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`ILMN EXHIBIT 1016ILMN EXHIBIT 1016
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`Page 1 of 5
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`ILMN EXHIBIT 1016
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` .
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`. _ .
`‘rt’
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`.7 M lbitinns
`‘
`iiid pjninen-.oiii.iia'i‘sre."
`-- as C-
`'1"i--
`..
`
`NATURE'REPORT$'
`
`.
`
`..
`
`
`
`US high-energy physics faces painful
`‘peace dividend’ I Soviet scientists
`JESSI in jeopardy? I Two aPP.l'0aches to": 7‘ : j_.
`.
`.1
`Japanese patent disputes I A Swiss showpiedgih
`lransitlon I The Ukraine will shut Chernobirl
`3
`A cry for help from Yugoslavia
`? ‘f
`
`CORRESPONDENCE _ ‘NI ;..._;,-.
`
`No ‘plus’ from AIDS I Species inlrodutions
`I Astronomical costs.
`
`
`
`I
`
`
`
`.;'I_
`
`
`
`COMMENTARY
`
`Quenching the wild wells of Kuwait
`R L Garwin & H W Kendall
`
`NEWS AND VIEWS
`
`Metamorphosis of a British laboratory
`John Maddox
`
`Molecular neurobiology: NMDA receptors cloned
`at last
`
`Mark L Mayer
`Cosmology: Quark soup, do not boil
`M Fukugita & C J Hogan
`Developmental psychology: Face to face with babies
`P E Bryant
`-
`Gamma-ray bursts: Astronomy versus astrophysics
`David Lindlcy
`Fluid tlynaniics: Forging the missing link
`John D S Jones
`
`Viral proleases: Molecular metamorphosis
`Dagmar Ringo & Gregory A Petsko
`Evolution: Sex, slime and selfish genes
`Laurence D Hurst
`
`Daedalus: Arresting waste
`
`SCIENTIFIC CORRESPONDENCE
`
`Hazard from volcanic ash A J Praia. I J Barton,
`R W Johnson. K Kama & J Kingwell I Calnollflaged
`DNA B E Griffin
`
`.
`
`Out on in limb S A Newman; Reply — J M W Slack I
`Lead correction D Bellinger
`
`BOOK REVIEWS
`
`
`
`.‘
`
`_'
`
`__‘ "tr __
`
`.
`
`30!"-
`
` '
`
`.
`
`. THIS WEEK .
`
`.
`
`..
`
`Whale conservation
`Genetic
`sequences
`flanking
`simple sequence length poly-
`morphisms (SSLPS} in whales
`seem to have been conserved to
`a remarkably high degree in
`whales of all kinds. This could
`reflect
`unprececlentedly
`slow
`evolution.
`or
`that modern
`whales are a product of a rel-
`atively recent. explosive specia-
`tion event. This finding will en-
`able researchers to use stan-
`dard probes on many — if not
`all -— whale species. Page 63.
`
`Viral core
`The crystallographic structure of
`the core protein of Sindbls virus
`reveals
`a
`polypeptide
`fold
`homologous
`to
`that
`of
`chymotrypsin-like
`serine
`pro-
`teases. This explains why its
`proteoiytic activity is blocked af-
`ter autocleavage from the spike
`protein. and predicts a vlrion
`structure with T=-1» symmetry.
`This
`research has interesting
`ramifications for the design of
`antiviral drugs active against
`togavimses,
`some
`of which
`cause serious diseases such as
`encephalitis and arthritis. Pages
`37 and 22.
`
`Hat trick
`Ultraviolet light from the flash of
`the explosion that generated
`supernova SN19BTA is
`now
`travelling through the circum-
`steilar material surrounding the
`supernova.
`creating the
`so-
`calied Nap-o|eon’s hat emission
`nebula. A new model to explain
`the nebula's structure makes
`predictions for the development
`of the emission structure over
`the next few months. Page 43.
`
`Building a library
`On pages 82 and 84 two related
`new approaches are described
`for constructing libraries of pep-
`tides
`Including
`all
`possible
`sequences. which can be used
`to screen for ligands to recep-
`tors. new antimicrobial agents
`and other bioactive peptides.
`
`Guide to Authors
`Page 8?.
`
`A History of Geology by G Gohau Peter J Smith
`Wanderers in Space: Exploration and Discovery in -
`the Solar System by K R Long J: C A Whitney
`'”‘
`Clark R Chapman
`jg‘
`The Biology of the Naked Mole-Rat ed: 1'’ W Shanna" '
`J’ Jarvis & A Alexander Brian Bertram I
`'
`Trafficliing of Proteins eds CJ Sreer & J A
`John Armstrong
`The Solid Earth: An Introduction to Global
`I
`'
`by C M‘ R Fowler Leigh Roydcn‘
`Mninnillan
`.
