`27606]
`t9ts)
`
`Reports
`
`AAAS Meetings
`
`Book Reviews
`
`Products & Materials
`
`OCT
`
`et
`
`
`
`459 (cid:9)
`461 (cid:9)
`
`464 (cid:9)
`
`467 (cid:9)
`
`470 (cid:9)
`
`474 (cid:9)
`
`476 (cid:9)
`
`479 (cid:9)
`
`483 (cid:9)
`
`490 (cid:9)
`
`494 (cid:9)
`
`s: R. M. HABERLE
`Interannual Variability of Global
`Gap Junctional Conductance and Permeability Are Linearly Related:
`V. VERSELIS, R. L. WHITE, D. C. SPRAY, M. V. L. BENNETT
`Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens:
`A. M. LLOYD, A. R. BARNASON, S. G. ROGERS, M. C. BYRNE, R. T. FRALEY,
`R. B. HORSCH
`Participation of c-myc Protein in DNA Synthesis of Human Cells:
`G. P. STUDZINSKI, Z. S. BRELVI, S. C. FELDMAN, R. A. WATT
`Shock and Tissue Injury Induced by Recombinant Human Cachectin:
`K. J. TRACEY, B. BEUTLER, S. F. LOWRY, J. MERRYWEATHER, S. WOLPE,
`I. W. MILSARIC, R. J. HARIRI, T. J. FAHEY III, A. ZENTELIA et al.
`Cultivation of Rhinosporidium seeberi in Vitro: Interaction with Epithelial Cells:
`M. G. LEVY, D. J. MEUTEN, E. B. BREITSCHWERDT
`Human Monoclonals from Antigen-Specific Selection of B Lymphocytes and
`Transformation by EBV: P. CASALI, G. INGHIRAMI, M. NAKAMURA,
`T. F. DAVIES, A. L. NOTKINS
`Synchronized Rearrangement of T-Cell y and 13 Chain Genes in Fetal Thymocyte
`Development: W. BORN, G. RATHBUN, P. TUCKER, P. MARRACK, J. KAPPLER
`
`Science and Security: The Future of Arms Control: Program • Advance Registration
`Form
`
`The Positive Sum Strategy, reviewed by R. C. LEVIN • Circulation, Respiration,
`and Metabolism, S. C. WOOD • Cell Motility, D. R. BURGESS • Books Received
`
`Expert System for Experimental Design • Microprocessor-Monitored Photometer
`• Kit for RNA Synthesis • Digital Oscilloscope Peripheral • Pyrolytic Analyzers
`• Literature
`
`Board of Directors
`Gerard Piel
`Retiring President,
`Chairman
`Lawrence Bogorad
`President
`Sheila E. Widnall
`President-elect
`
`Robert McC. Adams
`Robert W. Berliner
`Floyd E. Bloom
`Mary E. Clutter
`Mildred S. Dresselhaus
`Donald N. Langenberg
`Dorothy Nelkin
`Linda S. Wilson
`William T. Golden
`Treasurer
`William D. Carey
`Executive Officer
`
`Editorial Board
`David Baltimore
`William F. Brinkman
`Ansley J. Coale
`Joseph L. Goldstein
`James D. Idol, Jr.
