官能基選択的接触還元触媒「パラジウム−フィブロイン」
Contents
1. 120 Relative activity 96 Temperature C 1 Ib uS2. 0926 3 75 1194 H DKE CHS 5 79 Ostwald W Grosse Manner Studien zur Biologie des Genies Leipzig 1909 Ostwald W Farbkunde Leipzig 1923 Ostwald W Lebenslinien eine Selbstbiographie 3 Bande Berlin 1926 Walden P Wilhelm Ostwald Berichte der Deutsch Chem Gesell A 65 101 141 1932 Donnan F G Ostwald Memorial Lecture J Chem Soc 316 332 1933 Holt N R A Note on Wilhelm Ostwalds Energism Isis 61 386 389 1970 KRE 3 12 1949 1 1979 1 7 1998
3. Umicore AG amp Co KG Solvias AG Ws 1 ZA 4 IPr Pd allyl Cl IMes Pd ally CI IPr Pd NQ IMes Pd NQ 4
4. 50000 1 200 1 50 000 He a Fe Up a 5 a ee Ve VA ie RIFE 20C 1 Baker H et al J Comp Neurol 285 246 1989 2 Buiakova O I et al Genomics 20 452 1994 3 Cummings D M et al J Comp Neurol 421 362 2000 4 Keller A and Margolis F L J Neurochem 24 1101 1975 5 Koo J H et al J Neurochem 90 102 2004 6 Koo J H et al J Comp Neurol 487 1 2005 7 Rama Krishna N S et al Brain Res 593 295 1992 8 Verhaagen J et al J Neurosci Res 26 31 1990 Figure Immunofluorescence staining of adult mouse olfactory epithelium with goat anti OMP Wako Chemicals USA Codex 544 10001 Green OMP staining was visualized with Cy2 Jackson ImmunoResearch Data was provided by Dr Frank L Margolis and Dr Jae Hyung Koo Department of Anatomy and Neurobiology School of Medicine University of Maryland No 544 10001 Anti
5. Wako No Z TETE DNA Ligase thermostable recombinant solution 25 uk 30 000 030 19871 Cellulase thermostable recombinant solution 1 m4 30 000 Nm n 034 19891 Chitinase thermostable recombinant solution lima 037 19881 Cystein synthetase thermostable recombinant solution lm 071 05191 Glutamic acid decarboxylase thermostable recombinant solution lmg 090 05381 Inositol 1 monophosphate synthetase thermostable recombinant solution 1m4 Vol 74 No 2 2006 _ SERIES Eole B c S BERE LA VS 9f AN S I A X
6. Pd C en Pd Pd Fib Pd Fib Pd OAc Fig 1 b a c e R b 4 Cwe EHEZ e 2 5 96 Pd Fib Pd Fib b Pd OAc in MeOH Figure 1 Preparation of Pd Fib catalyst d Fibroin in solution 3 h
7. CEED 5 250 gg CIN Phospho MAPK Array 4 Array Buffer 3 21 me x 3 Lysis Buffer 21 m2 x 1 Wash Buffer 25 21 m x 2 Anti Phospho MAPK Detection Antibody Cocktail 1 Streptavidin HRP 4 Well Rectangular multi dish 1 Transparency Overlay Template 1 WesternGlo 557 72171 amp off 4 3 4 5 6 4 8 9 I0 1I 12 13 14 158 T6 14 18 19 20 21 22 Control Akt2 MSK2 Control ERK2 HSP27 ERK1 JNK2 p70 S6 Kinase JNK1 p38a Control JNK pan D38 Control p38 y RSK2 Control p38 GSK 3 Control RSKI Akt3 Control GSK 3 a Akt pan Control Aktl JNK3 Control BE WT A Untreated B HepG2 Cells C Untreated vs hlL 1B Treated mmmm Untreated hiL 1B Treated 8 8 8 E 8
8. G0 G1 G2 M 1 coptisme 2 coptisine 1 015 A E D O om eo Z coptisine M2 Vol 74 No 2 2006 OX EO EY AU F berberine coptisine palmatine 3 4 Copgs Japonica C chinensis C deltoidea
9. IQ IDE Mcr ID LAL aa a e eee cec a UI E EU ee SOG eg NS INOS ey Bus E MEER E Suc Ios blk BS
10. 1872 1866 DS CH 1878
11. 1 1887 Xll 44 EE b F4 CIS B 34 22 SS 1887 1906 1887 6
12. 1904 BOR 1905 1906 67 1919 T 82 1917 53
13. 250mg IPr Pd allyl CI IMes Pd ally CI SL J002 1 SL J009 1 Palladium II acetate 500mg Tris dibenzylideneacetone dipalladium 0 CD ROM No Pr Pd NQ l2 500mg IM es Pd N Q 2 SK CCO1 A SK CCO2 A 55979951 UPM55 7995 CX Vol 74 No 2 2006 92 000 Solvias esearch Olfactory marker protein OMP from PAGE band to structure and function An overview E Abstract The peripheral vertebrate olfactory system is com prised of the olfactory sensory neurons OSNs that reside high in the nasal vault within the olfactory neuroepithelium The axons of these neurons form Cranial Nerve I that innervates the olfactory bulb of the CNS These mature chemosensory neurons can be replaced from progenitor cells throughout
14. 3 LJe Buchwald Hartwig Buchwald Hartwig 3 IPr Pd NQ 100C lt 0 5mol Pd 50C Ne Catalyst 1mol Pd Me a ketone arylation KO tBu 1 1 mmol 110 C toluene 202 CI Hos D C 2 o IPA 0 5 1h Heck R ge S Rees A 2 COOBu rn on ee Heck reaction R Me OMe KPO 4 HOB A Roane J veo 2 Suzuki reaction NaOtBu M NR T 110 C toluene MA eee 1 1 C X Ene anes IPr
15. 0 fH ri AZ XH Wb ed UC oit 6 23 AC E 7 WABCO EM Mt Hc 2 Vol 74 No 2 2006 Tafel 1 Die unbunten Nermen Hergestellt von dom Verlag Unesma G m h M Leip 1 Ostwald Farbkurde M7 24 8 W Ostwald Farbkunde 1923
16. FOIE EIDE LED Jo 1 1904 87 x 44 cm 5 la COM TEVA ww SS 1881 1887 16 150 1885 193
17. Etre Wako em sa MED ow gt mam 016 18271 100me 3 100 Aluminium Standard Solution Al N0 in 0 5mol 2 HNO 016 15471 1 000 100m4 2 900 013 18281 100m 4 500 Antimony Standard Solution SbCl in 3mol 2 HCI 010 15491 1 000 100m4 3 100 Lea Arsenic Standard Solution As 0 and NaOH in water Wm 9 100 013 15481 1 000 PH 5 0 with HCI 100mt 2 900 027 15321 Barium Standard Solution 1 000 BaCO in 0 1mol 2 HNO 100m6 2 500 023 14201 n 100mt 4500 Bismuth Standard Solution Bi NO in 0 5mol 2 HNO 021 12661 1 000 100m4 3 100 030 16211 l mt 3 100 Cadmium Standard Solution Cd N0 in 0 1mol 2 HNO 036 16171 1 000 100m4 2 800 036 17891 l mt 3 100 Calcium Standard Solution CaCO in 0 1mol 2 HNO 039 16161 1 000 100m4 2 900 037 16221 100 100m4 3 100 Chromium Standard Solution K Cr 0 in 0 1mol 2 HNO 030 16191 1 000 100m4 2 900 039 17901 100 100m4 4 500 Cobalt Standard Solution Co N0 in 0 1mol 2 HNO 033 16181 1 000 100m4 3 000 034 16231 100 100m4 3 100 Copper Standard Solution Cu N0 in 0 1mol 2 HNO 033 16201 1 000 100m4 2 700 091 03851 100 j 100m4 3 000 lron Standard Solution FetN0sn O 1mol e
18. 1906 1932 TA WW ers T Hee ZU RECRE TTAF nA 1906 7 3 1909 1911
19. 2 1 30 s GRENDLINEN ue 7 ANORGANISCHEN CHEMIE SFE A 4216 4 y R amp x A ib A m Int Mod cs ot es So 3 NO 34r i DERIT 4 37 1904 1 1592 F 83 3 1900 72e IO009 30 60 70
20. T PGT Mlk OPE PPAR7 EEO 0 001 mol 15 A g E V e JV Quee QN d CO H 2 SS O C H 0 316 43 20 28 3 0 0lmol 4 15 AN les 2 e Da CO H 2 O C H 0 314 42 20 207 3 0 0lmol 15 A Jar CO H 2 SS O C H 0 318 45 20 30003 0 0lmol 2 ATEARI ZIIZ X7 Mi amp iit C H4 0 7334 45 20 30 4 0 Ol00 NN 1 ee CO H 2 C aH 0 332 43 20 28 4 Seem 20C No pisi Ae Mie A 0 01mol 4 15
21. Suzuki Heck gt 90 97 Ce eo TT eu OOo OO Heck Heck bo Heck 4 IMes Pd NQ 0 1mol ae e 88 ON 80 YN 97 MeO OD s SK CCO2 A CI R x H N R base PNA ARR 2 YILDEP AEO EET o A 3 5 SK CCOT A SK CCO2 A Buchwald Hartwig
