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Kuvumbura no gukoresha neza ibikomoka ku bimera karemano bishobora gufasha mu kunoza ubuzima bw'abantu. Imiti ibuza gukura kw'ibimera ikoreshwa cyane nk'imiti yica ibyatsi bibi. Bitewe n'uko hakenewe gukoresha ubwoko butandukanye bw'imiti yica ibyatsi, hakenewe kumenya imvange zifite uburyo bushya bwo gukora. Muri ubu bushakashatsi, twavumbuye imvange nshya ya N-alkoxypyrrole, coumamonamide, ikomoka kuri Streptomyces werraensis MK493-CF1 kandi dushyiraho inzira yuzuye yo gukora. Binyuze mu isuzuma ry'imikorere y'ibinyabuzima, twavumbuye ko aside urs-monoamic ari interuro y'ubukorano ya urs-monoamide kandi ikaba ishoboraibuza ibimera gukuraByongeye kandi, twakoze ibintu bitandukanye bikomoka kuri aside ya urbenonic, harimo na urbenyloxy derivative (UDA), ifite imikorere myinshi yo kwica ibyatsi bidafite ingaruka mbi ku mikurire y'uturemangingo twa HeLa. Twasanze kandi ko ibikomoka kuri aside ya urmotonic bibangamira mikorotubule z'ibimera; byongeye kandi, KAND igira ingaruka ku mitsi ya actine kandi igatera urupfu rw'uturemangingo; Izi ngaruka nyinshi zitandukanye n'iz'imitsi ikoreshwa mu gukumira mikorotubule kandi zigaragaza uburyo bushya bwo gukora aside ya ursonique, ikaba ari inyungu ikomeye mu iterambere ry'imiti mishya y'ibyatsi.
Kuvumbura no gukoresha neza ibikomoka ku bimera karemano n'ibikomoka kuri byo ni uburyo bwo kunoza imibereho y'abantu. Ibinyabutabire bya kabiri bikomoka ku dukoko, ibimera n'udukoko byatumye habaho iterambere rikomeye mu buvuzi no mu buhinzi. Imiti myinshi irwanya kanseri y'amaraso n'imiti irwanya kanseri y'amaraso yakozwe mu bimera karemano. Byongeye kandi, ubwoko butandukanye bwaimiti yica udukoko, imiti yica udukoko n'ibimera bivanwa muri ibi bimera karemano kugira ngo bikoreshwe mu buhinzi. By'umwihariko, imiti yica udukoko irwanya ibyatsi ni ingenzi mu kongera umusaruro w'ibihingwa mu buhinzi bwa none, kandi ubwoko butandukanye bw'ibintu bikoreshwa mu bucuruzi. Imikorere myinshi y'uturemangingo mu bimera, nko gukora fotosite, metabolism ya aside amine, gukora inkuta z'uturemangingo, kugenzura mitose, gutanga ibimenyetso bya phytohormone, cyangwa gukora poroteyine, bifatwa nk'ibibandwaho bisanzwe by'imiti yica udukoko. Ibintu bibuza imikorere ya mikorotubule ni ubwoko busanzwe bw'imiti yica udukoko igira ingaruka ku mikurire y'ibimera binyuze mu kugira ingaruka ku mitote2.
Microtubules ni ibice bigize cytoskeleton kandi bibungabungwa cyane mu turemangingo twa eukaryotic. Heterodimer ya tubulin igizwe na α-tubulin na β-tubulin birema protofilaments za microtubule zigororotse, hamwe na protofilaments 13 zirema imiterere ya cylindrical. Microtubules zigira uruhare runini mu turemangingo tw’ibimera, harimo kugena imiterere y’uturemangingo, igabanyuka ry’uturemangingo, no gutwara 3,4. Uturemangingo tw’ibimera tuba dufite microtubules munsi y’urukiramende rwa plasma, kandi izi microtubules zitwa cortical zifatwa nk’izigenzura imiterere ya microfibrils za cellulose binyuze mu kugenzura complexes za cellulose synthase4,5. Microtubules za cortical z’uturemangingo tw’imizi y’inyuma, tuba mu gace k’uburebure bw’umuzi, ziherereye ku ruhande, kandi microfibers za cellulose zikurikiza izi microtubules kandi zikagabanya icyerekezo cy’ubwiyongere bw’uturemangingo, bityo bigateza imbere ubwiyongere bw’uturemangingo twa anisotropic. Kubwibyo, imikorere ya microtubule ifitanye isano rya hafi n’imiterere y’ibimera. Gusimbuza aside amine mu turemangingo dufata tubulin bitera kugorama kwa microtubule arrays za cortical no gukura ku ruhande rw'ibumoso cyangwa iburyo muri Arabidopsis 6,7. Mu buryo nk'ubwo, impinduka muri poroteyine zifitanye isano na microtubule zigenga imigendekere ya microtubule zishobora no gutuma imizi ikura nabi8,9,10,11,12,13. Byongeye kandi, kuvura imiti yica udukoko nka disopyramide, izwi kandi nka pretilachlor, nabyo bitera gukura kw'imizi ya oblique ku ruhande rw'ibumoso14. Aya makuru agaragaza ko kugenzura neza imikorere ya microtubule ari ingenzi cyane mu kumenya icyerekezo cy'imikurire y'ibimera.
Ubwoko butandukanye bw'imiti ibuza udukoko twa mikorobe bwaravumbuwe, kandi iyi miti yagize uruhare runini mu bushakashatsi ku mitsi, ndetse no mu buhinzi n'ubuvuzi2. By'umwihariko, oryzalin, ibinyabutabire bya dinitroaniline, disopyramide, ibinyabutabire bifitanye isano na benzamide, hamwe n'ibindi bimera bingana nabyo bishobora kubuza imikorere ya mikorobe bityo bikabuza gukura kw'ibimera. Kubwibyo, bikoreshwa cyane nk'imiti yica ibyatsi. Ariko, kubera ko mikorobe ari igice cyingenzi cy'uturemangingo tw'ibimera n'inyamaswa, inyinshi mu miti ibuza udukoko twa mikorobe ni uburozi ku turemangingo twombi. Kubwibyo, nubwo byemewe ko ari imiti yica ibyatsi, hari umubare muto w'imiti ibuza udukoko twa mikorobe ikoreshwa mu bikorwa bifatika.