`Leon Knopoff
`Seymour Lipset
`Walter Massey
`Oliver E. Nelson
`Allen Newell
`Ruth Patrick
`David V. Ragone
`Vera C. Rubin
`Howard E. Simmons
`Solomon H. Snyder
`Robert M. Solow
`
`Board of Reviewing
`Editors
`Oais AI-Awqati
`James P. Allison
`Luis W. Alvarez
`Don L. Anderson
`C. Paul Bianchi
`Elizabeth H. Blackburn
`Floyd E. Bloom
`Charles R. Cantor
`James H. Clark
`Bruce F. Eldridge
`Stanley Falkow
`Theodore H. Geballe
`Roger I. M. Glass
`
`Stephen P. Goff
`Robert B. Goldberg
`Patricia S. Goldman-Rakic
`Corey S. Goodman
`Richard M. Held
`Gloria Heppner
`Eric F. Johnson
`Konrad B. Krauskopf
`Karl L. Magleby
`Joseph B. Martin
`John C. McGill
`Alton Meister
`Mortimer Mishkin
`Peter Olson
`Gordon H. Orians
`John S. Pearse
`Yeshayau Pocker
`Jean Paul Revel
`
`Frederic M. Richards
`James E. Rothman
`Thomas C. Schelling
`Ronald H. Schwartz
`Stephen M. Schwartz
`Otto T. Solbrig
`Robert T. N. 'Dan
`Virginia Trimble
`Geerat J. Vermeij
`Martin G. Weigert
`Harold Weintraub
`Irving L. Weissman
`George M. Whitesides
`Owen N. Witte
`William B. Wood
`Harriet Zuckerman
`
`24 OCTOBER 1986
`
`TABLE OF CONTENTS 405
`
`Ex. 1091 - Page 6
`
`(cid:9)
`

`

`8. R. A. Watt, A. R. Shatzman, M. Rosenberg, Mal.
`Cell. Biol. 5, 448 (1985).
`9. N. Sullivan, C. Green, M. Pasdar, R. Watt. Curr.
`Top. Microbial. Immunol., in press. (cid:9)
`10. W. M. LeStourgeon, A. Forer, Y-Z Yang, J. S.
`Bertram, H. P. Rush, Biochim. Biophys. Acta 379, (cid:9)
`529 (1975).
`11. U. Sheer, H. Hinssen, W. W. Franke, B. M. Jock-
`usch, Cell 39, 111 (1984).
`12. S. Ikegami et al., Nature (London) 275, 458 (1978). (cid:9)
`13. J. A. Huberman, Cell 23, 647 (1981).
`14. R. H. Reeder and R. G. Roeder, J. Mol. Bid. 67, (cid:9)
`433 (1972).
`15. R. Dalla-Favera, F. Wong-Staal, R. C. Gallo, Nature
`(London) 299, 61 (1982).
`16. E. H. Westin et al., Proc. Natl. Acad. Sci. USA. 79, (cid:9)
`2490 (1982).
`17. The amount of c-myc protein in 2 x 10s nuclei was (cid:9)
`estimated by immunoblot to be approximately 10 ng (cid:9)
`(Fig. 3B, lane 1). Therefore, in the DNA synthesis
`
`assay, approximately 100 ng of myc protein from
`2 x 106 nuclei was incubated with 800 ng of anti-
`body—an approximately tenfold excess.
`G. S. McKnight and R. D. Palmiter, J. Bid. Chem.
`254, 9050 (1979).
`S. Seki and G. C. Mueller, Biochim. Biophys. Acta
`435, 236 (1976); G. P. V. Reddy and A. P. Pardee,
`Proc. Natl. Acad. Sci. U.S.A. 77, 3312 (1980); B. W.
`Stillman, Cell 35, 7 (1983).
`F. A. Baglia and G. G. Maul, Proc. Natl. Acad. Sci.
`USA. 80, 2285 (1983).
`P. Donner, I. Greiser-Wilke, K. Moelling, Nature
`(London) 296, 262 (1982); S. R. Hann, H. D.
`Abrams, L. R. Rochrschneider, R. N. Eisenman,
`Cell 34, 789 (1983).
`L. Kaczmarek, J. K. Hyland, R. A. Watt, M.
`Rosenberg, R. Baserga, Science 228, 1313 (1985).
`N. Sullivan et al., in preparation.
`G. P. Snidzinski, A. K. Bhandal, Z. S. Brelvi, Proc.
`Soc. Exp. Bid. Med. 179, 288 (1985).
`
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`24.
`
`25. Z. S. Brelvi and G. P. Studzinski, J. Cell Bid. 102,
`2234 (1986).
`26. T. Maniatis, E. F. Fritsch, J. Sambrook, Molecular
`Cloning: A Laboratory Manual (Cold Spring Harbor
`Laboratory, Cold Spring Harbor, NY, 1982).