22. by UC wA TS Cot esso as E o 1 2
23. 1894 IS NHN N 1898 1898 1 3 35 ee Physikalisch chemisches Institut 1906 hr NR 1893
24. MD MDGRAPE 3 2 PC PC PC eMD MD eMD MDGRAPE 3 MDGRAPE 3 12 967 154 5 1 B Intel Pentium4 3 0GHz without MDGRAPE 3 Intel Xeon 2 8GHz with MDGRAPE 3 1394 7 2486 83 100 1 000 10 000 100 000 Cytochrome C MD 1 000 step No m AMARA 303 17151 MD AC1Std eMD 1 500 000 300 17161 MD CP1Std
25. Table 2 3 84077 FORRM ARETE Pd C Pd Fib Fibroin rt ESI OAC ONG ED Pd O Fib HCHO 2AcOH MeOH rt MeOH e Scheme 1 Mechanism of Pd Fib generation Pd C AC mg H Pd Fib Ar R OH Pd C en a M H Ar R 2 Scheme 2 Reduction of aromatic carbonyls Vol 74 No 2 2006 Table 1 Chemoselective hydrogenation of olefin in the presence of aromatic carbonyl Entry Substrate Time h m d CHO gt s o vA Product Me O Br 100 7 Br Br functionalities using 2 5 Pd Fib Yield Entry 100 quant a5 atm of pressure of hydrogen PThe reaction was performed at 50
26. B 85C 3 Vol 74 No 2 2006 2 IU ERE 4eropyrum pernix O PP ch THERE EJO Pe EE a AE PE Bre IUE
27. im 2 oe LU 1908 Ze 1909 4 1912
28. ms ooo Cy 6 Lae eMD BRAT OEY Yi ave va
29. EE Ao kN 6 2 61 3 3 6 2 4 L GOCE XR TP PORC IE 8 3 24 7 1920
30. 1 2 85C 3 an oC rd ee B Pyrococcus horikoshii 1204 FREF Y T ill n TT Qt M E unn 2 Alt
31. Pd C Pd C en Z benzyloxycarbonyl 3 O TBDMS t butyldimethylsilyl F 9 odi LIA 5 HER 1 ERRIA 59 109 2001 2 Sajiki H Hattori K and Hirota K J Org Chem 63 7990 1998 3 Hattori K Sajiki H and Hirota K Tetrahedron 56 8433 2000 4 Hattori K Sajiki H and Hirota K Tetrahedron Lett 41 5711 2000 5 Sajiki H Hattori K and Hirota K Chem Eur J 6 2200 2000 6 Sajiki H Hattori K and Hirota K J Chem Soc Perkin Trans 1 4043 1998 No ZAARA 163 21441 Palladium Activated Carbon Ethylenediamine 4 000 16921443 Complex Pd 3 5 6 5 13 500 Vol 74
32. 1 B Pyrococcus furiosus po KET W 2 Fo 3 4 2
33. f 2 5 Pd Fib No Reaction shia 2 5 Pd Fib Scheme 2 2 5 Pd Fib Table 1 Entries 1 6 Entries 7 and 8 2 Pd C Pd C en 7 Pd 2 5 Pd Fib
34. in situ CN J Z 5L 002 1 SI IO009 1 SES Pd OAc Pd dba vidi in sz Yale ohn F Hartwig C N OMe H OMe H Cy eae 0 05 24h 05 24h 99 94 ace O em eon 0 05 48h 0 05 48h 0 5 24h 95 82 8 100C DME ey ey Y 50ppm 36h 50 100 ppm osiphos SL T009 1 ie Mise
35. MT OO ie SUAE e B 2 500ppb HU KUL B 1 800ppb B HPLC 315nm HORE 415nm B 268nm ORR 440nm Co9H35N5 0 533 62 057 07391 2 B 5 Cy2HssNO7 685 89 No SB F208 A 057 07391 Ergovaline Tartrate Img 30 000 122 05071 Lolitrem B 1 3 g 40 000 Vol 74 No 2 2006 Products Wako 6
36. Scheme 4 LALA 2 5 Pd Fib N Cbz Table 4 Chemoselective hydrogenation of olefins in the presence of aromatic N Cbz ps Entry Substrate Solvent Time h Product Yield 96 1 or MeOH 44 woos 64 2 THF 5 92 Ph Z Phe 5 NF ROD E y Scheme 3 Cbz Cbz PAs L 4 CO Ph THF 24 Co Ph 48 Noe CX Cx bn NHCbz THF 34 NHCbz 99 Scheme 4 6 coh MeOH 25 co 50 7 J MeOH 22 CbzHN 100 NHCbz 8 MeOH 92 Z Entry 1 THF Entry 2 W Cbz Entries 2 3 5and 7 Entry 8 DAT 3 5 HP Entries band 8 4 Pd Fib
37. 95C 1 PR 95 3 E ee 4 7ococcgs horikoshii 000 000 0 000 0 000 0 000 0 000 10 30 60 120 180 KLORWSNEAWY 3 YRKE y 7 3 RR GABA pH 4
38. 2 98 Aconitum carmichaeli Debeaux Aconitum japonicum Thunberg Ranunculaceae CAS No 126266 38 4 OH O OCH3 mui uu m L d X H XPO C iH aNO HCI xH 0 C iH NO HCI 626 13 Benzoylmesaconine 022 15491 Hydrochloride Wako L D mote HPLC 95 0 K 0 HO OMe Y w y O NH 0 C H N 0 294 30 15 000 010 20401 L a Aspartyl D phenylalanine 200mg 016 20403 Methyl Ester lg 50 000 Vol 74 No 2 2006 Products J OWako PG COX
39. Wako No m S 1 SEZ 81 eee Tere lg 4 500 163 292183 Palladium Fibroin ES 14 000 Vol 74 No 2 2006 Umicore Solvias C X Avy ZU VY SREY Bb C C C N B Buchwald Hartwig HAA Y PU YT Re Heck AIi 1
40. N Vcr cu cud EG TE Li TIARE 6 38 232e v DIT T Talking of LAL 10 WAGED WicH AIC LotTLy NGBMAMOG 6 aoe oD SABO hn PE sp use 3 PO CGM aera de 24 IS eee 07 lee US OD NU E QD UA IM D d ES 24 357 1996 64 LAL No m 293 51601 Endotoxin Extracting Solution
41. CHO COO O 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 O Wako No T 022 07681 Berberine Chloride Standard 20mg 7 000 036 11311 Coptisine Chloride 20mg 19 600 166 17641 Palmatine Chloride Standard 20mg 23 000 Vol 74 No 2 2006 Products Wako CPME CPME lk SESE THF OCH3 C H 20 100 16 iz 27 9 k B 0856 0 864g m6 K 001 HIO ELT 0 005 Quee atate O4 OX pis CPME or THF 1 2 Yield of Products Sel of Products Solvent 1 2 1 2 THF 44 8
42. MAC Array BAC DNA WP BAC FISH ODA REIT ORR USAID E Y Wako n DNA 4 DNA TIFF WY 547 WY 647 BMP OAT raw Excel Text OLog2 BMP Chromosome ALL Female W Y 647 Male W Y 547 112222222233333644466655666666 7777 788880 9 910901018 191919 1202031916 1418181616171717181019202021212222 X X X X X Y CD R 10 fsa BAC
43. GelRed M dsDNA ssDNA RNA 20 E 1 00 0 80 i 0 60 B 0 40 M ki s 0 20 0 00 0 5 10 15 20 25 h 1 x PBS GelRed M Vol 74 200 300 400 500 600 700 nm DNA 1 x PBS 300nm 595nm EtBr DNA 19 GelRed EtBr EDEA CHANTO Do lane 1 200ng lane 2 100ng lane
44. Zingiber officinale Roscoe Zingiberaceae CAS No 555 66 8 O p HO OCH CH2403 276 37 No m Ae iti RT 199 14111 6 Shogaol 17 000 7 7X Powdered Processed Aconite Root Aconitum carmichaeli Debeaux Aconitum japonicum Thunberg Ranunculaceae CH20CH3 aconitine CoHs OH H jesaconitine CoHs OH OCH3 mesaconitine CH3 OH H hypaconitine CH3 H H Nm 0 05me 0 05me 0 lms 0 lmer a UA Hin CF o 1 1 5bm Aconitum Diester 2 Alkaloids Standard
45. 1907 1909 T Jeep Var 6 6
46. Be SUE OWES Ae CLG SG JRO Dee Sear Pe UDI SR GEO amv I pe eee eG Nee le SUE 96 Pet GS ENS GE up eae v Ie NE eee 0 ME ce MU Ee WPA SA LAL IEA pat SUUS UA soe yb bay Ve mL BUCH ae
47. eMD Empowered Molecular Design Dynamics MD 2A Ye Paws eS MD PP TI Le d eu CATED eMD MD eMD
48. C Wako S po7D g VELTSP Schizosaccharomyces pombe 96 2 CHE NIR nc ST OD 250 cfu ug 96 9 os d as i3 B5 c ON PZA M969 BDF 7E 96
49. 50 100ppm DME HA NaO Bu 25 100C A 6 7 ppm 8 0 01 36h 0 5 24h Vol 74 No 2 2006 Cy oom N SN N N y RS PO HoNR NaOtBu CT M N NHoctyl 50 ppm 90 0 0196 90 50 ppm 90 9396 8396 9696 H H en a N aN Ph N 0 01 100 1 0 70 0 05 100 79 67 91 95 ee 7
50. 96 96 6 CIM 16 CIM16 Vol 74 No 2 2006 Products GPCR IgE EGF NK Biotium GelRed 10 000 DMF EtBr CHE
51. MeO O e AR lt Se S OMe OMe palmatine berberine E ON ER rud duet oi itii 032 So win oisi Bui 16 C 8 fk
52. E mail bacarray wako chem co jp VAN acra RT CES RT CES 16