Streptomyces ni ubwoko bw'ubwoko bwa Streptomyces, burimo bagiteri zikora aerobic, gram-positive, filamentous kandi izwi cyane kubera ubushobozi bwayo bwo gukora ubwoko bwinshi bwa metabolites. Kubwibyo, ifatwa nk'imwe mu masoko y'ingenzi y'ibicuruzwa bishya by'ibinyabuzima bikora. Mu bushakashatsi buriho ubu, twavumbuye ikintu gishya cyitwa coumamonamide, cyakuwe muri Streptomyces werraensis MK493-CF1 na S. werraensis ISP 5486. Dukoresheje isesengura rya spectral n'isesengura ryuzuye rya spectral, imiterere ya coumamonamide yaragaragajwe kandi hagenwa imiterere ya N-alkoxypyrrole skeleton. Acide Ursmonic, intermediate ya ursmonoamide n'ibikomoka kuri yo, byagaragaye ko ibuza gukura no kumera kw'ikimera gikunzwe cyane cyitwa Arabidopsis thaliana. Mu bushakashatsi bwakozwe ku miterere n'imikorere, twasanze ikintu gifite C9 cyahinduwe muri aside ursonic, cyitwa nonyloxy derivative ya aside ursonic (KAND), cyongera cyane ingaruka zo kubuza gukura no kumera. Ikigaragara ni uko iyi mashini ibuza gukura kw'ibimera yavumbuwe yagize ingaruka ku mikurire y'itabi na liverwort kandi ntiyangizaga bagiteri cyangwa uturemangingo twa HeLa. Byongeye kandi, bimwe mu bikomoka kuri aside urmotonic bitera isura idahwitse y'imizi, bivuze ko izi nkomoko zigira ingaruka ku mikorobe. Dukurikije iki gitekerezo, ibyo twabonye kuri mikorobe zanditseho immunohistochemical cyangwa poroteyine zitanga urumuri bigaragaza ko uburyo bwo kuvura bwa KAND bukuraho polymeri ya mikorobe. Byongeye kandi, uburyo bwo kuvura hakoreshejwe aside kumamotonic bwahungabanyije microfilaments za actin. Bityo, twavumbuye uburyo bushya bwo kubuza gukura kw'ibimera bufite uburyo bwihariye bwo gukora burimo gusenya cytoskeleton.
Ubwoko bwa MK493-CF1 bwakuwe mu butaka i Shinagawa-ku, muri Tokyo. Ubwoko bwa MK493-CF1 bwakoze stromal mycelium ifite amashami meza. Igice cy'uruhererekane rwa gene ya 16S ribosomal RNA (1422 bp) cyarabonetse. Ubu bwoko busa cyane na S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: ubwoko busanzwe, 99.93%). Hashingiwe kuri ibi bisubizo, byagaragaye ko ubu bwoko bufitanye isano ya hafi n'ubwoko bwa S. werraensis. Kubwibyo, twise ubwoko bwa S. werraensis MK493-CF1 by'agateganyo. S. werraensis ISP 5486T nayo ikora ibintu bimwe bifatika. Kubera ko habayeho ubushakashatsi buke bwa mbere ku kubona ibintu karemano bivuye muri ubu bwoko bw'ibinyabuzima, hakozwe ubundi bushakashatsi bwa chimique. Nyuma yo guhinga S. werraensis MK493-CF1 ku gipimo cy’ingano ya sayiri hakoreshejwe ubushyuhe bukabije kuri 30°C mu gihe cy’iminsi 14, icyo gipimo cyakuwemo 50% EtOH. Mililitiro 60 z’icyitegererezo zaramishijwe kugira ngo haboneke 59.5 mg y’igipimo cy’ingano. Igipimo cy’ingano cyashyizwe muri HPLC kugira ngo habeho N-methoxy-1H-pyrrole-2-carboxamide (1, yitwa coumamonamide, 36.0 mg). Igiteranyo cya 1 ni hafi 60% by’igipimo cy’ingano. Kubera iyo mpamvu, twahisemo kwiga birambuye imiterere ya kumamotoamide 1.
Coumamonamide 1 ni ifu yera idahinduka kandi ifite ubushobozi bwo gupima uburemere (HRESIMS) yemeza C6H8N2O2 (Ishusho ya 1). Igice cya pyrrole cyasimbuwe na C2 cy'iki kinyabutabire kirangwa na δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH muri 1H NMR spectrum: 4.5 Hz, H-5) na δH 6.78 (1H, d, J = 2.5 Hz, H-6), kandi spectrum ya 13C NMR igaragaza ko hari atome enye za sp2 za karuboni. Kuba hari itsinda rya amide ku mwanya wa C2 byapimwe hifashishijwe isano ya HMBC kuva kuri protoni ya C-3 na karuboni ya amide carbonyl kuri δC 161.1. Byongeye kandi, 1 H na 13 C NMR peaks kuri δH 4.10 (3H, S) na δC 68.3 bigaragaza ko hari amatsinda ya N-methoxy muri molekile. Nubwo aho itsinda rya methoxy riherereye hataramenyekana neza hakoreshejwe isesengura rya spectroscopic nka enhanced difference spectroscopy na nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide yabaye imvange ya mbere.
Kugira ngo hamenyekane imiterere nyayo ya 1, hakozwe isesengura ry’imiterere y’ibintu byose (Ishusho ya 2a). Gutunganya 2-aminopyridine 2 igurishwa hamwe na m-CPBA byatumye haboneka N-oxide 3 ijyanye nayo mu musaruro w’ingano. Nyuma yo gupima 2-aminoazidation ya 2, igisubizo cya cyclocondensation cyasobanuwe na Abramovich cyakozwe muri benzene kuri 90°C kugira ngo haboneke 1-hydroxy-1H-pyrrole-2-carbonitrile 5 muri garama. Umuvuduko 60% (intambwe ebyiri). 15,16. Methylation na hydrolysis ya 4 byatanze 1-methoxy-1H-pyrrole-2-carboxylic acid (yiswe "aside cumotonic", 6) mu musaruro mwiza (70%, intambwe ebyiri). Amaherezo, amidation binyuze muri aside chloride intermediate 6 hakoreshejwe ammonia y’amazi yahaye Kumamoto amide 1 mu musaruro wa 98%. Amakuru yose ya spectral ya 1 yakozwe yari asa na 1 yihariye, bityo imiterere ya 1 yaragenwe;
Gutunganya no gusesengura imikorere rusange ya urbenamide na aside urbenic. (a) Gutunganya Kumamoto amide yose. (b) Imbuto z'ibiti byo mu bwoko bwa Arabidopsis Columbia (Col) zo mu gasozi zimaze iminsi irindwi zatewe ku masahani ya Murashige na Skoog (MS) arimo coumamonamide 6 cyangwa coumamonamide 1 ku gipimo cyagaragajwe. Umwanya w'ibipimo = cm 1.