`Ltd (-1 Little Esau Strcn. London \\"('.'2R
`'
`'_l'oI' USA aid
`093133.51 it
`(hard:
`_
`rue). U l35{indi'vidi.iii[ lI‘l.Iki.l!l| pcrsodcl payrncnt). USandCanadmi 0I15£rSl.0INl'll.ilN. 5%lD‘_iL_'fl[)llD|| Dept. PO B;ox‘1T13. Rm.-riou.
`_
`,
`_
`
`vi-eesfily on Thursday. slow! the last neck or December. by
`_
`_
`_
`_
`use.
`' Brunei Rand. Bnsinmoke. I-limit RG2! ZXS. UK. Second class postage paid at New York. NY 10012. and additional iiuiting olfiocii. iiturliormuon to phoiocopy ianncnal lorhilcrliul or
`. Other ordrim in
`Nai-are to lfbwarics and others
`registered with the Copyrl
`1. Clearance cenire ICCC) '!‘zsnni.-rlcinnl R:
`ning
`provided the has: ice of ll.l;l2l
`uocofspodfic clients. lsgriuilcd by
`_
`_
`' or
`lflapnpis
`paid direct in COL‘. 21 Congress Street. Salem. MA Ell9'i'iJ. USA. identification node or Name: tllzii-llfllffll SLIII + 50.10. US
`_
`_
`_
`Pglrnutci-scnd=rddreiIclia_:igcsldiNaiurc.c5
`. New York. NY lmlz. Puhticlied in Japan by Dianne Japan K.K.. Shirl-Mitsiikc Bldg. Jo lcliigsyii Tanuclu. Sliinnilru-lro. Tokyo I62. Japan 0 H91. Mnuiiilloo lolapniics Lid.
`U’
`
`7 Noiiember 1991
`Vol. 354 Issue no. 6348
`1 Astronauts aboard the space shuttie
`have described the curious phenomenon
`of ‘shuttle glow’ from around the shuttles
`tail-end. which faces into the oribltal wind
`as the shuttle circles the Earth. The cover
`shows a simulated shuttle giow caused by
`nitric oxide release from the shuttla's
`payload bay. part of an experiment to
`find an explanation for the phenomenon.
`Page 48.
`. . . THIS WEEK .
`
`
`
`3
`
`- Ive leamlng
`z.
`te is an important ex-
`neurotransmitter in the
`..
`--‘ nervous system that acts
`mber of different classes
`Dior. one of which. the
`receptor,
`is thought
`to
`a key role in processes
`ng learning and mem-
`page 31. Moriyoshi er
`it the cloning of a rat
`whose product functions
`'I.!. NMDA receptor, while on
`__"(O Kumar et al. describe a
`t
`clone
`encoding
`a
`is binding protein which
`-
`-_: present a subunit of an
`1-
`receptor complex. News
`ews page 16. on page 66..
`ng and expression of a
`in transporter from rat
`‘-is reported.
`
`.. Iar graphite
`type of carbon structure
`sting
`of
`needle-like
`ite rnicrotubules. made up
`al tubes of between 2
`0 graphite sheets.
`is de-
`-
`on page 56. These
`. are up to a few tens of
`etres in diameter. sug-
`:
`that
`carbon-based
`engineering
`at
`a
`Iar
`greater than that of the
`_ es is a possibility.
`
`.;i
`‘I
`
`.
`
`__
`
`doused
`weal-i's Commentary de-
`-
`the various proposals
`by scientists earlier this
`that contributed to putting
`the hundreds of fires in
`it's oil wells. The fires
`be under control by mid-
`ber. much earlier
`than
`ginal estimates. Page 11.
`
`drift
`to assessing future world
`i is an understanding of
`Antarctic ice sheet will
`"d to climate change. New
`e from a group of sites
`lites Land points to a re-
`large increase in snow
`atiori rates to the high-
`- is for 170 years. Intensi-
`dnic activity since 1958
`possible cause. Page 58.
`
`_
`
`'
`
`
`
`Page 2 of 5
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`Page 2 of 5
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`

`

`o 'lilA".'Ul:RE’
`
`" T
`
`libraries of longer or more diverse peptides should they be
`required for any given application.
`We have expanded the applications of our peptide library
`approach by modifying the synthesis procedure to incorporate
`cleavable linkers on each bead. After exposure to the cleaving
`agent. such beads can then release a portion of their peptides
`into solution for biological assay while still retaining sulficient
`peptides on the beads for subsequent structure determination.
`The one«bead, one-peptide concept and its applications dis-
`cussed above demonstrate that this approach provides important
`new tools with which to search for specific ligands of potential
`diagnostic or therapeutic value. Such information should also
`enhance fundamental understanding of interactions between
`ligands and acceptor molecules.
`U
`
`t-teceweu 30 May; accented mseetnnllier 1991.