53. EN RAAF 1764 623 1889 hl ik 1893 20 vC hit SE FR ARSE E dah Lc 4r 18904 Fs Reo FS ha 7 oO Aan 1900 1903 1894 96 CT
54. JT RENE TBA PhiL CA n 515nm 553nm 2 Y Sc CH aie Pr HHO K CH CH CH S TBA Malondialdehyde TBA pigment 0 23 Oo 60 100 21nmol m 5 CV 10 0 40nmolm6 55mg x 1 N 12 330m x 1 10 45m x 1 TBA 60ml x 1 n 300m x 1 TEW 1 1 3 3 5 nmol m 10mg x 1 ETT Ex usan 298 62901 45 000 F X Wako
55. Vol 74 No 2 2006 1896 2 3 e ei Rif L 1898 Hg MHAI L AER SARI sein amp ta maces 3 1900 9 6 1 fr Hii AAS 5 PR 3
56. EU E Pd OAc QR Pd Fib Pd 0 Fig 1 e 72 90 Scheme 1 ESBESESGEER IE RR ZU 1 Pd C Pd C en
57. 90M NII M japonica var japonica japonica var dissecta 2 japonica var major 3 ROMS CU CRS INTOA KH PRUE PEO DS Ole L CHR EE I E URN C chinensis a C deltoides C teeta po He berberine palmatine berberine Growth percentage coptisine Concentration uM 3 coptisine berberine palmatine 0 8 0 6 0 4 10 0 4 0 2 10 4 eU Ch a MESS
58. CX em 1 PdClz PhCN j 3 200 cer ee room gc 0C 74 Nec ee veo _hLocw 74 RT 1h R Ph 87 80 C 1h 97 SL JOO2 1 9 7 85 RT 2h 71 80 C 4h 88 80 C 2h 92 RT 10h 1 a Tsuji J Palladium Reagents and Catalysts John Wiley and Sons Ltd Chichester 1995 b Diederich F and Stang P J Metal Catalyzed Cross Coupling Reactions Wiley VCH Weinheim 1998 c Stark G Riermeier T H and Beller M Transition Metals for Organic Synthesis ed by Beller M and Bolm C Wiley VCH Weinheim Vol 1 p 208 1998 solvias 7 2 Umicore t Solviastt CX ea d Old D W Wolfe J P and Buchwald S L J Am Chem Soc 120 9722 1998 e Fox J M Huang X Chieffi A and Buchwald S L J Am Chem Soc 122 1360 2000 D Kawatsura M and Hartwig J F J Am Chem Soc 121 1473 1999 g Roncali J J Chem Rev 92 711 1992 Navarro O Kelly R A Il and Nolan
59. 1889 232 1907 Chemie ohne Stoffe 7 RJ 1908 1904 ED Vol 74 No 2 2006 1895
60. 10mg x 4 15 000 Vol 74 No 2 2006 echnical Report B 2 m D ER wath CW HL SAAS PMC s BAR CWI CRBS A
61. 40 1907 852 Ja AIS a TP KO DY LAA C 7e 1901 1923 1900 1904 4 X HL AB i KD EPA E 7g o 1899 1903 4 1903 1904 1927 3 40 1907 852 5 1900
62. 10 000 290 62704 40 000 VoL 74 No 2 2006 4 H 15 H FTE PHAR T E mail jiho wako chem coJp OH MICNTAZBHATII CES E CHAPS el O20 052 099 ZU AEQ 806 E mail labchem tec wako chem co jp T 540 8605 1 2 TEL 06 6203 3741 URL http www wako chem co jp
63. 60C 130C R SK CCO2 A Np R isolated yields 80 99 Ketone arylation H Me MeO tested functional groups R COOEt CN OMe Me Buchwald Hartwig 89 E mos us up to 96 N CFs Amines i OE JC O HN HN 0 4 SK CC01 A SK CC02 A 0 5mol 5 F SK CCO2 A 2 Vol 74 No 2 2006 0005 1 mol Pd OAoz NHR Y 0 005 1 mol L Tor aS ol ze Sx N NHocty NaO7Bu DME HNRR 2570 2 16h aed MIE Da OC O VO HO C HO NHBn 2 100 C 72 SO UO nO T XO ZEE Por 10 ppm 100 86 N N E roe 0 01 100 95 0 d 100 C 0 5 70 C 0 5 100 C 0 05 100 C 92 o 67 85 6 Dioxane 3
64. 210 50 1764 235 1885 1887 Lila 2 Vol 74 No 2 2006 NCHA
65. F LE u J 37 7747 556nm OGR 574nm Fho zo Phalloidin Rhodamine Vol 74 No 2 2006 Products MAPKs RD Proteome Profiler RAR MAPK MAP MAPK MAP MAP HRP
66. Pd Fib Pd C Pd C en W Cbz NHCbz O Pd Fib Pd C en Pd C NO MEE 0 Pd Fib Pd C en MIT Buchwald 1 a feta Ashi RRB E Bias 99 109 2001 b fete kA MM Organic Square WAKO 12 1 2004 4 1978 a Sajiki H Ikawa T and Hirota K T
67. coptisine 5 BUB CUR coptisine tx tr 3 amp TFA DARED HPLC berberine n chloride 4 2 1 5 aoc berberine TLD coptisine LI JST Exe D HH EH
68. me MS pal palmatine cop coptisine berberine chloride berberine O 01334 EEDENI cop pal min pal 20 min HPLC Wakosil II SC18 HG 4 6 mm X 150 mm 400 mL 600 mL 3 4g 1 7 g 345 mm W 5 HPLC berberine chloride berberimne H Phellodendron amurense vif 210 4 23 WIT OD BR E OO ERR Z D UL HE C Elias 20014 Mid 2 18 85
69. 2 AN O Christophe Le Ret Marc Thommen Ph D B mes 0 2 JW Hise amp LC NaOt Bu 2
70. V YES 25 uf YES 2m Lome S24 e e 28 30C 21 24 ODeoo 0 2 100 ug A 25u2 BARS 37C 2 FN ee Ey EED 20 300 0Dan 60 70 28 800 5 7 B 46C 2 ba BERET A AYT om Dm M WT k 28 30 C 5 7 1 ee a yz EU nu Sp Transformation Reagent 90 ug DNA i Carrier DNA Auk 42 C 4 37C 6 WAK C 100 20 100 500 ee Sp Transformation Reagent 2 25m x1 dono od 5 7m x1 20 C MF Carrier DNA 5 mg m f 0 1m x1 05m x1 1 25m x2 No 290 64301 20 4 800 296 64303 S pombe Direct Transformation Kit wako 100 NOOO 294 64304 500 40 000 en sq No m FJ 296 62701 4 800 292 62703 S cerevisiae Direct Transformation Kit wako
71. Heck SK CC01 A 4 C CC SK CC02 A 4 SK COUIJN SKCCOO0 8 0 05mol 5 NaOtBu Cs CO K3 PO Toluene Dioxane DME
72. MAPK 24 19 R amp D WesternGlo 24 I SHAR IPLRhkX LMBBIBH ee 13 DNA te LIP amen Seton o bos 15 eee ole 17 BAC 26 JCSS ee 19 Biotium GelRed 27 oo 20 S pombe 39 T had bbc Hp Mc 20 E AXES T C BRA mem 21 o OGRA EE E 25 DEEA 26 JOR Bae J Joano o oai o oo oo 22 DP FIV ANI OY RB cece cece eee eee eens 22 tT eee MISES 23 X REO CO ATTORE ETE 23 Bae Aiea V22774 7 42u0 T71 m Pd C Pd C en 19 Cbz TBDMS
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74. 369 2 264 76 1996 5 Kim H H Puche A C and Margolis F L Odorant deprivation reversibly modulates NR2B mediated CREB phosphorylation in mouse piriform cortex A Chem S abstract 2006 Schiffman S S and Zervakis J Taste and smell perception in the elderly effect of medications and disease Adv Food Nutr Res 44 247 346 2002 Margolis F A brain protein unique to the olfactory bulb Proc Nat Acad Sci 69 1221 1224 1972 Keller A and Margolis F L Immunological studies of the rat olfactory marker protein J Neurochem 24 1101 1106 1975 Monti Graziadei G A Margolis F L Harding J W and Graziadei P P C Immunocytochemistry of the olfactory marker protein J Histochem Cytochem 25 1311 1316 1977 Farbman A I and Margolis F L Olfactory marker protein during ontogeny Immunohistochemical localization Dev Biol 74 205 215 1980 11 Margolis F L Olfactory marker protein OMP Scand J Immunol Suppl 9 181 99 1982 12 Kim H and Greer C A The emergence of compartmental organization in olfactory bulb glomeruli during postnatal development J Comp Neurol 422 2 297 311 2000 13 Kasowski H J Kim H and Greer C A Compart mental organization of the olfactory bulb glomerulus 3 Ta 4 6 lir d 7 N 8 a 9 10 VY J Comp Neurol