Ubwa mbere, twasuzumye imikorere y’ibinyabuzima bya urbenamide n’ibiyigize kugira ngo tumenye ubushobozi bwabyo bwo guhindura imikurire y’ibimera. Twongeyeho ibipimo bitandukanye bya ursmonamide 1 cyangwa aside ursmonic 6 kuri MS agar medium hamwe n’imbuto za Arabidopsis thaliana zahinzwe kuri ubu buryo. Ibi bipimo byagaragaje ko ibipimo byinshi (500 μM) bya 6 byabujije imikurire y’imizi (Ishusho ya 2b). Hanyuma, twakoze ibikomoka ku bimera bitandukanye dusimbuza umwanya wa N1 wa 6 kandi dukora inyigo ku miterere n’imikorere yabyo (uburyo bwo guhuza analogue busobanurwa muri Supporting Information (SI)). Imbuto za Arabidopsis zahinzwe ku buryo burimo 50 μM aside ursonic derivatives, kandi uburebure bw’imizi bwapimwe. nk’uko bigaragara ku ifoto. Nkuko bigaragara ku Ishusho ya 3a, b, na S1, aside coumamo zifite uburebure butandukanye bw'iminyururu ya alkoxy iri ku murongo (9, 10, 11, 12, na 13) cyangwa iminyururu minini ya alkoxy (15, 16, na 17) ku mwanya wa N1. Ibikomoka kuri iyo minyururu byagaragaje ko imizi idashobora gukura neza. Byongeye kandi, twasanze gukoresha 200 μM 10, 11, cyangwa 17 byabujije kumera neza (Ishusho ya 3c na S2).
Inyigo ku miterere n'imikorere bya Kumamoto amide n'ibindi bifitanye isano. (a) Imiterere n'uburyo bwo guhuza ibisa nabyo. (b) Gupima uburebure bw'imizi y'imbuto z'iminsi 7 zatewe ku miterere ya MS hamwe cyangwa nta n'ibikomoka kuri coumamonamide bya 50 μM. Inyenyeri zigaragaza itandukaniro rikomeye mu kuvura ibinyoma (ikizamini cya t, p< 0.05). n>18. Amakuru agaragazwa nk'ikigereranyo cya ± SD. nt bivuze "bitapimwe" kuko imbuto zirenga 50% ntizashibutse. (c) Ingano y'umuvuduko w'imikurire y'imbuto zavuwe zashyizwe mu gihe cy'iminsi 7 mu buryo bwa MS hamwe cyangwa nta coumamonamide ya 200 μM n'ibindi bifitanye isano. Inyenyeri zigaragaza itandukaniro rinini mu kuvura ibinyoma (ikizamini cya chi-square). n = 96.
Igishimishije ni uko kongeramo iminyururu ya alkyl miremire kurusha C9 byagabanyije imikorere yo gukumira, bigaragaza ko imvange zifitanye isano na aside kumamotoic zisaba iminyururu y’impande ifite ingano runaka kugira ngo zigaragaze imikorere yazo ya biyoloji.
Kubera ko isesengura ry’imiterere n’imikorere ryagaragaje ko C9 yahinduwe ihinduka aside ursonic kandi ko ikomoka kuri aside ursonic (izwi nka KAND 11) ari yo yari inzitizi ikomeye yo gukura kw’ibimera, twakoze isesengura rirambuye rya KAND 11. Kuvura Arabidopsis hakoreshejwe 50 μM KAND 11 byabujije kumera neza, mu gihe ingano nto (40, 30, 20, cyangwa 10 μM) ya KAND 11 byabujije gukura kw’imizi mu buryo bushingiye ku gipimo (Ishusho ya 4a, b). Kugira ngo tumenye niba KAND 11 igira ingaruka ku miterere y’imizi, twasuzumye meristems z’imizi zandujwe na propidium iodide (PI) kandi twapimye ingano y’ubuso bwa meristem. Ingano ya meristem y'ingemwe zahinzwe ku gipimo kirimo 25 μM KAND-11 yari 151.1 ± 32.5 μm, mu gihe ingano ya meristem y'ingemwe zahinzwe ku gipimo cy'ubugenzuzi kirimo DMSO yari 264.7 ± 30.8 μm (Ishusho ya 4c, d), bigaragaza ko KAND-11 igarura ibikorwa bya selile. Imizi ya meristem. Dukurikije ibi, uburyo bwo kuvura bwa KAND 11 bwagabanije ingano y'ikimenyetso cyo kugabanya uturemangingo CDKB2; 1p::CDKB2; 1-GUS ikimenyetso mu mizi ya meristem (Ishusho ya 4e) 17. Ibi bisubizo bigaragaza ko KAND 11 ibuza gukura kw'imizi igabanya ibikorwa byo kwiyongera kw'uturemangingo.
Isesengura ry'ingaruka zo kubuza ibikomoka kuri aside ya urbenonic (derivatives ya urbenyloxy) ku mikurire. (a) Imbuto za Col zo mu bwoko bwa wild-type Col z'iminsi 7 zakuriye ku masahani ya MS zifite igipimo cya KAND 11. Umugozi w'igipimo = cm 1. (b) Ingano y'uburebure bw'imizi. Inyuguti zigaragaza itandukaniro rikomeye (Ikizamini cya Tukey HSD, p.< 0.05). n>16. Amakuru agaragazwa nk'ikigereranyo cya ± SD. (c) Mikroskopi ya Confocal y'imizi ya Col ya propidium iodide yakuwe ku mabara ya MS ifite cyangwa idafite 25 μM KAND 11. Uduce tw'umweru tugaragaza mizi ya meristem. Uduce twa scale = 100 µm. (d) Ingano ya meristem y'imizi (n = 10 kugeza 11). Itandukaniro ry'imibare ryagaragajwe hakoreshejwe ikizamini cya t (p)< 0.05). Utubati tugaragaza ingano mpuzandengo ya meristem. (e) Mikroskopi y'itandukaniro ry'itandukaniro (DIC) ya meristem y'umuzi irimo CDKB2; 1pro: CDKB2; 1-GUS yasizwe ibara kandi irangi ku mbuto z'iminsi 5 zakuwe ku masahani ya MS hamwe cyangwa nta gipimo cya KAND cya 25 µM.