`27. P. L. Ey, S. J. Prowse, C. R. Jenkin, Immunochemis-
`try 15, 429 (1978).
`28. We thank A. Bhandal and J. Dong for excellent
`technical assistance, M. Pasdar for monoclonal anti-
`bodies B3 and F5, B. Lubit for the anti-actin, R.
`Weinmann for the anti—RNA polymerase II, A.
`Alama for the anti—DNA polymerase a, N. Ponzio
`for the conventional rabbit antisera, J. Shipman and
`P. Chambers for secretarial assistance, and the New
`Jersey State Commission on Cancer Research for
`financial support (to G.P.S.).
`
`29 May 1986; accepted 15 August 1986
`
`Shock and Tissue Injury Induced by Recombinant
`Human Cachectin
`
`KEVIN J. TRACEY, BRUCE BEUTLER,* STEPHEN F. LOWRY,
`JAMES MERRYWEATHER, STEPHEN WOLPE, IAN W. MILSARK,
`ROBERT J. HARIRI, THOMAS J. FAHEY III, ALEJANDRO ZENTELLA,
`JAMES D. ALBERT, G. TOM SHIRES, ANTHONY CERAMI
`
`Cachectin (tumor necrosis factor), a protein produced in large quantities by endotox-
`in-activated macrophages, has been implicated as an important mediator of the lethal
`effect of endotoxin. Recombinant human cachectin was infused into rats in an effort to
`determine whether cachectin, by itself, can elicit the derangements of host physiology
`caused by administration of endotoxin. When administered in quantities similar to
`those produced endogenously in response to endotoxin, cachectin causes hypotension,
`metabolic acidosis, hemoconcentration, and death within minutes to hours, as a result
`of respiratory arrest. Hyperglycemia and hyperkalemia were also observed after
`infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent
`on gross and histopathologic examination, along with ischemic and hemorrhagic
`lesions of the gastrointestinal tract, and acute renal tubular necrosis. Thus, it appears
`that a single protein mediator (cachectin) is capable of inducing many of the
`deleterious effects of endotoxin.
`
`closely related, endotoxin-sensitive strain
`C3H/HeN (9). Similarly, irradiated C3H/
`HeN mice reconstituted with C3H/HeJ
`marrow are endotoxin resistant (9). Thus,
`a host factor expressed by cells of hemato-
`poietic origin appears to confer endotoxin
`sensitivity.
`The macrophage appears to be the princi-
`pal cell involved in mediating the effects of
`endotoxin. Infectious agents capable of
`stimulating reticuloendothelial hyperplasia
`render animals exquisitely sensitive to the
`effects of endotoxin (10). Moreover, macro-
`phages activated by endotoxin in vitro pro-
`duce a soluble factor that is capable of killing
`endotoxin-resistant animals (11).
`Cachectin (12, 13) is a macrophage-de-
`rived polypeptide hormone known for its
`ability to modulate adipocyte metabolism
`(12, 14), lyse tumor cells in vitro (15, 16),
`and induce hemorrhagic necrosis of certain
`transplantable tumors in vivo (15). It consti-
`tutes between 1 and 2% of the total secre-
`
`B ACTERIAL ENDOTOXIN (LIPOPOLY-
`
`saccharide, LPS) is highly toxic to
`most mammals. When administered
`intravenously, it evokes a "shock" state,
`characterized by fever, hypotension, and
`multi—organ system failure (1-5). Fatality is
`associated with injuries involving the lungs
`(shock lung syndrome), kidneys (acute tu-
`bular necrosis), and gastrointestinal tract
`(mesenteric ischemia) (1, 2, 4, 6). This
`syndrome frequently occurs in the course of
`invasive Gram-negative infections and is as-
`sociated with a high mortality (2, 5, 7).