75. 094 03841 1 000 HNO 100m 2 700 127 04301 100 100m4 2 900 Lead Standard Solution Pb NO in 0 1mol 2 HNO 124 04291 1 000 100m4 2 700 129 05221 Lithium Standard Solution 1 000 Li CO in 0 2mol 4 HNO 100m 2 400 136 1 3601 Magnesium Standard 1 00 Mg NO 0 imol 2 HNO 100m4 Cal 00 136 12121 Solution 1 000 Mi 100m0 2 700 133 12131 Solution 1 000 pon 100m6 2 700 135 13671 100m 3 100 Mercury Standard Solution HgCL in 0 1mol 2 HNO 138 13661 1 000 100m4 2 900 Molybdenum Standard Mo in 0 4mol 2 HCI 1390 14961 sci tion 1 000 oo HNO a CT su Ni NO in 0 1mol 2 HNO one ckel Standard Solution in 0 1mo 14706461 i 1 000 4 2 700 162 19941 100m 3 100 Potassium Standard Solution KCI in Water 165 17471 1 000 100m4 2 700 188 01951 Rubidium Standard Solution 1 000 RbCI in Water 100m4 4 900 192 13861 Selenium Standard Solution 1 000 Se in 0 1mol 2 HNO 100m4 2 500 191 12111 100 100m4 3 100 Sodium Standard Solution 100 NaCl in Water 199 10831 1 000 100m4 2 700 199 13871 Strontium Standard Solution 1 000 SrCO in 0 1mol 2 HNO 100m 2 500 205 16301 Thallium Standard Solution 1 000 TINO in 1mol 2 HNO 100m4 2 900 202 16311 Tin Standard Solution 1 000 Sn in 3mol 2 HCI 100m4 2 400 261 01431 _ IOm0 3 100 Zinc Standard Solution Zn N0 in 0 1mol 2 HNO 264 01 421 1 000 100m4 2 700 m E MES
76. 3 1 Aeropyrum pernix 1 6 1 A d 1 EP WP HO p o cH Son OH NH 3 Ape DNA 2AT Y X 4 Ape DNA U 10 30 60 120 180 95C B DNA Aeropyrum pernix DNA ATP DNA DNA
77. 407 2 261 74 1999 14 Kosaka K Toida K Margolis F L and Kosaka T Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb II Prominent differences in the intraglomerular dendritic arborization and their relationship to olfactory nerve terminals Neuroscience 16 3 775 86 1997 Koo J H Gill S Pannell L Menco B and Margolis F L The interaction of Bex and OMP reveals a metabolically active covalent dimer of OMP J Neurochem 90 102 116 2004 16 Baker H Grillo M and Margolis F L Biochemi cal and Immunocytochemical Characterization of Olfactory Marker Protein in Rodent Central Nervous System J Comp Neurol 285 246 261 1989 Koo J H Saraswati M and Margolis F L Immunolocalization of Bex protein in the mouse brain and olfactory system J Comp Neurol 487 1 1 14 2005 18 Danciger E Mettling C Vidal M Morris R and Margolis F L The OMP gene Its structure and olfactory neuron specific expression in transgenic mice Proc Natl Acad Sci USA 86 8565 8569 1989 19 Buiakova O Rama Krishna N S Getchell T V and Margolis F L Human and Rodent OMP genes Conservation of structural and regulatory motifs and cellular localization Genomics 20 452 486 1994 20 Wang M M Tsai R Y Schrader K A and Reed R R Genes encoding
78. No HH mg 9 AE S3 A FA Ammonium lon Standard ee NH NO in 0 02mol 2 019 15461 Solution NH 1 000 HNO 50m4 3 900 024 15331 a lon Standard amp 1 000 KBr in Water 50m 4 500 032 16151 in lon Standard ci 1 000 NaCI in Water 50m4 3 900 066 03401 Rois lon Standard 000INaF in Water 50m 3 800 143 06441 SN Standard INO 1000INaNo in Water 50m4 3 900 140 06451 ae Standard 0 1 000 NaNO in Water Xmb 4000 168 17461 NO lon Standard 56 3 1 990 NaH PO in Water 5me 4000 192 10821 Sulfate lon Standard jco 2 4 ooo Na SO in Water 50m8 4 000 Solution CES zd B pH No pH 25C s MTS A 151 01845 Oxalate pH Standard Solution 1 68 500m 2 500 168 12145 Phthalate pH Standard Solution 4 01 500m4 2 400 165 12155 Phosphate pH Standard Equimolal Solution 6 86 500m 2 400 166 17445 Phosphate pH Standard Solution 7 41 500m4 3 300 205 08775 Tetraborate pH Standard Solution 9 18 500m 2 400 037 16145 Carbonate pH Standard Solution 10 01 500m4 2 600 EHBESSGEHRB TE Rb TC RUE Wako Pd C en Pd C 1 1
79. 3 50ng lane 4 25ng GelRed EtBr BYU ATA VE 1 50m0 OF WU AWM AHS ul 2 EKA AFAN VE 1 Kml5ul z 50ml 2 30 amp 3 e Nox m t fi A GelRed Nucleic Acid Gel Stain 10 000x in DMF 9559 78731 41000 22 000 No 2 2006 402 1853 9 2 1932 4 4 1853 1881 3 Da uU DD TEKITA
80. 8 8 HH rhIL 1 30 Hep G2 200 rg A B rhIL 1 30 C m TELE Proteome Profiler i 99491471 ARYO02 Human Phospho MAPK Array Kit 0 RD WesternGlo HRP A B 1 8 5 cm x 6 5 cm 50 2700 cm ELE temic pe A B 1 20 dE 2 WesternGlo A ii 100 m x 1 WesternGlo B if 100 m g x 14 No OC TM 557 72171 AROOA WesternGlo Chemiluminescent 1PK 21 000 Detection Standard Vol 74 No 2 2006 Products INFOGRAM
81. C TYFRATAZRALAY CC Bis Table 3 Entries 1 3 5and 7 THF Entries 2 4 6and 8 Entries 9 12 T 2 5 Pd Fib THF 9 4 CBZ Pd C Substrate a b 992 97 r O Time h Product Yield Et 24 Kx 100 Cl O 20 coer 98 Cl O 21 aol 99 Cl
82. Deoxy A prosta d glandin J Ethanol Solution Img 0 01mol 2 15 Deoxy A prostaglandin A J Acetylene Analog Ethanol Solution 500ug 0 01mol 4 15 Deoxy A RAS prostaglandin J Ethanol Solution 7 0 01mol 2 A Prostaglandin sc eee Jo Ethanol Solution 500ug 0 01mol 2 A Prostaglandin Js 5 Ipa aaan Acetylene Analog Ethanol Solution 500g Wako N N NN 20 W M N lt 1 ee oases
83. S P J Am Soc 125 16194 2003 Navarro O Oonishi Y Kelly R A Stevens E D Briel O and Nolan S P J Organomet Chem 689 3722 2004 Goossen L J Paetzold J Briel O Rivas Nass A Karch R and Kayser B Synlett 2 275 2005 5 ad 6 2 Selvakumar K Zapf A and Beller M Org Lett Vol 4 No 18 3031 2002 a Indolese A F and Schnyder A EP 113 2361 2000 assigned to Solvias AG b Schnyder A Indolese A F Studer M and Blaser H U Angew Chem Int Ed 41 3668 2002 c Nettekoven U Naud F Schnyder A and Blaser H U Synlett 14 2549 2004 Shen Q Shekar S Stambuli J P and Hartwig J F Angew Int Ed 44 1371 2005 8 Roy A H and Hartwig J F J Am Chem Soc 125 8704 2003 umicore Umicore Solvias CX 6 Josiphos 2 Pd Acetate Pd CD ROM dba
84. eMD 1 1 500 000 634 08061 MDGRAPE 3 PCI X 1 1 200 000 Hk m Vol 74 No 2 2006 Products CGH BAC BAC DNA BAC 2006 4 BAC Comparative genomic hybridization CGH Macrogen MAC Array ze f ff Cg 2 e O70 TDNA CGH
85. glomeruli in the olfactory bulb Eur J Neurosci 22 10 2649 54 2005 Koos D S and Fraser S E The Grueneberg gan glion projects to the olfactory bulb Neuroreport 16 17 1929 32 2005 Buiakova O I Baker H Scott J W Farbman A Kream R Grillo M Franzen L Richman M Davis L M Abbondanzo S Stewart C L and Margolis F L Olfactory Marker Protein OMP Gene Deletion Alters Physiological Activity Of Olfactory Neurons Proc Nat Acad Sci 93 9858 9863 1996 Youngentob S and Margolis F L OMP gene causes an elevation in behavioral threshold sensitivity Neuroreport 10 15 19 1999 31 Ivic L Pyrski M Margolis J W Richards L J Firestein S and Margolis F L Adenoviral vector mediated rescue of the OMP null mouse Nature Neuroscience 3 1113 1120 2000 26 28 N NY 29 30 hd 32 Youngentob S L Kent P F and Margolis F L OMP gene deletion results in an alteration in odorant induced mucosal activity patterns J Neurophysiol 90 6 3864 73 2003 Epub 2003 Aug 13 Youngentob S L Margolis F L and Youngentob L M OMP gene deletion results in an alteration in odorant quality perception Behav Neurosci 115 3 626 31 2001 Kwon H J Leinders Zufall T Koo J H and Margolis F L The absence of OMP causes compromised NCX activity in the mouse olfactory rece