Uburozi bwa KAND 11 bwagenzuwe cyane hakoreshejwe ikindi kimera cy’itabi (Nicotiana tabacum), n’ikimera cy’ingenzi cyo mu butaka, liverwort (Marchantia polymorpha). Nk’uko byagenze kuri Arabidopsis, imbuto za SR-1 z’itabi zatewe ku gipimo kirimo 25 μM KAND 11 zatanze imizi migufi (Ishusho ya 5a). Byongeye kandi, imbuto 40 muri 48 zatewe ku masahani arimo 200 μM KAND 11, mu gihe imbuto zose 48 zatewe ku gipimo cya KAND, bigaragaza ko KAND yari nyinshi cyane (p).< 0.05; chi test -square) byabujije kumera kw'itabi. (Ishusho ya 5b). Byongeye kandi, ubwinshi bwa KAND 11 bwabujije gukura kwa bagiteri muri liverwort bwari busa n'ubwinshi bufatika bwa Arabidopsis (Ishusho ya 5c). Ibi byavuye mu bushakashatsi bigaragaza ko KAND 11 ishobora kubuza gukura kw'ibimera bitandukanye. Hanyuma twakoze ubushakashatsi ku burozi bushoboka bw'ibinyabutabire bifitanye isano na monoamide by'idubu mu bindi binyabuzima, ni ukuvuga uturemangingo twa HeLa tw'abantu na Escherichia coli strain DH5α, nk'ibihagarariye uturemangingo twinshi tw'inyamaswa na bagiteri, uko bikurikirana. Mu bushakashatsi bwakozwe ku kwiyongera kw'uturemangingo, twabonye ko coumamonamide 1, aside coumamonamidic 6, na KAND 11 bitagize ingaruka ku mikurire y'uturemangingo twa HeLa cyangwa E. coli ku gipimo cya 100 μM (Ishusho ya 5d,e).
Kubuza KAND 11 gukura mu binyabuzima bitari Arabidopsis. (a) Imbuto z'itabi zo mu bwoko bwa SR-1 zo mu gasozi zimaze ibyumweru bibiri zatewe ku mabara ya MS ahagaze neza arimo 25 μM KAND 11. (b) Imbuto z'itabi zo mu bwoko bwa SR-1 zo mu gasozi zimaze ibyumweru bibiri zatewe ku mabara ya MS ahagaze neza arimo 200 μM KAND 11. (c) Imbuto z'itabi zo mu bwoko bwa Tak-1 zo mu gasozi zimaze ibyumweru bibiri zatewe ku mabara ya Gamborg B5 zifite ingano ya KAND 11. Utwambi dutukura tugaragaza utubuto twahagaze gukura mu gihe cy'ibyumweru bibiri byo gutera. (d) Isuzuma ry'ubwiyongere bw'uturemangingo twa HeLa. Umubare w'uturemangingo dushobora kubaho wapimwe mu gihe gihamye hakoreshejwe uburyo bwo kubara uturemangingo 8 (Dojindo). Nk'igenzura, uturemangingo twa HeLa twavuwe na actinomycin D D ya 5 μg/ml (Act D), ibuza transcription ya RNA polymerase kandi igatera urupfu rw'uturemangingo. Isesengura ryakozwe mu byiciro bitatu. (e) Isuzuma ry'ubwiyongere bw'uturemangingo twa E. coli. Iterambere rya E. coli ryasesenguwe hapimwa OD600. Mu rwego rwo kugenzura, uturemangingo twavuwe na ampicillin ya 50 μg/ml (Amp), ibuza imikorere y'urukuta rw'uturemangingo twa bagiteri. Isesengura ryakozwe mu byiciro bitatu.
Kugira ngo dusobanukirwe uburyo uburozi buterwa n’ibinyabutabire bifitanye isano na uramide bukora, twongeye gusesengura ibintu bikomoka kuri aside urbenique bifite ingaruka zo gukumira. Nk’uko bigaragara ku ishusho. Nkuko bigaragara ku Ishusho ya 2b, 6a, ingemwe zatewe ku masahani ya agar arimo ibipimo byinshi (200 μM) bya aside urmotonic 6 byatanze imizi migufi kandi ifite uburebure bw’ibumoso (θ = – 23.7 ± 6.1), mu gihe ku ngemwe zatewe ku buryo bwo kugenzura, ingemwe zatanze imizi hafi ya yose (θ = – 3.8 ± 7.1). Uku gukura kw’inyuma kuzwiho guturuka ku mikorere mibi ya microtubules14,18. Dukurikije ibi byavumbuwe, imiti igabanya ubushobozi bwa microtubules disopyramide na oryzalin yateje imizi isa n’iyo mu gihe cy’imikurire yacu (Ishusho ya 2b, 6a). Muri icyo gihe, twagerageje ibintu bikomoka kuri aside urmotonic kandi twatoranyije byinshi muri byo, ku bipimo bimwe na bimwe, byatumye imizi ikura. Ibinyabutabire 8, 9, na 15 byahinduye icyerekezo cy'imikurire y'imizi kuri 75 μM, 50 μM, na 40 μM, uko bikurikirana, bigaragaza ko ibi binyabutabire bishobora kwangiza mikorotubule neza (Ishusho ya 2b, 6a). Twanagerageje aside ursolic ikomeye cyane, KAND 11, ku gipimo gito (15 µM) maze dusanga gukoresha KAND 11 byabujije imikurire y'imizi kandi ko icyerekezo cy'imizi cyari kitari gihwanye, nubwo byakundaga kumanuka ibumoso (Ishusho ya C3). Kubera ko ibinyabutabire byinshi by'imiti igabanya imikurire y'imizi rimwe na rimwe bibuza imikurire y'ibimera aho gutera imizi igorama, twaje gusuzuma ko bishoboka ko KAND 11 igira ingaruka kuri mikorotubule twitegereje mikorotubule mu turemangingo tw'imizi y'imizi. Immunohistochemistry ikoresheje antibodies za anti-β-tubulin mu turemangingo tw'imizi y'imbuto zavuwe na 25 μM KAND 11 byagaragaje ko mikorotubule hafi ya zose mu turemangingo tw'imizi y'imizi mu gace k'uburebure (Ishusho ya 6b). Ibi bisubizo bigaragaza ko aside kumamotonic n'ibiyikomokaho bikora ku mikorobe mu buryo butaziguye cyangwa butaziguye kugira ngo biyihungabanye, kandi ko ibyo bintu ari ibintu bishya bibuza mikorobe.