`. Ruby. V. J.. Nvfltlieid. F. It Kazrnlnruti. W. Hdfidtll. J! 1. 24fi—$2 (19901.
`.
`lfulliy. lit. J. & $nan'ne.S.D. Rom CH”. 51015011 3. 599-905l'.1991l.
`. Geysen_H. M..Metven.R.l'l.ItB81i0lll'I.S. 1. PW: Mm tIGlIi‘.S€'llU.£-R. 81.3998-4lOCl2i19B4i.
`. Factor. 5. P el‘ at science 2|!-. 76T'7l3 l1991l.
`. Parmleir. S. F 8i 5i'rIi'.'h. G P. Eerie 1'3. H164-I18 {ISHL
`. Scott. J. K. at smith. 8. P. Science ace. 3ae—39u (1990:.
`(MII'|a. s. E. Peters, E. A..samnt.l1 lit. at um-er.w.1 Prue min .-‘iced Sci. usx. er.s31s—ess2
`l1990i.
`. Devlin]. J..Pang.Il'lll:|bl"i. L C. St Ddldlrt. P. E. Scheme‘ 33. ‘D4--ilfil195Ul.
`. Niall, H. D.. Tregeer. G. ‘N. d: $138.1. lfl C'hdl'I'It-sin’ QMBJOIHI‘ DI‘ Wntltks. led. Meieroofer. J.l.
`B95 lilm Arbor. hlidiiifil. 1972}.
`
`Amm We thorli F. Al-Coelda. M. Floss and R. l-tlrsehmarni for valudsic suuostiorts
`BnlIE.l.l'itletlol'txI1'itl'Idi'llof|l!2I'rlfl‘I.IIil:rlpt. K.SL i5i5DEGlU Fellow olllIeI.el.ltnt1'il.ISn:lntyol
`America. The DEBT-lde svnlrnsis rid scroenim process is referred to as the Selection Process and
`is the sthlodt of pntnftl. This work was supported by the National Institutes of i‘h9i1t'l_ the Arizona
`Disease Control Fllesnorott commission and the Selection Corporation.
`
`Generation and use of
`
`synthetic peptide
`combinatorial libraries
`
`for basic research and
`
`drug discovery
`
`Rich-d A. I-lnufiIItelI.—¢lernonc|n Plnlla,
`Sylvie E. Blondole, Jon n. Appol, Colette r. Dooley
`at Julio I-I. cuervo
`
`Torrey Pines Institute for Molecular Studies. 3550 General Atomics Court.
`San Diego, California 92121. LEA
`
`EXISTING methods for the synthesis and screening of large num-
`bers of peptides are limited by their inability to generate and
`screen the requisite number (millions) of individual peptides”
`andfor their inability to generate unmodified free peptides in
`quantities able to interact in soluIlon“". We have circumvented
`these limitations by developing synthetic peptide combinatorial
`libraries composed of mixtures of free peptides in quantities which
`can he used directly in virtually all existing assay systems. The
`screening of these heterogeneous libraries. along with an iterative
`selection and synthesis process. permits the systematic identi-
`fication of optimal peptide ligands. Stlrting with a library com-
`posed o!’ more than 34 million hex:-peptides, we present here the
`precise identification of an antigenic determinant recognized by a
`monoclonal antibody as well as the straightforward development
`of new potent antimicrobial peptides.
`library (SPCL)
`The initial synthetic peptide combinatorial
`prepared and used in this work consisted ofsix-residue peptide
`sequences with acetylated N terminals and emidatecl C ter~
`minals. The first two positions in each peptide were individually
`and specifically defined, whereas the last four positions consisted
`of equimolar mixtures of 18 of the 20 natural L-amino acids
`(for ease of synthesis, cysteine and tryptophan were omitted in
`
`this initial library). Such libraries can be generally repres
`by the sequence Ac-0,03XX)(X-NH; (where Ac -—:-'3'
`acetyl) (see legend to Fig. 1).
`:-
`Using a competitive enzyme-linked immunosorbent
`(ELISAJ, each ofthe 324 different peptide mixtures ofthe S
`(Ac-0l0,XXXX-NH1J was assayed to determine its abili "
`inhibit the interaction of a monoclonal antibody with a zg
`I3-residue peptide {Ac-YPYDVPDYASLRS-NH}; single- -
`amino-acid code). Of the 324 peptide mixtures examined '3
`ll. Ac~DVXXXX~Nl-lg caused the greatest inhibition of
`body binding [Table 1]. Twenty new peptide mixtures were 5
`synthesized in which the third position of the peptide <_i_
`Ac-DVXXXX-NI-lg was defined (Ac-DVOXXX-NI-I2,
`-g
`tophan now included in the X positions). Each new a
`'
`mixture contained 6,859 (193) individual peptides (137.1
`total). The most effective inhibiting peptide mixture was .