86. the life of the animal and are characterized by the expression of the olfactory marker protein OMP that is a unique hallmark of them The identification and characterization of this protein will be the primary subject of this essay after we address the biology of the olfactory system Excellent overviews of the anatomy neurobiology function and clinical aspects of the olfactory system are found in Doty R L et al n Introduction The vertebrate olfactory neuroepithelium OE is a generative neuroepithelium that is a site of continuing neurogenesis throughout life Figure 1 This is an unusual property of OSNs as the vast majority of central nervous system CNS neurons are not capable of being replaced and if damaged or destroyed as degeneratin 9 tq AL Q neuronal precursors University of Maryland Baltimore sustentacular cells immature zm olfactory bulb gomme a result of trauma or disease are irrevocably lost By contrast in all vertebrates mature OSNs that die or degenerate for whatever reason are replaced from mitotically active progenitors residing in this generative neuroepithelium These newly formed neurons undergo maturation and can correctly reform synaptic connections with their neuronal targets in the olfactory bulb OB of the CNS that were lost as a result of the degeneration of the dying OSNs This progenitor mitotic activity and subsequent neuronal maturation is as
87. toluene 50 KOH aq O 0 C 1 2h HN S ons 1M citric acid H Ny Ri THF r t 10h R OBn Y 81 98 ee opn Ri H Y 83 98 ee O R R 3 5 Bistrifluoromethyl Ph henyl NAS Bromide 2mol PhCH CH CHO N a ee Ee H toluene 196 NaOH ag O C 2h OH O OH O 1N HCl cae PhHzCHzC OBut PhHzCHzC OBut NH2 NH2 Y 80 73 27 erythro isomer 90 ee threo isomer GZH 1 Ooi T Kameda M Tannai H and Maruoka K Tetrahedron Lett 41 8339 2000 2 Ooi T Taniguchi M Kameda M and Maruoka K Angew Chem Int Ed 41 4542 2002 No 201 16401 S S 3 4 5 30 000 207 16403 Trifluorophenyl NAS Bromide HEN 201 15921 R H 3 4 5 30 000 207 15923 Trifluorophenyl NAS Bromide a ex R R 3 5 na ne Bistrifluoromethylphenyl NAS Bromide Vol 74 No 2 2006 o Products 1 8 JCSS Japan Calibration Service System pH
88. 000 017 19921 Acetonitrile Solution 2 8 4 500 014 19931 Acetonitrile Solution 3 7 CH CN H 0 3 7 4 500 011 19941 Acetonitrile Solution 4 6 CH CN H 0 4 6 5 000 018 19951 Acetonitrile Solution 5 5 CH CN H 0 5 5 5 000 015 19961 Acetonitrile Solution 6 4 CH CN H 0 6 4 5 500 012 1997 1 Acetonitrile Solution 7 3 CH CN H 0 7 3 si 5 500 019 19981 Acetonitrile Solution 8 2 CH CN H 0 8 2 S 6 000 016 19991 Acetonitrile Solution 9 1 CH CN H O 9 1 ZHY 6 000 No m 214 01301 1 600 5140 01303 Ultrapure Water LC MS 3 000 016 19854 1 900 012 19851 Acetonitrile LC MS 5 600 018 19853 13 000 132 14524 1 050 138 14521 Methanol LC MS 1 600 134 14523 3 450 018 20061 Acetic Acid LC MSH 50m2 5 500 067 045311 Formic Acid LC MS 50m2 9 000 Wako B BSE
89. 33 1 57 5 42 5 THF CPME 1 1 vol 66 8 14 7 82 0 18 0 CPME 81 9 1 6 98 0 2 0 No RAWAM A 031 19845 a 3 500 yclopentyl Methy DER IRE Ether with Stabilizer OOD 037 19847 BHT 0005 FoR Wako Maruoka catalyst S S 3 4 5 NAS A H 3 4 5 NAS H 35 NAS o o gw amp gw F F F F CssHa BrF N 914 77 CssHa BrF N 914 77 S S 3 4 5 Trifluorophenyl NAS Bromide A R 3 4 5 Trifluorophenyl NAS Bromide T O se Ri R R 3 4 5 Trifluorophenyl NAS Bromide 1mol san Dy OOE ester n e e onu T OBn
90. 34 000 n Hydrochloride 017 20411 N N Diacetylspermidine 40mg 36 000 047 29711 ci N Acetylspermine d n Hydrochloride 40mg 38 000 N N Diacetylspermin n Hydrochloride 045 2951 1 40mg 42 000 Vol 74 No 2 2006 Products Wako TBARS TBA ee ie ooze 1 ae TBA iB
91. Josphos SLJ002 1 SL IO09 1 A 9 a CX
92. MP exhibits the beta clam fold formed from eight beta strands and a pair of helical domains and two loops One of the loops is in the so called omega loop configuration that is postulated to participate in protein protein interaction This loop is highly flexible and could adopt a more ordered structure on interaction with a protein partner This js consistent with the postulated role of OMP in transduction and in o 15 17 37 its interaction with the Bex protein b Conclusion These brief comments are intended to illustrate the potential importance of the OMP in olfactory function and the value of the antiserum for studies of the degeneration regeneration and function of the olfactory system in all vertebrates including humans Literature cited 1 Handbook of olfaction and gustation ed by Doty R L Marcel Dekker Inc New York 2003 2 Baker H Kawano T Margolis F L and Joh T H Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat J of Neurosci 3 69 78 1983 Margolis F L Verhaagen J Biffo S Huang F L and Grillo M Regulation of gene expression in the olfactory neuroepithelium a neurogenetic matrix Prog Brain Res 89 97 122 1991 Cho J Y Min N Franzen L and Baker H Rapid down regulation of tyrosine hydroxylase expression in the olfactory bulb of naris occluded adult rats J Comp Neurol
93. No 2 2006 Q Products Wako 0 1vol9 0 1vol96 0 1vol926 HPLC LC MS LC MS CT UV HPLC LUV Ei HOLDS Hf Ti ull LC MS No SER 9 011 20551 O 1vol96 Acetic Acid 10 5 700 017 20553 Acetonitrile LC MSH 39 13800 062 04721 O 1vol96 Formic Acid 5 700 068 04723 Acetonitrile LC MSRI 13 800 206 16451 O 1vol96 Trifluoroacetic 6 400 202 16453 Acid Acetonitrile 34 16 000 No zs garth 010 1991 1 Acetonitrile Solution 1 9 4