Aside ya Ursonique n'ibiyikomokaho bihindura microtubules za cortical muri Arabidopsis thaliana. (a) Inguni yo kumera kw'imizi yapimwe imbere y'ibikomoka kuri aside urmotonic bitandukanye ku gipimo cyagaragajwe. Ingaruka z'ibintu bibiri bizwiho gukumira microtubules: disopyramide na oryzalin nazo zasesenguwe. Ishusho yerekana igipimo gikoreshwa mu gupima inguni yo gukura kw'imizi. Inyenyeri zigaragaza itandukaniro rikomeye mu kuvura ikinyoma (ikizamini cya t, p< 0.05). n>19. Umugozi w'igipimo = cm 1. (b) Udukoko tw'inyuma mu turemangingo tw'inyuma mu gace k'uburebure. Udukoko tw'inyuma mu mizi ya Arabidopsis yo mu bwoko bwa wild-type Col yakuriye ku dupande twa MS dufite cyangwa tudafite 25 μM KAND 11 twagaragaye hakoreshejwe irangi rya immunohistochemical hakoreshejwe β-tubulin primary antibodies na Alexa Fluor-conjugated secondary antibodies. Umugozi w'igipimo = 10 µm. (c) Imiterere ya mitotubules muri meristem y'umuzi. Udukoko tw'inyuma twagaragaye hakoreshejwe irangi rya immunohistochemical. Imiterere ya mitotubule, harimo prophase zones, spindles, na phragmoplasts, yabazwe uhereye ku mashusho ya confocal. Imyambi igaragaza imiterere ya mitotubules. Inyenyeri zigaragaza itandukaniro rikomeye mu kuvura sham (ikizamini cya t, p< 0.05). n>9. Umwanya w'igipimo = 50 µm.
Nubwo Ursa ifite ubushobozi bwo kubangamira imikorere ya microtubule, imikorere yayo byitezwe ko itandukanye n’iy’imiti isanzwe ya microtubule depolymerizing. Urugero, ubwinshi bw’imiti ya microtubule depolymerizing nka disopyramide na oryzalin butera kwaguka kwa anisotropic kwa selile z’uruhu, mu gihe KAND 11 itabikora. Byongeye kandi, gukoresha hamwe KAND 11 na disopyramide byatumye habaho igisubizo cyo gukura kw’imizi giterwa na disopyramide hamwe kandi hagaragaye ko hari ukwirinda gukura kwa KAND 11 (Ishusho ya S4). Twanasesenguye uburyo disopyramide 1-1 (phs1-1) itameze neza kuri KAND 11. phs1-1 ifite imiterere idashingiye ku buziranenge bwa tubulin kinase kandi ikora imizi migufi iyo ivuwe na disopyramide9,20. Imiti ya phs1-1 itameze neza yakuriye ku buryo bwa agar irimo KAND 11 yari ifite imizi migufi isa n’iyakuriye kuri disopyramide (Ishusho ya S5).
Byongeye kandi, twabonye imiterere ya mitotubule ya mitotike, nka prophase zones, spindles, na phragmoplasts, mu mizi y'imbuto zavuwe na KAND 11. Dukurikije ibyagaragaye kuri CDKB2; 1p::CDKB2; 1-GUS, hagaragaye igabanuka rikomeye ry'umubare wa mitotubule ya mitotike (Ishusho ya .6c).
Kugira ngo tumenye uburozi bwa KAND 11 ku turemangingo duto tw’itabi, twavuje uturemangingo twa BY-2 dukoresheje KAND 11 kandi twitegereza uko twabyitwaramo. Twabanje kongeramo KAND 11 ku turemangingo twa BY-2 tugaragaza TagRFP-TUA6, dushyira ikimenyetso ku turemangingo duto tw’amashanyarazi, kugira ngo dusuzume ingaruka za KAND 11 ku turemangingo duto tw’ingirabuzimafatizo. Ubucucike bwa turemangingo duto tw’ingirabuzimafatizo bwapimwe hakoreshejwe isesengura ry’ishusho, ryapimye igipimo cya pixel za cytoskeletal muri pixel za cytoplasmic. Ibisubizo by’isuzuma byagaragaje ko nyuma yo kuvurwa na 50 μM cyangwa 100 μM KAND 11 mu gihe cy’isaha 1, ubucucike bwagabanutse cyane bugera kuri 0.94 ± 0.74% cyangwa 0.23 ± 0.28%, mu gihe ubucucike bw’uturemangingo twavuwe na DMSO, bwari 1.61 ± 0.34% (Ishusho ya 7a). Ibi bisubizo bihuye n'ibyagaragaye muri Arabidopsis ko uburyo bwo kuvura bwa KAND 11 butera gukuraho polymerization ya microtubules za cortical (Ishusho ya 6b). Twanasuzumye umurongo wa BY-2 hamwe na filaments za actin zanditseho GFP-ABD nyuma yo kuvurwa hakoreshejwe ingano imwe ya KAND 11, maze tubona ko uburyo bwo kuvura bwa KAND 11 bwabangamiye filaments za actin. Ubu buryo bwo kuvura hakoreshejwe 50 μM cyangwa 100 μM KAND 11 mu gihe cy'isaha 1 bwagabanyije cyane ubucucike bwa filaments za actin bugera kuri 1.20 ± 0.62% cyangwa 0.61 ± 0.26%, mu gihe ubucucike mu turemangingo twavuwe na DMSO bwari 1.69 ± 0.51% (Ishusho ya 2). 7b). Ibi bisubizo bitandukanye n'ingaruka za propyzamide, itagira ingaruka ku mikorobe za actin, na latrunculin B, actin depolymerizer itagira ingaruka ku mikorobe (Ishusho ya SI S6). Byongeye kandi, kuvura hakoreshejwe coumamonamide 1, aside coumamonamide 6, cyangwa KAND 11 ntacyo byagize ku tunyangingo duto mu tunyangingo twa HeLa (Ishusho ya SI S7). Bityo, uburyo KAND 11 ikora bugaragara ko butandukanye n’uburyo abantu bazwiho kuvumbura amagufwa ya cytoskeleton. Byongeye kandi, kureba kwacu muri microscope ku tunyangingo twa BY-2 twavuwe na KAND 11 byagaragaje ko ingirabuzimafatizo zapfuye mu gihe cyo kuvurwa na KAND 11 byatangiye, kandi byagaragaje ko igipimo cy’utunyangingo twapfuye twa Evans kitigeze cyiyongereye cyane nyuma y’iminota 30 yo kuvurwa na KAND 11, mu gihe nyuma y’iminota 90 yo kuvurwa na 50 μM cyangwa 100 μM KAND, umubare w’utunyangingo twapfuye wiyongereye ugera kuri 43.7% cyangwa 80.1%, uko bikurikirana (Ishusho ya 7c). Iyo mibare yose hamwe igaragaza ko KAND 11 nshya ikomoka kuri aside ursolic ari ikintu gikingira cytoskeletal mu bimera gifite uburyo kitazwi mbere.