`Recently, it has become clear that endo-
`toxin does not injure host tissues directly,
`but does so through the action of an endog-
`enous mediator or mediators. C3H/HeJ
`mice, which are highly resistant to the lethal
`effect of endotoxin as the result of a genetic
`lesion (the /psd allele on chromosome 4) (8),
`are rendered endotoxin-sensitive if subjected
`to total body irradiation and transplanted
`with marrow obtained from mice of the
`
`470
`
`tory protein produced by endotoxin-activat-
`ed macrophages in vitro (12). Copious
`quantities of the protein are also produced
`in vivo in response to endotoxin. Approxi-
`mately 5.5 mg of cachectin are present per
`liter of rabbit serum 1.5 hours after endo-
`toxin administration (17). With consider-
`ation given to the half-life of the hormone in
`vivo (18), this would suggest the net pro-
`duction of milligram quantities of cachectin
`per kilogram of body mass.
`Recently, Beutler et al. (19) suggested
`that cachectin itself might initiate the tissue
`injury elicited by endotoxin. This suggestion
`was based on the observation that animals
`passively immunized against cachectin were
`protected against the lethal effect of endo-
`toxin (19). However, direct analysis of ca-
`chectin as a critical mediator of organ injury
`in sepsis required the isolation of large
`amounts of purified hormone, devoid of
`pharmacologically active quantities of endo-
`toxin.
`Accordingly, an artificial gene, encoding
`the amino acid sequence of cachectin, was
`constructed by means of an Applied Biosys-
`tems model 380A gene synthesizer. The
`gene was synthesized in 25 overlapping
`segments, which were annealed and ligated
`in a single reaction. Codons were chosen so
`as to optimize expression of the protein
`product in yeast. At the 5' end, additional
`codons were added to specify the signal
`sequence of yeast a-factor, in order to ob-
`tain a secreted product (20). The synthetic
`
`K. J. Tracey, S. F. Lowry, R. J. Hariri, T. J. Fahey III, J.
`D. Albert, G. T. Shires, Department of Surgery, New
`York Hospital—Cornell Medical Center, New York, NY
`10021.
`B. Beutler, S. Wolpe, I. W. Milsark, A. Zentella, A.
`Cerami, Laboratory of Medical Biochemistry, Rockefel-
`ler University, New York, NY 10021.
`J. Merryweather, Chiron Research Laboratories, Emery-
`ville, CA 94608.
`
`*To whom correspondence should be addressed at How-
`ard Hughes Medical Institute, University of Texas
`Health Science Center at Dallas, 5323 Harry Hines
`Boulevard, Dallas, TX 75235.
`
`SCIENCE, VOL. 234
`
`Ex. 1091 - Page 7
`
`

`

`— 200,000
`
`— 97,400
`
`— 68,000
`
`— 43,000
`
`— 25,000
`
`— 18,400
`
`— 14,300
`
`Fig. 1. Recombinant human cachectin, purified as
`described in the text, was subjected to electropho-
`resis in a 10 to 15% polyacrylamide gel under
`denaturing conditions and was stained with Coo-
`massie blue. Molecular weights are indicated.
`
`sequence was placed downstream from a
`fused alcohol dehydrogenase/glyceralde-
`hyde-phosphate dehydrogenase (ADH2/
`GAPDH) promoter.
`Cachectin biosynthesis and secretion was
`induced in cultures of transformed Saccharo-
`myces cerevisiae by glucose deprivation. Ap-
`proximately 30% of the secreted protein was
`
`cachectin. The sample was desalted by pas-
`sage over a Sephadex G-25 column equili-
`brated with 5 mM tris-chloride buffer, pH
`8.4. The protein-rich fraction was separated
`over a Mono-Q column, with a tris-chloride
`gradient, pH 8.4. Cachectin eluted as the
`major peak, at a concentration of 0.19M
`tris-chloride. The Mono-Q purified material
`(approximately 85% cachectin) was then
`mixed with an equal volume of 3.4M
`(NH4)2SO4 and separated further on a phe-
`nyl Superose column. Proteins were eluted
`by means of a declining salt gradient [1.7M
`to OM (NH4)2SO4 buffered with 50 mM
`Na-PO4, pH 7.0]. The endotoxin content of
`the sample was less than 0.4 jiz per milli-
`gram of protein, as assessed by Limulus
`amebocyte lysate tests. The final product
`(Fig. 1) was electrophoretically and chro-
`matographically homogeneous and behaved
`as a dimer on high-performance gel filtra-
`tion analysis.