94. Ns that express olfactory marker protein OMP and odor receptors Immature neurons express GAP43 Additional markers permit identification of various stages of OSN differentiation and of the various cell types in the OE Axons of OSNs project to the olfactory bulb where they synapse with mitral and tufted cells and juxtaglomerular interneurons The expression of dopamine DA its biosynthetic enzyme tyrosine hydroxylase and fos in tufted cells and interneurons is regulated by OSN activity Modified from G Ronnett Vol 74 No 2 2006 Department of Anatomy and Neurobiology Program in Neuroscience School of Medicine Frank L Margolis Ph D ang Jae Hyung Koo Ph D together all these studies confirm that the central and peripheral portions of the olfactory system are extremely plastic and responsive to manipulations of the external odor environment In addition olfactory function 1s modulated by administration of various systemically administered drugs These include agents used in chemotherapeutic treatments of cancer chronic alcohol abuse and other therapeutic and environmental agents Clearly it is critical to identify unique molecular reagents that will facilitate the study of this system bl Discovery of OMP Therefore we began to search for neuronal cell specific examples of gene expression in this pathway The thought was that such proteins could serve as reagents to study the physiol
95. O Et 12 100 OCH CO H CI OCH CO H CI NH 24 OX 91 CI Et 24 JL Jf 100 Br coz 3 94 Br Br CO5Pr Br CO5Pr Br Table 2 Chemoselective hydrogenation of olefin and azide Table 3 Chemoselective hydrogenation of olefins and azide in the presence of benzyl esters Entry Substrate Solvent Time h Product Yield 1 CD30D 6 81 A CO Bn EtCO Bn 2 THF ds i 91 3 CDsOD 6 69 p iPrCOsBn 4 CO Bn THF ds 7 93 5 ma MeOH 23 Me 50 6 COA MeOH 18 Ecouan 77 if MeOH 24 33 En En pn CO2Bn 8 THF 24 98 9 COsBn MeOH 8 PrO CO Bn 99 YQ CO Bn CO Bn 10 eax MeOH 12 XT 100 CO Bn CO Bn CO Bn CO Bn 1 wor MeOH 6 Kx 97 Et CO Bn CO Bn 12 JW MeOH 17 ST 100 N3 HN Cbz Cbz Pd C en THF k W Cbz W Cbz Vol 74 No 2 2006 Scheme 3 ArCbz Pd C en
96. Olfactory Marker Protein Goat 100 u 43 000 Vol 74 No 2 2006 lh SERIES I 63 Gy Ne N QD 2 To NG OU Se eques ge oe UL2INie ot UNL a Ge SE 16 55 EE UTE UD TCU VIS Che 1990 l7 223b SU Ee CORO Tim Deci AC L HOA heyy
97. Pa allyl CI IMes Pd allyl C M X W A he SES IPr Pda NQ 2 Bs As dee RS Yield 50C Yield LL IMes Pd NQ 2 ou y fa _ J OY Vol 74 No 2 2006 Aas GC average of 2 runs Catalyst 1mol KO7Bu crdop C IPA 1h C 3 9596 SK SARI 02388 F 71 2 3 Yado Yd 0 E N oO 95 6 D 27 48 X e loc d lun Conditions 1 00mmol arylchloride 1 50mmol amine 0 50mol cat 3 00mmol KOH 4mL dioxane 100C 16h isolated yields with NaOt Bu as base 530 0mmol arylchloride 40 0mmol amine O 05mol96 cat 5 00mmol KOH 40mL dioxane KOH 2 3 IPr Pd NQ KOH NaOrBu
98. actory function The OMP null mice were responsive to odors but required 50 100 times higher concentrations to achieve the same behavioral responses as the wild type intact mice In addition Figure 4 OMP immunostaining in mouse hypothalamus visualized by confocal microscopy The ventricle is apparent as a vertical dark area to the left of the figure A subset of neurons stained with OMP are evident as are numerous axonal processes throughout the figure Koo J H et al 2005 their electrophysiological responses exhibited a delayed recovery to baseline compared to controls indicating that they were somehow compromised in their ability to recover after odor stimulation Other studies demonstrated that the OMP null mice were generally compromised in several parameters associated with their ability to respond to odors Curiously although the expression of OMP protein is stringently regulated and highly conserved phylogenetically its effect on olfactory transduction processes is quite subtle We expected dramatic effects on various aspects of olfactory mediated behavior such as those associated with mating maternal care food finding etc but instead its role 1s more of a modulator Ongoing studies to identify exactly where in the overall sensory transduction process are underway Where in the process was not clear as the molecular steps in the transduction cascade appear to be all well characterized without requiring the pr
99. components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression Mol Cell Biol 13 9 5805 13 1993 Kudrycki K Stein Izsak C Behn C Grillo M Akeson R and Margolis F L Olf 1 binding site characterization of an olfactory neuron specific promoter motif Mol Cell Biol 13 5 3002 14 1993 15 Ta 17 NY 21 d 22 Behrens M Venkatraman G Gronostajski R M Reed R R and Margolis F L NFI in the development of the olfactory neuroepithelium and the regulation of olfactory marker protein gene expression Eur J Neurosci 12 4 1372 84 2000 23 Walters E Grillo M Oestreicher A B and Margolis F L LacZ and OMP are co expressed during ontogeny and regeneration in olfactory receptor neurons of OMP promoter lacZ transgenic mice Int J Dev Neurosci 14 7 8 813 22 1996 24 Margolis F L Regulation of olfactory neuron gene expression Cytotechnology 11 17 22 1993 25 Mombaerts P Wang F Dulac C Chao S K Nemes A Mendelsohn M Edmondson J and Axel R Visualizing an olfactory sensory map Cell 87 4 675 86 1996 Storan M J and Key B Septal organ of Gruneberg is part of the olfactory system Comp Neurol 494 5 834 44 2006 27 Fuss S H Omura M and Mombaerts P The Grueneberg ganglion of the mouse projects axons to
100. e organization of the olfactory bulb during 12 13 14 and its response to disturbances of development the olfactory system Immunocytochemistry with this antiserum has facilitated these studies by both light and electron microscopy Curiously the OMP was also identified in a small subpopulation of neurons in 16 17 the hypothalamus Figure 4 a Figure 2 Cloning and mapping of the OMP gene In addition to the interest in understanding the function of the OMP another gnawing question was to learn why it is so highly restricted to the mature OSNs The growing availability of recombinant DNA technology provided opportunities to learn more about the mechanisms regulating the highly selective expression of this protein We determined the amino acid sequence of OMP from HPLC purified tryptic peptides using semi automated peptide sequencing Knowing the protein sequence facilitated new directions in the study of this protein and its gene Peptide sequence information permitted prediction of degenerate oligo nucleotides that could then be used to identify OMP cDNA clones from an olfactory neuroepithelium library With this information it was possible to generate additional reagents to study the distribution of the OMP mRNA confirming its virtual restriction to expression in mature OSNs Further it allowed the mapping of the OMP gene to a defined area of mouse chromosome 7 and human chromosome 11 tha