KAND igira ingaruka ku tunyangingo tw’uturemangingo tw’uturemangingo, imiyoboro ya actin, n’ubushobozi bw’utunyangingo twa BY-2 tw’itabi. (a) Kureba neza utunyangingo tw’uturema ...< 0.05). Umwanya w'igipimo = 10 µm. (b) Uduce twa actine ya cortical mu turemangingo twa BY-2 twagaragaye imbere ya GFP-ABD2. Uturemangingo twa BY-2 twavuwe na KAND 11 (50 μM cyangwa 100 μM) cyangwa DMSO twasuzumwe hakoreshejwe mikorosikope ya confocal. Ubucucike bwa filaments ya actine ya cortical bwabazwe hashingiwe kuri mikorografi z'uturemangingo 25 twigenga. Inyuguti zigaragaza itandukaniro rikomeye (Ikizamini cya Tukey HSD, p< 0.05). Umugozi w'igipimo = 10 µm. (c) Kwitegereza uturemangingo twa BY-2 twapfuye hakoreshejwe irangi ry'ubururu rya Evans. Uturemangingo twa BY-2 twavuwe na KAND 11 (50 μM cyangwa 100 μM) cyangwa DMSO byasuzumwe hakoreshejwe mikorosikopi y'umurima w'ubugari. n=3. Umugozi w'igipimo = 100 µm.
Kuvumbura no gukoresha ibintu bishya karemano byatumye habaho iterambere rikomeye mu bice bitandukanye by'ubuzima bw'abantu, harimo ubuvuzi n'ubuhinzi. Ubushakashatsi bw'amateka bwakozwe kugira ngo haboneke ibintu by'ingirakamaro bivuye mu mutungo kamere. By'umwihariko, actinomycetes zizwiho kuba ingirakamaro nk'imiti yica udukoko twangiza udukoko bitewe n'ubushobozi bwazo bwo gukora ibintu bitandukanye bya kabiri nka avermectin, imvange y'ingenzi ya ivermectin na bleomycin n'ibikomoka kuri yo, ikoreshwa mu buvuzi nk'umuti urwanya kanseri21,22. Mu buryo nk'ubwo, ibintu bitandukanye byangiza ibyatsi byavumbuwe muri actinomycetes, bimwe muri byo bikaba bikoreshwa mu bucuruzi1,23. Kubwibyo, isesengura rya actinomycete metabolites kugira ngo hamenyekane ibintu karemano bifite ibikorwa by'ibinyabuzima byifuzwa rifatwa nk'ingamba nziza. Muri ubu bushakashatsi, twavumbuye ikintu gishya, coumamonamide, gikomoka kuri S. werraensis kandi twakigize neza. Aside ya Ursonic ni igice cy'ubukorano cya urbenamide n'ibikomoka kuri yo. Ishobora gutera guhindagurika kw'imizi, ikagaragaza imikorere iri hagati kugeza ku ikomeye yo kwica ibyatsi, kandi ikangiza microtubules z'ibimera mu buryo butaziguye cyangwa butaziguye. Ariko, uburyo aside urmotonic ikora bushobora gutandukana n’uburyo aside urmotonic ikora busanzwe, kuko KAND 11 inabangamira filaments za actin igatera urupfu rw’uturemangingo, ibyo bikaba bigaragaza uburyo aside urmotonic n’ibikomoka kuri yo bigira ingaruka ku miterere myinshi ya cytoskeletal.
Isesengura rirambuye rya aside urbenonic rizafasha gusobanukirwa neza uburyo aside urbenonic ikora. By'umwihariko, intego ikurikiraho ni ugusuzuma ubushobozi bwa aside ursonic bwo gufatana na microtubules zagabanutse kugira ngo hamenyekane niba aside ursonic n'ibikomoka kuri yo bikora ku mikorotubules no kuzikuraho polymer, cyangwa niba ibikorwa byabyo bitera microtubules kudakomera. Byongeye kandi, mu gihe microtubules atari intego itaziguye, kumenya aho aside ursonic ikorera n'intego za molekile za aside ursonic ku turemangingo tw'ibimera bizafasha gusobanukirwa neza imiterere y'ibinyabutabire bifitanye isano n'uburyo bushoboka bwo kunoza imikorere y'ibyatsi. Isuzuma ryacu ry'imikorere y'ibinyabuzima ryagaragaje ubushobozi bwihariye bwa aside ursonic mu gukura kw'ibimera nka Arabidopsis thaliana, itabi na liverwort, mu gihe nta turemangingo twa E. coli cyangwa HeLa byagizweho ingaruka. Uburozi buke cyangwa nta na bumwe ku turemangingo tw'inyamaswa ni inyungu y'ibikomoka kuri aside ursonic iyo byakozwe nk'imiti yica ibyatsi ikoreshwa mu mirima y'ubuhinzi. Koko rero, kubera ko microtubules ari imiterere isanzwe muri eukaryotes, kuzibuza mu bimera ni ikintu cy'ingenzi gisabwa ku miti yica ibyatsi. Urugero, propyzamide, umuti ugabanya uburozi bw’uturemangingo duto dufatana na tubulin kandi ukabuza polymerization, ukoreshwa nk'umuti w’ibyatsi bitewe n’uburozi buke ku turemangingo tw’inyamaswa24. Bitandukanye na disopyramide, benzamides zifitanye isano nazo zifite imiterere itandukanye. Uretse uturemangingo duto tw’ibimera, RH-4032 cyangwa benzoxamide inabuza uturemangingo duto tw’uturemangingo tw’inyamaswa cyangwa oomycetes, uko bikurikirana, na zalilamide ikoreshwa nk'umuti urwanya fungi bitewe n’uburozi buke bw’ibimera25,26,27. Idubu riherutse kuvumburwa n’ibikomoka kuri ryo bigaragaza uburozi bwihariye ku bimera, ariko ni ngombwa kumenya ko izindi mpinduka zishobora guhindura imiterere yabyo, bishobora gutanga ibindi bikomoka ku bimera mu kugenzura ibihumyo cyangwa oomycetes.