`DVPXXX—NH; (50% inhibitory concentration, lCm=4l -'
`Table lb]. The above iterative process, which reduces the I1
`ber of peptide sequences by 20-fold each time it
`is 2:?
`was then carried out for the remaining three positions (T
`1, C-9).
`It should be noted that on defining the fifth posi
`(Ac-DVPDOX-NH2, Table 1d},
`the
`IC,.,
`found for
`':_
`DVPDYX-NH: (0.38 pM) was at
`least 3,500-fold lower i‘
`any ofthe other 19 peptide mixtures. Also, the peptide mixt
`Ac-DVPDXX-NH: and Ac-DVPXXX-NH; had |C5o
`lower than all of the peptide mixtures with the fifth pas’
`defined, with the exception of Ac-DVPDYX-NH3. This -—-'
`
`TABLE 1
`
`Identification of the antigenic determinant recognized by
`clonal antibody 19810
`
`Peptide mixture
`la)
`Ac—Dvxxxx.NH2
`Ac-oixxxx.NH,
`Ac-oMx><x><.NH,
`Ac.oLxxxx.Ni-1,
`
`tel
`Ac—DvPxxx-Ni-i,
`Ac-DvExxx-NH,
`Ac-ovQxxx-NH,
`xi:-ovxxxx-ill,
`Ac-ovMxxx.Nl-l,
`Ac—Dvaxxx-NH,
`Ac—DvAxxx-NH,
`Ac-DVCXXX-Nl-t._,
`
`(cl
`Ac-DVPDXX -NH,
`Aisovrxxx-H-I,
`Ac-DVPAXX-NH2
`
`(oi
`Ac-D\i'PDYX—Nl-12
`no-ovnoxx-in-I,
`Ac-DVPDAX-Nhlg
`
`EC5‘,
`(phi;
`
`250
`313
`752
`>1.4oo
`
`41
`145
`215
`250
`451
`905
`1:0?
`>1.4oo
`
`4.4
`41
`>1.4oo
`
`0.33
`4.4
`> 1.400
`
`Peptlcie
`
`lei
`Ac.ovPDYA-NH,
`Ac-DVPDYS-NH;
`no-ovpovx-in-I,
`Ac.ovPovc.NH,
`itc.ovPi:wv-NH,
`Ac-ovrovr-NH,
`Ac-DVPDYG-NH:
`Ai:.ovPo~rE.NH,
`Ac.ovi=ovi.i-in,
`tic-ovpovm-NH,
`Ac.ovl>o~rQ-i-iii,
`Ac-DVPDYH-NH,
`Ac-DVPDYL-NH,
`Ac-DVPDYR-Ni-I2
`Ac-DVl3DYF-NI-I,
`Ac-ov‘PorN—i~iH,
`Ac-DVPDYK-NH,
`Ac-DVPDYY-NH,
`Ac.pvi=o~rl=.nH,
`Ac-DVPDYW-NH,
`Ac-DVPDYD-NH:
`
`:i*!"!"""9°S3‘.°
`§5mmm~m
`
`l-4.
`
`._—.._.,4._._._
`
`
`..'-e.."—‘.':'i.-.'_.
`
`:_
`The icws of the most effective inhibitory peptide mixtures obtai
`each iterative step are illustrated for: a peptide mixtures from the :'_.
`screening of the SPCL D. the third position defined {AC-D\t'0XXX.hll-la}:
`fourth position defined (Ac-DVPO>iX—NH,): ct the fifth position dcfined=__
`D\e‘PDO><~NH,): and e. the sixth position defined (Ac-DVPDYO-Ni-l,l. The
`of the peptide mixture derived from the previous iterative step is In I.
`for comparison. Peptide mixtures were assayed by competitive ELISA ._
`Fig. 1). The concentration of each peptide mixture necessary to inhibit ‘I
`of the antibody binding to the control peptide on the plate was obi
`serial dilutions of the peptide mixture. The titans were calculated usi
`soitware GRAPPPAD (ISL San Diego). The four-step iterative screeni
`synthesis process takes approximately 4 weeks. This time frame will
`depending on the assay being used and the number of cases moved i-
`at each iterative step.
`
`_
`1
`J‘
`
`NATIJRE - VOL 354 - 7 NOVEMBER
`
`
`
`Page 3 of 5
`
`

`

`arc-
`
`‘tsrrslis ro NATURE‘
`
` ...,__—_..
`
`Pseudomonas cremgirrosa (Gram-negative bacteria). and the
`yeast Candida albicans was examined in microdilution assays
`using the same set of 324 peptide mixtures making up the
`Ac-0.03XXXX-NH; peptide library used above. Although
`many antimicrobial lead peptide sequences derived from this
`SPCL have been followed in detail (manuscript in preparation),
`a single example (Ac-RRxxXx—NH;} found effective against
`the above microorganisms is presented here. Positions three to
`six of Ac-RRXXXX-NI-I2 were defined using the iterative pro-
`cess described above (the data for S. aureus are shown in Table
`2}. The minimum inhibitory concentrations (MIC) of the 20
`individual peptides obtained on defining the sixth position of
`Ac-RRWWCX-NHL as well as the C-terminal amide fornt of
`the naturally occurring antimicrobial peptide magairtitt, are
`shown in Table 3. The hexa-peptide Ac-RRWWCR-NH; was
`the most active of‘ this set.