101. eo Reducible functionalities R OTBDMS TES Benzyl alcohol R CO Bn epoxide ArCOR aromatic N Cbz alkyl N Cbz Ar X olefin acetylene Ar NO R N G 2 Umicore Solvias C X Christophe Le Ret Marc Thommen 5 Olfactory marker protein OMP from PAGE band to structure and function Frank L Margolis and Jae Hyung Koo 9 are 16 gt Fis aac 14 Talking of LAL 6335 EAEN E 13 ERK qu 28 qrir Ne a a Z 0 00 0 0 ce 4 H Olfactory Marker Protein decere re ee e TA 12 Umicore Solviastt CX 8 ete eo Uc a 15 EISE FI X FI TL T Jb ER MR 18 LA AINIVFI D DS VH Sec eed ee RIS o Tos 18 R amp D Proteome Profiler
102. esence of an additional component Furthermore two olfactory sub systems the main olfactory system and the vomeronasal system both express OMP but they utilize very different transduction cascades Since OMP is abundant in both subsystems it is difficult to see how it fits in these pathways One option 1s that it plays a role not in the primary response transduction pathway but that it plays a role in regulating the mechanisms associated with return of intracellular calcium to basal levels To date these questions while still unanswered are under investigation Nevertheless all the evidence indicates that OMP is a participant in the overall olfactory transduction cascade E Structure of OMP Another complementary approach to learn something of OMP function was being pursued simultaneously Possibly the primary amino acid sequence of the protein is not per se a direct determinant of function but is the basis of a conserved element of the three dimensional structure of OMP that is critical to its function in olfac tory transduction Therefore we undertook to determine the 3 dimensional structure of OMP in solution To address this we utilized heteronuclear NMR spectroscopy to determine the structure of OMP in solution Another group independently determined the structure by 39 X ray crystallography Both structures are essentially Vol 74 No 2 2006 identical and demonstrate that O
103. et rahedron Lett A4 171 2003 b Ikawa 2 Bd 4E 27 7 3 aal 5 Pd C H balloon poc NH MeOHor THF 24 h rt 100 conversion 5 Pd C en H balloon ee NCbz THF 17 h rt aa 92 5 Pd C en H balloon Ph NIR THF 4 h rt 96 Reducible functionalities R OTBDMS TES Benzyl alcohol R CO Bn epoxide aromatic N Cbz ArCOR Pari alkyl N Cbz Ar X olefin acetylene ArNO RN 9 Figure 2 T Sajiki H and Hirota K Tetrahedron 61 2217 2005 40 Pd Akabori S Sakurai S Izumi Y and Fujii Y Nature 178 323 1956 5 6 a Sajiki H Tetrahedron Lett 36 3465 1995 b Hevea ahd 120 1091 2000 a Sajiki H Kume A Hattori K and Hirota K Tetrahedron Lett 43 7247 2002 b Sajiki H Kume A Hattori K Nagase H and Hirota K Tetrahedron Lett 43 7251 2002 8 Sajiki H Ikawa T and Hirota K Tetrahedron Lett 44 8437 2003 Pete ACoA ia 63 1218 2005 10 ERRIAL 7 42 140 2006 4 iod 7 cag 9 lir d
104. ied and it became clear that at least one such element was present in several genes whose expression pattern was highly selective for regulating genes preferentially expressed in the OSNs 77 2 OMP transgenic and knock out mice This set the stage to take advantage of the then novel technology for generating transgenic mice to perform promoter analyses in vivo in OSNs The OMP promoter was then used by many laboratories to drive expression of various genes into mature OSNs In vivo to analyze the biology of the olfactory system One of the most powerful ways to do this was to utilize the technique of homologous recombination in mice zn vivo by which a known gene can be inserted into the exact genomic locus normally occupied by OMP This allowed the normal genomic regulatory elements that regulated the expression of OMP to drive the expression of an ectopic gene that could be used a probe of function In one such example the OMP was replaced by the fluorescent protein EGFP leading to the identification of a group of OMP expressing olfactory neurons in an anatomical site that was previously unknown This technology also offered an opportunity to overcome one glaring deficit that of the function of OMP After all of these studies we still did not have any insight as to the function of this protein whose expression was developmentally regulated and phylogenetically conserved Indeed Figure 3 Immunostaining for OMP in hu
105. ions It rapidly became apparent the protein was conserved across many vertebrate species and was restricted almost exclusively to the As a result and in the ab mature olfactory neurons sence of any information about its function we called the protein OMP Olfactory Marker Protein Even now over 30 years later its function 1s only beginning to be unraveled The OMP has been demonstrated to be present in all mammals tested including humans in several species of fish in marsupials in amphibia and as best as we can tell is present in mature olfactory neurons of all vertebrate species The antisera to OMP gave no evidence of immunologically cross reactive materials in any invertebrate species The critical question that remained unanswered was that of function However even in the absence of knowledge of its function the nearly unique specificity of the antiserum for mature olfactory neurons has proven to be an extremely valuable reagent It has been invaluable for studies of olfactory neurogenesis in developing animals and in response to surgical and chemical lesions of the olfactory system Its broad phylogenetic cross reactivity has made this antiserum valuable for immunocytochemical studies of the olfactory system In species as diverse as mice Figures 2a b and humans Figure 3 In addition the presence of the OMP throughout the cytoplasm of the OSN has also made the antiserum a valuable reagent for the study of th