Imiterere yihariye ya aside urbenonic n'ibiyikomokaho ni ingirakamaro mu iterambere ryayo nk'imiti yica udukoko no kuyikoresha nk'ibikoresho by'ubushakashatsi. Akamaro k'amagufwa ya cytoskeleton mu kugenzura imiterere y'uturemangingo tw'ibimera kazwi cyane. Ubushakashatsi bwa mbere bwagaragaje ko ibimera byahinduye uburyo bugoye bwo gutunganya microtubule ya cortical binyuze mu kugenzura imikorere ya microtubule kugira ngo bigenzure neza imiterere yayo. Umubare munini w'uturemangingo dushinzwe kugenzura imikorere ya microtubule waragaragaye, kandi ubushakashatsi bujyanye nabyo buracyakomeje3,4,28. Ubu buryo dusobanukiwe ku mikorere ya microtubule mu turemangingo tw'ibimera ntibusobanura neza imikorere ya microtubule ya cortical. Urugero, nubwo disopyramide na oryzalin byombi bishobora gukuraho polymeri ya microtubule, disopyramide itera guhindagurika gukomeye kw'imizi mu gihe oryzalin igira ingaruka ntoya. Byongeye kandi, impinduka muri tubulin, ituma microtubule zituza, nazo zitera dextrorotation mu mizi, mu gihe paclitaxel, inatuza microtubule, ntabwo ibikora. Kubwibyo, kwiga no kumenya intego za molekile za aside ursolic bigomba gutanga ubumenyi bushya ku mikorere ya microtubule ya cortical y'ibimera. Mu buryo nk'ubwo, kugereranya ibinyabutabire bizaza mu gihe kizaza bifite akamaro mu guteza imbere imikurire idahwitse, nka disopyramide, n'ibinyabutabire bidahwitse, nka oryzalin cyangwa aside kumamotoric, bizatanga ibimenyetso by'uburyo imikurire idahwitse ibaho.
Ku rundi ruhande, gusubiranyamo kwa cytoskeletal bifitanye isano n’ubwirinzi ni ubundi buryo bwo gusobanura uburozi bwa aside ursonic. Kwandura kwanduza indwara cyangwa kwinjiza encitor mu turemangingo tw’ibimera rimwe na rimwe bitera kwangirika kwa cytoskeleton ndetse n’urupfu rw’uturemangingo 29. Urugero, cryptoxanthin ikomoka kuri oomycete byavuzwe ko yangiza microtubules na actin filaments mbere y’uko uturemangingo tw’itabi dupfa, nk’uko bibaho mu kuvura KAND30,31. Kusa hagati y’ubwirinzi n’ubwirinzi buterwa na aside ursonic byatumye dutekereza ko bitera inzira zisanzwe z’uturemangingo, nubwo ingaruka zihuse kandi zikomeye za aside ursonic kurusha cryptoxanthin zigaragara. Ariko, ubushakashatsi bwagaragaje ko guhungabana kwa filaments za actin bituma uturemangingo dupfa mu buryo bwihuse, bitajya biherekezwa no guhungabana kwa microtubules29. Byongeye kandi, haracyari ngombwa kureba niba iyo pathogen cyangwa encitor itera gukura nabi kw’imizi, nk’uko ibintu bikomoka kuri aside ursonic bibikora. Bityo, ubumenyi bwa molekile buhuza ubwirinzi n’amagufwa ya cytoskeleton ari ikibazo gikurura gukemurwa. Mu gukoresha ibintu bifite uburemere buke bwa molekile bifitanye isano na aside uronic, hamwe n'ibindi bintu bitandukanye bifite imbaraga zitandukanye, bishobora gutanga amahirwe yo kwibasira imikorere itazwi y'uturemangingo.
Muri rusange, kuvumbura no gukoresha ibintu bishya bihindura imikorere ya microtubule bizatanga uburyo bukomeye bwo gukemura imikorere y’uturemangingo tw’ibimera. Muri uru rwego, aside urmotonic iherutse gutegurwa, igira ingaruka ku mikorotubule na filaments za actin kandi igatera urupfu rw’uturemangingo, ishobora gutanga amahirwe yo gusobanukirwa isano iri hagati yo kugenzura microtubule n’izi nzira. Bityo, isesengura rya chimique na biologique hakoreshejwe aside urbenonic rizadufasha gusobanukirwa imikorere ya molekile igenzura cytoskeleton y’ibimera.
Shyira S. werraensis MK493-CF1 mu icupa rya Erlenmeyer rya mL 500 rigizwe na mL 110 z'imbuto zigizwe na galactose ya 2% (w/v) 2% (w/v) Essence paste, 1% (w/v) Bacto composition. -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) ibigori bivamo (KOGOSTCH Co., Ltd., Ubuyapani), 0.2% (w/v) (NH4)2SO4 na 0.2% CaCO3 mu mazi yakuwemo iyoni. (pH 7.4 mbere yo gusukura). Imbuto zashyizwe mu cyuma gishyushya amazi (180 rpm) kuri 27°C mu gihe cy'iminsi 2. Guhinga hakoreshejwe uburyo bwo gushyushya amazi. Imbuto zo mu bwoko bwa fermentation (7 ml) zashyizwe mu icupa rya K-1 rifite garama 500 z'uburyo bwo gukora bugizwe na garama 15 za sayiri (MUSO Co., Ltd., mu Buyapani) na garama 25 z'amazi yakuwemo iyoni (pH ntirahindurwa mbere yo koroshya). Gushyushya byakozwe kuri dogere selisiyusi 30 mu mwijima mu gihe cy'iminsi 14. Ibikoresho byo gushyushya byakuwemo mililitiro 40/icupa rya EtOH hanyuma bishyirwamo (1500 g, 4°C, iminota 10). Supernatant yo gushyushya (60 ml) yakuwemo uruvange rwa 10% MeOH/EtOAc. Urusobe rw'ibinyabuzima rwahinduwe umwuka bitewe n'igitutu gito kugira ngo haboneke ibisigazwa (59.5 mg), byashyizwe muri HPLC hakoreshejwe uburyo bwo gupima ubuziranenge (iminota 0–10: 90%) ku gice cyo hagati (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × uburebure 250 mm) H2O/CH3CN, iminota 10–35: 90% H2O/CH3CN kugeza 70% H2O/CH3CN (ubuziranenge), iminota 35–45: 90% H2O/EtOH, iminota 45–155: 90% H2O/EtOH kugeza 100% EtOH (ubuziranenge (ubuziranenge), iminota 155–200: 100% EtOH) ku gipimo cy'amazi cya 1.5 ml/min, coumamonamide (1, 36.0 mg) yakuwemo ifu yera idahinduka.