`Its MIC against S. aureus was
`3.2-6.5 pg ml“. Preliminary data indicate that this peptide is
`bacteriocidal. The haemolytic activity of Ac-RRWWCR-NH;
`was less than 0.2% at 500 pg ml”.
`It
`is noteworthy that the
`antistaphylococcal activities of 17 of the 20 sequences were
`greater than rnagainin".
`The use of SPCLS has been illustrated here For both the precise
`identification of a linear antigenic determinant recognized by‘!
`monoclonal antibody and For the development of new, highly
`effective antimicrobial peptides. A number of other libraries,
`
`FIG. 1 Initial screening of the SPCL (Ac-D,02XXXX-NH2l for ability to inhibit
`the binding of monoclonal antibody 19B10. Each of the 324 peptide mixtures
`of the SPCL was assayed by competitive ELISA for its abitlty to inhibit the
`binding of monoclonal antibody 19310 (ref. 201 to the plate-adsorbed peptide
`Ac-YPYDVPDYASLRS-NH;,. The individual bar graphs are segregated by first
`amlno acid (01). with the individual bars in each graph representing the 18
`individual amino acids melting up the second position (0,). The y~axis
`represents optical density (OD) at £92 nm. The horizontal line in each bar
`graph represents the average CD of the 324 peptide mixtures. 01 and 0,
`are specific indivldual amino acids: that is G,02=AA. AD. AE. and so on
`through to W. W. for a total of 324 combinations at positions 01 and 0,
`i187}. Each X position represents an equimolar mixture of the 18 amino
`acids A. D. E. F. G. H, I, K. L. M, N, F’. Q, R. S. T. V. Y for a total of 10-€l.9if6
`combinations l18“l. Each of the 324 different peptide mixtures consists of
`104,976 individual hexamers. which represent 34.012224 peptides in total
`l324><1o4,9':'6). Assuming an average relative molecular mass for Ac-
`OLOQXXXXNH: of 735. then a mixture of 104,976 peptides (18‘l at a total
`final concentration of 1.0 mg ml" yields a concentration of every peptide
`in each mixture of 9.53 ng ml” l12.1 nrnol l"‘l-.
`METHODS. The synthetic peptide library was prepared using methylbenzhy-
`drylamine (MEI-tn} polystyrene resin and standard t-Boc chemistry in combl«
`nation with simultaneous multiple peptide synthesis(5MPS3)_ A divide. couple
`and recombine lDCRl process was used to synthesize the XXIX-peptide
`resin. This process assures equimolarity of the peptides on the resin. Briefly,
`18 porous polypropylene packets, each containing 4.65 mmol (5.00 g} of
`MBHA resin. were coupled to each of the protected N-it-I-Boo amino acids
`of interest. All coupling reactions proceeded to completion 999.596). as
`assessed by Gisln‘s plcric acid“ or Kaiser's ninhyclnn tests”. The resulting
`resins from each packet were then combined and thoroughly mixed. This
`resin mixture was separated into 18 portions of equal weight which were
`placed into porous polypropylene packets. followed by N-rr-t—B0c Dtatectiflfi.
`group removal and neutralization of the resulting amine TFA salts. The resin _
`packets were then reacted with solutions of the individual activated ar_nin_o
`acids to yield the 324 dipeptide cornblnatlons l18’l. The above DCR process
`was repeated twice more. yielding a final mixture of 104,976 protected
`tetrapeptide resins (18-‘J. This XXXX-resin was divided into 324 aliquots
`l150mg each) and placed in numbered. porous polypropylene packets.
`Synthesis of the next two defined positions was carried out by SMPS. The
`peptide mixtures were deprotected and cleaved from their respective reslns
`using low—high hydrogen fluoride (I-Fl” as described for individual peptides
`earller"3“ in a multiple HF cleavage apparatus (Multiple Peptide Systems.
`San Drego. California). Extraction of the individual peptide mixtures was
`carried out with H,0. The time frames for synthesis, and the amounts of
`each peptide mixture obtained. are the same as described earlier for this
`number of individual peptidf-.‘S3. The competitive ELISA used is a modification
`of the direct ELISA technique described previously“. It differs only ln the
`antibody addltion step in which 25 pl each peptide mixture of the SPCL was
`added. with a fixed dilution of antibody 19810 (25 in per welt).