106. man olfactory tissue Mature olfactory neurons are stained brown in the middle of the olfactory epithelium with their dendritic processes projecting to the surface of the epithelium where they end in dendritic knobs OMP stained axon bundles are visible deep in the epithelium Buiakova O et al 1994 with the advent of extensive gene cloning and with the sequencing of whole genomes it became clear that the gene for OMP was present in every vertebrate species analyzed The predicted amino acid sequences are gt 50 identical across all vertebrate species The gene for this protein is absent from the genomes of invertebrate species including Drosophila C elegans as well as from unicellular eucaryotes such as yeast Nevertheless it became apparent that comparison of its amino acid sequence with all the sequences in the databases did not identify any functional domains that might provide information as to its function a Function of OMP Therefore we utilized the technique of homologous recombination noted above to generate mice in which the OMP gene was deleted This promised to provide an entr e to function as any novel phenotype observed in the absence of any OMP in vivo would provide clues as to function of the OMP We were not disappointed in this hope The OMP mice appeared superficially normal but careful electrophysiological and behavioral analyses demonstrated that the OMP null mice were deficient in their olf
107. ogy and function of individual classes of neurons in the presence of their cellular neighbors This may seem somewhat naive today but we refer to a time when many of the standard techniques of contemporary biology were still undreamed of and in the future Thus it is important to recall that this investigation began before the advent of monoclonal antibodies 2D gel electro phoresis recombinant DNA PCR transgenic mice etc Indeed we have adopted these various techniques as they arose and have applied them in order to unravel the role of this protein in the olfactory system We began by using the low resolving power of 1D non denaturing gel electrophoresis to search for small acidic proteins that exhibited CNS regional specificity Extracts of mouse brain regions exhibited quantitative and qualitative differences in staining patterns across CNS regions and in the olfactory system an apparently unique protein band was observed that was absent from other regions of the CNS To study this in detail and to study the function of this novel protein it was necessary to obtain purified homogeneous protein and generate antisera to it Olfactory tissue was collected from many rats and the protein was purified to homo geneity using the mobility in PAGE as an assay This purified protein was then used to generate polyclonal antisera These antisera to the protein were used to characterize its immunoreactivity in several species and many brain reg
108. ptor neurons Soc for Neurosci Abstract 2005 35 Lucas P Ukhanov K Leinders Zufall T and Zufall F A diacylglycerol gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice mechanism of pheromone transduction Neuron 40 3 551 61 2003 Berghard A Buck L B and Liman E R Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs Proc Natl Acad Sci USA 93 6 2365 9 1996 Baldisseri D M Margolis J W Weber D J Koo J H and Margolis F L Olfactory marker protein OMP exhibits a beta clam fold in solution implications for target peptide interaction and olfactory signal transduction Mol Biol 319 3 823 37 2002 Wright N T Margolis J W Margolis F L and Weber D J Refinement of the solution structure of rat Olfactory Marker Protein OMP Biomol NMR 33 1 63 8 2005 39 Smith P C Firestein S and Hunt J F The crystal structure of the olfactory marker protein at 2 3 A resolution Mol Biol 319 3 807 21 2002 40 Behrens M Margolis J W and Margolis F L Identification of members of the Bex gene family as olfactory marker protein OMP binding partners J Neurochem 86 1289 1296 2003 33 od 34 36 NS 37 38 No RAPER NIE RAWiNt jy Olfactory Marker Protein F Olfactory Marker Protein OMP
109. sociated with changes in gene expression in the OE The accompanying process of degeneration of the synaptic terminals in the olfactory bulb is also associated with transynaptic alterations in gene expression Thus olfactory bulb neuron phenotype is modulated by degeneration and regeneration of the OSNs and their associated synaptic activity in the OB In addition to alterations in con nectivity the olfactory bulb neuronal phenotype can be manipulated by alterations in afferent OSN activity This has been demonstrated by altering or restricting peripheral odor stimulation resulting in modulation of gene expression in target neurons in the olfactory bulb Current studies in my lab demonstrate that gene expression in deeper cortical areas that receive olfactory input can also be modulated by manipulation of afferent input to the OB 4 Km E Ea hoe Taken mature neuron neuron REOS en mitral cell Figure 1 Schematic of olfactory neuroepithelium The olfactory neuroepithelium is avascular and rests on a highly vascularized substratum studded with glands whose ducts project to the surface Non neuronal sustentacular supporting cells are interspersed among the mature olfactory sensory neurons OSNs Neuronal precursor cells lie at the base of the olfactory epithelium OE The panel illustrates the life cycle of the OSN Precursor globose basal cells GBC undergo mitosis migration maturation and differentiation to mature OS
110. t was near the locus for an hereditary auditory defect called shaker l in mouse and Usher syndrome in humans respectively The OMP gene was isolated and characterized leading to the demonstration that the entire OMP gene was contained in a single exon and that only one copy of it existed in the genome This information and the determination of the nucleotide sequence of this genomic region enabled us to analyze the genomic elements that were responsible for the highly restricted pattern of OMP expression Using zn vitro DNA binding gel shift assays we b a OMP immunostaining of mouse olfactory neuroepithelium visualized by confocal microscopy The OSN cell bodies are apparent with their dendritic processes projecting to the surface of the epithelium where they terminate in dendritic knobs The OSN axons project centrally and gather into bundles seen in round cross sections Koo J H et a 2005 b OMP immunostaining of mouse olfactory bulb OB visualized by confocal microscopy The OSN axons are seen penetrating the surface of the olfactory bulb where they gather into globular neuropil containing structures called glomeruli No staining is seen deeper in the OB indicative of the specificity of the antiserum Koo J H et a 2005 Vol 74 No 2 2006 were able to identify and characterize the upstream regulatory elements of the OMP gene Several of the promoter elements of the OMP gene were identif
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