`To establish the acute toxicity of cachec-
`tin, we infused the highly purified hormone
`into unanesthetized female Sprague-Dawley
`rats (225- to 250-g body weight) via the tail
`vein in various doses over 5 minutes (Table
`1). The animals were observed for 12 hours
`after infusion. Those animals that suc-
`cumbed to the effects of the infusion were
`necropsied immediately, and all survivors
`were killed for necropsy at the end of the
`experimental period. The animals receiving
`
`200
`
`A
`
`2
`
`-
`= • 0
`E
`
`160
`
`120
`
`co ▪ 2
`
`o .E 80
`en
`o
`• 0
`rQ
`
`40
`
`0 (cid:9)
`
`
`-1
`
`200
`
`160
`
`120
`
`80
`
`40
`
`E
`
`0. al 2
`
`o
`O
`I; 0 Q
`
`600
`
`400
`
`:r2
`
`200
`
`150
`
`120
`
`0
`0° 9
`a.
`
`
`
`E
`0 " 60
`0.
`
`30
`
`....
`
`7.6
`
`7.4
`
`7.2
`
`6.
`
`.7.0
`
`6.8
`
`0 — _1 0 1 2 3 4 5 6
`
`6.6
`
`I=1
`
`0 1 2 3 4 5 6
`
`0
`
`600
`
`150
`
`120 •
`
`4 ••••••.
`
`
`
`N
`0
`fa, 0 .-.. ry, 90
`
`.. i c E
`
`400 o (cid:9)
`1-3 (cid:9)
`
`E
`Yi ' N --"
`O
`0
`i Q.
`200
`
`Sm
`
`60 •
`
`30
`
`7.6
`
`7.4
`
`7.2
`
`7.0
`
`6.8
`
`a.
`
`0 (cid:9)
`
`3 (cid:9)
`2 (cid:9)
`1 (cid:9)
`Time (hours)
`
`4
`
`5
`
`6.6
`0'
`-1 0 1 2 3 4 5 6
`Time (hours)
`
`Fig. 2. Vital signs and arterial pH, P02, and PCO2 monitored in individual rats. Animals were
`carmulated at t = — 1 hour. Top (A and B), infusion of isotonic saline via the superior vena cava (open
`bars); bottom (A and B), infusion of 1.8 mg of cachectin per kilogram of body weight (solid bars). (A)
`(Line 1), heart rate (beats per minute); (line 2), systolic blood pressure (BP); (line 3), respiratory rate
`(respirations per minute). (B) (Line 4), pH; (line 5), P02; (line 6), PCO2.
`
`24 OCTOBER 1986
`
`Fig. 3. Gross appearance of the gastrointestinal
`tract in an animal that died 4 hours after cachectin
`infusion (1.8 mg per kilogram of body weight).
`
`doses in excess of 0.6 mg of cachectin per
`kilogram of body weight became lethargic
`within minutes after infusion. This was fol-
`lowed by piloerection without chills, bloody
`diarrhea, and tachypnea. The 12-hour mean
`lethal dose was estimated to be 0.7 mg of
`cachectin per kilogram of body weight by
`the method of Bliss and Litchfield (21).
`We studied several physiological parame-
`ters to further characterize the response to
`cachectin administration. Pentobarbital-
`anesthetized rats were monitored via arterial
`cannula before, during, and after cachectin
`infusion into the superior vena cava. Arterial
`glucose, Na+, K+, and hematocrit were
`recorded in animals receiving various doses
`of cachectin (Table 2). Vital signs, as well as
`arterial pH, P02, and PCO2, are compared
`graphically for experimental and control ani-
`mals (Fig. 2).
`Control animals (receiving no cachectin),
`maintained stable hemodynamic and vital
`functions over 6 hours of continuous moni-
`toring. Similarly, no significant changes
`were observed in any of the other measured
`parameters except for hematocrit, which fell
`as a result of serial blood sampling with
`volume replacement (cubic centimeter for
`cubic centimeter) with lactated Ringer's so-
`lution. This decline in hematocrit induced
`no significant changes in hemodynamic or
`respiratory function throughout the 6-hour
`study period.