`
`85
`
`tea the importance of the presence of specific peptide
`:2: in each peptide mixture. Among the 20 peptides in
`"the sixth position was defined, Ac-DVPDYA-NH; had
`west IC5,. (0.03 p.M; Table 12). This sequence exactly
`.
`s the antigenic determinant found in earlier studies to
`gnized by this monoclonal antibody’''’'"'. With other
`_-= ng library procedures”. such precise sequence determi-
`-'u or the identification ofdifierent sequences with alfinities
`"_o or exceeding existing sequences, was not accomplished.
`ults presented here confirm our earlier work in which
`l_.- peptides°"' or chemically synthesized heterogeneous
`_' mixtures”'” were used to establish that each position
`‘near antigenic determinant has a specific, quantifiable
`rder of importance. The use of this SPCL pennits the
`determination ofthe specific peptide sequence that bound
`‘ii antibody out of a total of 34,012,224 possible hexa«
`3 s. Note that no information about the sequence of the
`in or antibody is required to carry out determinations of
`d.
`.' development of new. potentially useful therapeutic pep-
`"*9 requires the synthesis and screening of hundreds to
`{. d5 of analogues of an original, often serendipitously
`ered active sequence. The potential of SPCLs for the
`pment of new antimicrobial peptides against Staphylo-
`.our-etrs (Gram-positive bacterial. Escherichia coli and
`
`aoxxxx-u
`
`E
`
`IIIIIEIIYVI‘
`v -
`
`Wtntulnvdllrvv
`_'—
`or u-
`
`Al:-‘DDXT-
`
`Inilnrullnnsrtanlrvr
`AcwGOJ00(X-
`
`anrlantntunlonilrr
`
`ilD[ItI|lllLIIPflIIF‘f"
`Ae—l-IDBOCXXA
`
`Ac—E0flXX—Mfli
`
`
`nnl.HIurnt_lnlnIlIrI-V
`
`Ac—KOXXX.X—Nl-41
`
`AI:-LOXXXX-IE
`
`tltlurnllrvr IEIE FIIUIIIIIIIOIIVVV
`
`ac-nomo:-nu,
`
`Atllrelcllttlltrnllrer
`Ac-noxxxx-NH,
`
`
`
`Auxlnnuxulrur-unsrlrv
`N:-RDXKXX-N
`
`antluurutuurnllrvv
`At:—SOXX'XX—
`
`aollrlulluururulrlrvr
`lIc—VOKXXX«NH,
`
`ndirorttntulilollrvv
`
`fl fllffillflrr
`TOM!!-
`
` I Ilrl
`
`IIlIlPBIII'VV ADEIIIIIILIIIIIUIIIVY ADIIIIDIIKLIIHIDIIYVV
`
`._;fVOL 354 - 7 NOVEMBER 1991
`
`Page 4 of 5
`
`
`
`Page 4 of 5
`
`

`

`Tb rinrutirsi
`
` 3. Hoirgtiten, R. A. Price. hem. Acact spr. USA. 32, 51315135 usasr. us Patent 4.53:.
`TAB1.E 2 Antimicrobial activity against S. atrreus obtained in the iterative
`‘- ‘°“'"i 5- "- ‘- 9' "" 5°"-"’°" 351- 757-773 ‘199’-1
`5, Bisysan. H. M., Hodcla. s. J a. Mason. 7. J, Maniac; rrrrrrrun 23. ros-r1st19aa:.
`p'°°°55
`s, scan. J, K, r. SmlLl1.G, P, .Sr:i'oime 1-Is. sea-390 (1990).
`r, Dmrlln. J. J,. Parrgeniban. L, c, s Dentin, P, E Sderrcie us. dD4l—&O6 (1990:,
`3, Cwirla. S. E. Peters. E, 1.. Barrett R. W,&Dcwer. W. J. Prac. naln Acad Sci. £13.11. 81.33
`;
`(1990:,
`9, Hnugmen, R A,. Hoffman. 5, R, an Niman. I-L L lfarruries 56 (ads Brown. F.. Cha'iods._
`10 $"r“.r"irii.;’i‘§‘:E.?.”$'i”r?.TE’.f’;.r‘;?.”7.T‘L°'f.'i,£::f.;.“°rIr’.I‘;"¥irf$”;ree..
`.
`11. Pinl|1a.t'1.AppeF,J.R s Houghtpn R. A. Peprraes Proc. 21st Eur. Peptide Syrup. {eds ‘
`rincireu. 9.: aso lEscom scrence. Leiden. the Netherlands. 1991i.
`-
`tein
`search ounclatlon.
`inoh
`i. came. 1
`.
`r
`12. ;icéi;gr;t;r;t ApFfi. J. R. E}r:Pi'n|Iln. c. P"b'1ptide_s(:Im1isrry 1?;Jleds snipe T. s.
`13. Housman R. A.. ripper, J R. or Pinllla. c.