`Rats infused with cachectin at low (0.2 to
`
`REPORTS 471
`
`Ex. 1091 - Page 8
`
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`0.6 mg/kg) doses exhibited mild to moder-
`ate tachypnea and hypotension followed by
`recovery. At higher doses, an overwhelming
`metabolic acidosis occurred, leading to a
`preterminal decline in arterial pH to 6.99 ±
`0.11 (SD; n = 10). Plasma lactate levels
`were elevated by a factor of 3 to 5 when
`compared to concentrations before infusion.
`Death occurred after respiratory arrest. The
`physiological derangements that occurred in
`a typical animal infused with cachectin are
`detailed in graphic form in Fig. 2.
`At necropsy, several gross abnormalities
`were readily apparent in cachectin-infused
`rats. Diffuse hyperemia and punctate areas
`of hemorrhage were evident in the lungs.
`Rats treated with cachectin in doses exceed-
`ing 0.6 mg/kg exhibited segmental ischemia
`of the bowel with regions of frank hemor-
`rhage or necrosis (Fig. 3); the cecum ap-
`peared particularly sensitive to the hormone,
`and was invariably infarcted. The kidneys
`were uniformly distended and hyperemic.
`The pancreas was edematous and contained
`regions of focal hemorrhage. Adrenal hem-
`orrhage was also observed in several ani-
`mals.
`
`Histopathologic examination was per-
`formed on tissues fixed with 10% formalde-
`hyde in phosphate-buffered saline. Sections
`obtained from areas of the lung (Fig. 4A)
`that were hemorrhagic in appearance re-
`vealed occlusion of large arteries by thrombi
`composed primarily of polymorphonuclear
`leukocytes. An intense margination response
`was observed, with migration of polymor-
`phonuclear cells through the walls of the
`pulmonary vessels. A severe interstitial and
`peribronchiolar pneumonitis was also ap-
`parent, with marked thickening of the alveo-
`lar membranes.
`Histological sections taken through non-
`necrotic regions of the intestinal tract (Fig.
`4B) also showed inflammatory changes,
`with invasion of the submucosa and muscu-
`laris mucosa by polymorphonuclear leuko-
`cytes. The epithelium was denuded in a focal
`distribution throughout the bowel.
`The renal glomeruli appeared mildly hy-
`percellular. In addition, acute tubular necro-
`sis was evident, with disruption of both
`proximal and distal elements of the nephron
`(Fig. 4C). In longitudinal section, numer-
`ous red cell casts, as well as occasional
`
`inflammatory cells, were seen within the
`tubules. Histological examination of rats
`infused with phosphate-buffered saline (the
`solvent in which cachectin was adminis-
`tered) revealed none of the changes charac-
`teristic of cachectin-treated rats (Fig. 4, D to
`F).
`The pathophysiological and histological
`findings reported here mimic changes
`evoked by administration of lethal doses of
`endotoxin (1-4, 6). The inflammatory pneu-
`monitis, mesenteric ischemia, and acute tu-
`bular necrosis characteristic of rats treated
`with cachectin are also observed in endotox-
`in-treated animals. The cannulation and in-
`fusion of the rats in these studies may have
`led to the introduction of small quantities of
`bacterial endotoxin or other contaminants.
`However, no detectable physiological or
`histopathologic changes occurred with en-
`dotoxin infused in doses as high as 10
`µg/kg. Moreover, preparations of cachectin
`that were boiled to destroy the biological
`activity of the hormone also failed to induce
`any changes.
`We obtained further evidence implicating
`cachectin as the toxic agent by administering
`
`Fig. 4. Histological appearance of tissues obtained from a single rat treated
`with cachectin at a dose of 1.8 mg per kilogram of body weight. Sections
`were stained with hematoxylin and eosin. (A) Section taken through a region
`of confluent hemorrhage observed on gross examination of