`i/spcrrres as {eds Ginsberg H.. BI'owri_ F.. L:
`chahoeir. R M.) 9-12 iccld spring Harbor Laboratory Press. New vent 1988!.
`14. Baman. H G. is. Huitrnene. o. .4. new. it-irprpbrar. 4:. 1.03-1.26 user}.
`15. resorts-i. Pros. rram Acad Sci. use. an. 5449-5453 i1.987l.
`
`-
`-
`Peptide mlxture
`‘*9’
`‘°'°‘RRWxxx‘NH2
`Ac-RRYXXX-NH,
`,r.r,-_nRnxxx.Ni-ta
`'Ac'RRHxxx'NH1
`A0-RRCXXX-NH:
`no-RRXXXKII-I,
`
`I050
`-1
`ml
`
`)
`
`215
`239
`275
`236
`333
`450
`
`-
`Pepude mixture
`‘‘’i
`A°‘RRwwcx'NH2
`Ac-RRWWWX-NH:
`A.;.nRwwRx_NH2
`'A°'RRwwFx'NH2
`A¢'RRWW'X'NH2
`Arc-flftwwxx-til-I,
`
`ICE”
`-1
`‘Hg ml
`
`)
`
`8-?
`9.9
`93
`12
`14
`32
`
`_
`
`i
`
`-.
`
`'=
`,
`I
`
`3.4
`4.1
`
`M)
`an
`Ac.RRWWCR-NH,
`36
`A¢'RRWWxX-NR2
`Ac-RRWWCWJNIHR
`58
`Ac-RRWFXX-NH;
`_RRw
`_
`_
`£6
`ELI2
`g;
`:6
`-
`9'
`'
`2
`‘ H2
`C‘
`5.5
`Ac-RRWWCK-NH,
`1?S
`Ac-RRWLXX-NI-I2
`an
`An.RRwwrcx.p..|,
`zfa
`A¢.Rnwxxx_“.|R
`The five lowest [Cans obtained are illustrated for the peptide mixtures on
`defining; a_ the mug posmgn (gc_Rfiox;(x_N|.|2}; Q the fgunh pgsmon (gc.
`RRWOXX-NH2lt c. the fifth position (Ac-Rriwwox.NI-I2); and :1 the sixth
`position (Ac-RRWWDD-M-lg). The IC5.-, of the peptide mixture derived from
`the previous iterative step is in hold for comparison. The antimicrobial
`activity of eacl1 peptide mixture against S. aureus ATCC 29213 was deter-
`mined as described earner“. Briefly, in 96-weil tissue culture plates. peptide
`mixtures were added to the bacterial suspension (1-5 X 105 colony-forming
`units ml") at concentrations derived from serial twofold dilutions ranging
`from 1.5mg' ml“ to 2.9 pg mI’1. The plates were incubated overnight at
`3? ‘C. and the growth determined at each concentration by the optical
`density at 620 rim. The relative per cent of growth found for each set of
`U 3 e U
`.
`its“.1?5‘5:3:‘.:2ri*:::f:i.:":';:."r:;.iri.".i2:i§°isJ“
`-
`"g
`" g
`
`1?. Cucrvo. J. H.. Rodriguez. B. .5. l-iougnten. R. A. Hsotide Res. 1. 81-86 (1958).
`16. Selsted. M, E..Brcwn. o. M.,oei.ange.n. J. a. Ler-rer.n I. at trial. Chem. :50. 1-1--I-85-14-4$_
`18. sipnoeiie. s. s a. Houfllten. R. A. Peptide Res. 4. 12-18 (19911.
`19. sipnoeiie. s. s it Hpugnten. R. ii. sracrrernisrry ao. 4-an--1573 (19911
`2o. WIl5on,l. rt. ezat tartar. ?67—?T8{19B4l.
`_.
`21. Glsrn, B. F. Analytic: crum Acts 58. 248-249 (19‘r2l.
`22. Kaiser. E. T.. Coiescou. R. L.. Bossiriger. C. D. or Cook. P. i. Angry: Biocnem. 34. 595-598 i‘
`23. Tam. J. F.. Heath. w. r. tr iuerrirreiti. R. s. .i Arrr. chem. soc 1os.a-142-s4s5i19s3i.
`‘
`24. E-|lo£;Len, R. A.. Bray. M. K. Decraw, 5. T. ar Kirby, C. J. int 1 Peptide Protein Res 21. B
`ACKNOIMEDCERENTS. we them P arena. 0. aurern 5. Mcvherecrt iv. Ssiazar. L. 5imo|I'lis'
`wirlds rer their technlcat assistance. 5. siiua tor manuscript preparation and H. Recon-art ' '
`3;} ‘r “'°“‘;’"; “';"_'
`'“'g;“',“f'““"‘* 5"“ ""”"“""“ “''‘*i W“ ‘"5 '“”"°" °>"
`'""”"”"“ ‘* “" ""°'
`‘°'"”
`
`I
`
`I
`
`-
`
`On Mars
`
`V- '*- ==*°'**- "- '-'- 5*=°"-*- '- °- W-“*»
`I. 5. Israel‘, or. KorrIatsu"' or v. s. Karat
`
`j “ Lunar and Planetary Laboratory. University of Arizona. Tucson.
`TABLE-3 Antimicrobial activity of Ac-RRWWCO-NH, against S aureus
`Minna 35721_ [55
`1‘ Department of Geosciences, University of Arizona. Tucson.
`'
`Arizona 35721. usn
`t Department of Geography. University of Poona. Pune 411 007. inma-
`
`MIC
`ins nil“!
`
`Sequence
`
`Mi‘? 1
`it-is ml‘ l
`
`Sequence
`
`Ac-R=RlN\lilCR-NH,
`Ac-RRWWCV-NH,
`Ac-RRWWCW-NH,
`Ac_RRwwcy,NH_z
`Ar;.amrvwcK.NH2
`Ac-RRWWCT-NH,
`Ac-RRWWCH-NH,
`Ac-RRWWCQ-NH:
`Ac-RRWWCS-NH,
`Ac-RRWWCX-til-I,
`Ac-RRWWONI-NH2
`
`3.24-3.5
`33-7.?
`4.5-9.0
`4_7_g_5
`43.9.5
`4.9-10
`5.5-11
`6-12
`7-14
`1-14
`B-16
`
`Ac-RRWWCA-N!-I,
`Ac-RRWWCP-M-I2
`Ac-RRWWCM-NH2
`,tc_RRwwc|__NH2
`Ac_nawwc|_NH2
`Ac-RRWWCF-NH,
`Ac-RRWWCC-NH,
`Magairlln-IHII2
`Ac-RRWWCE-NH2
`Ac-RRWWOG-NH,
`Ac-RRWWCD-NH,
`
`9-18
`10-19
`14- 2?
`1447
`17-34
`17-3:1
`19-38
`32-64
`> 250
`>500
`>1.00-0
`
`Nature 352’ 589_5g4 (1991).
`
`_.
`_
`,
`_
`_
`_
`IN this Article In the 15 August 199] issue, an errorm the -«__-
`office led to the omission of a line from Table 3. In --=‘_'
`some corrections noted by the authors were not made --'
`publication. Corrected versions of the relevant passages
`below and in reprints,
`
`TABLE 3 Possible sources of 002 related to catastrophic ou -
`ocean formation on Mars
`
`The Mics for the 20 peptides in which the sixth position is defined
`(Ac-RRWWCO-NH,lareshown.TheM|Clsdefin-edasthelowestconcentration
`of peptide at which no growth is detected after 21 h incubation at 37 “C.
`
`Source
`
`The Mics of the previous peptide mixture and magainii-i.II are hold for
`°°”"pa"5°"'
`such as one composed entirely of D-amino acids, have been
`prepared which in total permit the systematic screening of hun-
`dreds of millions of‘ peptides. A fundamental feature of SPCLs
`is that free peptides can be generated and used in solution in
`virtually all existing assay systems at a concentration of each
`peptide most applicable to the assay. This approach has also
`been successfully used in radio-receptor assays (opioid peptides)
`and plaque inhibition assays (human immunodeficiency virus
`(HIV-1) and herpes simplex virus (HSV). SPCLs, as described,
`greatly aid all areas of drug discovery and research involving
`peptides.
`El
`
`Received 31 July: accepted 16 September 1991.
`
`1. Marrilleid. R. a. 1 Am. charrr. Soc 35. 2149-2154 £1963).
`2. Goyaeh.H.M..Meloari.R.H.&Barte:ling.S..'|.F'i'oc1natn Apart ScHrf.5.A s1.ao9a-4oo2i19o4:.
`86
`
`Page 5 of 5
`
`'
`
`r_
`
`C02 partial ore -
`(rnbar)
`
`~20
`~1OD
`
`S1300
`54000
`
`"mm polar cap
`Massive volcanism
`Msomed °nreg°"th"|°::jan bag"
`Groundwater
`C02 clamrate
`
`_
`F“-“"'3' hi3t°'Y
`in firs! paragraph:
`The very high infiltration capacities of common martian _-;
`face rocks (lava flows and impact-brecciatecl rcgolith) .-
`allow subsurface aquifers to be replenished easily, so that r-.-
`dificrentials could be sustained and drive prolonged gro
`water flow. The resulting sapping“ would then produce
`obscrvcdzpattem of structurally controlled, low-density =53
`“°t“"Prk5 '
`_
`_
`_
`Although Noachian valleys are consistent with atrnosp ._'
`NATURE - VOL 354 - 7 NOVEMBER
`
`
`
`Page 5 of 5
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`