Kumamotoamide(1); 1H-NMR (500 MHz, CDCl3) δ 6.93 (t, J = 2.5 Hz, 1H), 6.76 (dd, J = 4.3, 1.8 Hz 1H), 6.05 (t , J = 3.8 Hz, 1H). ), 4.08 (s, 3H); 13C-NMR (125 MHz, CDCl3) δ 161.1, 121.0, 119.9, 112.2, 105.0, 68.3; ESI-HRMS [M+H]+: [C6H9N2O2]+ agaciro kabazwe: 141.0659, agaciro kapimwe: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Imbuto za Columbia (Col-0) zavuye mu kigo cyita ku buzima bw’ibinyabuzima cya Arabidopsis (ABRC) ku ruhushya rwo kuzikoresha mu bushakashatsi. Imbuto za Col-0 zarakwirakwijwe kandi zibungabungwa mu buryo bwa laboratwari yacu kandi zikoreshwa nk'ibimera bya Arabidopsis byo mu gasozi. Imbuto za Arabidopsis zasizwe umuti wo mu butaka kandi zihingwa mu buryo bungana na Murashige na Skoog bufite 2% ya sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic aside (MES) (Fujifilm Wako Pure Chemical). ) na 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, kuri 23 °C n'urumuri rudahinduka. Imbuto za phs1-1 mutant zatanzwe na T. Hashimoto (Ikigo cya Siyansi n'Ikoranabuhanga cya Nara).
Imbuto z’ubwoko bwa SR-1 zatanzwe na T. Hashimoto (Ikigo cya Siyansi n’Ikoranabuhanga cya Nara) kandi zikoreshwa nk’ibimera by’itabi byo mu gasozi. Imbuto z’itabi zasizwe umuti uvura hejuru hanyuma zijugunywa mu mazi yanduye mu gihe cy’iminsi itatu kugira ngo zitere imbere kumera, hanyuma zishyirwa mu gikoresho cy’ingufu ebyiri kirimo 2% ya sucrose, 0.05% (w/v) MES, na 0.8% ya gellan gum (Fujifilm Wako Pure Chemical) Murashige. na Skoog medium) ifite pH ya 5.7 hanyuma zishyirwa ku bushyuhe bwa 23°C munsi y’urumuri rudasibangana.
Strain Tak-1 yatanzwe na T. Kohchi (Kaminuza ya Kyoto) kandi yakoreshejwe nk'igice gisanzwe cyo kugerageza ubushakashatsi bwa liverwort. Gemma yakuwe mu bimera byakuwemo imiti isukuye hanyuma ishyirwa ku gipimo cya Gamborg B5 (Fujifilm Wako Pure Chemical) kirimo 1% ya sucrose na 0.3% ya gellan gum hanyuma ishyirwa ku bushyuhe bwa 23°C munsi y'urumuri rudashira.
Uturemangingo twa BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) twatanzwe na S. Hasezawa (Kaminuza ya Tokyo). Uturemangingo twa BY-2 twavanzwe inshuro 95 mu buryo bwa Linsmeier na Skoog kandi twongerwamo aside 2,4-dichlorophenoxyacetic 32 buri cyumweru. Uduce twavanzwe ku cyuma gihindura ikirere ku muvuduko wa 130 rpm kuri 27°C mu mwijima. Oza uturemangingo inshuro 10 z'uburyo bushya hanyuma wongere utwongere muri ubwo buryo. Uturemangingo twa BY-2 duhindura imiterere y'uturemangingo twerekana neza ikimenyetso cya microtubule TagRFP-TUA6 cyangwa ikimenyetso cya actin filament GFP-ABD2 munsi ya virusi ya cauliflower mosaic 35S promoter byakozwe nkuko byasobanuwe33,34,35. Iyi mirongo y'uturemangingo ishobora gukomeza no guhuzwa hakoreshejwe uburyo busa n'ubwakoreshejwe ku murongo w'uturemangingo twa BY-2 w'umwimerere.
Uturemangingo twa HeLa twatewe mu buryo bwa Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) hiyongereyeho 10% by'amaraso y'inka yo mu nda, 1.2 U/ml ya penisiline, na 1.2 μg/ml ya streptomycin mu cyuma gishyushya ubushyuhe bwa 37°C gifite 5% bya CO2.
Igerageza ryose ryavuzwe muri iyi nyandiko ryakozwe hakurikijwe amabwiriza n'amabwiriza agenga umutekano w'ibinyabuzima mu Buyapani.
Ibinyabutabire byashongeshejwe muri dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) nk'ibisubizo by'inyama hanyuma bivangwa mu buryo bwa MS kuri Arabidopsis n'itabi cyangwa Gamborg B5 medium kuri liverwort. Ku isuzuma ry'imizi ribuza gukura, imbuto zirenga 10 kuri buri sahani zatewe ku buryo bwa agar burimo ibinyabutabire byagaragajwe cyangwa DMSO. Imbuto zashyizwe mu cyumba cyo gukuramo imyaka mu gihe cy'iminsi 7. Imbuto zafotowe maze uburebure bw'imizi burapimwa. Ku isuzuma ry'imikurire ya Arabidopsis, imbuto 48 kuri buri sahani zatewe ku buryo bwa agar burimo 200 μM medium cyangwa DMSO. Imbuto za Arabidopsis zatewe mu cyumba cyo gukuramo imyaka kandi umubare w'imbuto zavutsemo warabazwe nyuma y'iminsi 7 (dag). Ku isuzuma ry'imikurire y'itabi, imbuto 24 kuri buri sahani zatewe ku buryo bwa agar burimo 200 μM KAND cyangwa DMSO. Imbuto z'itabi zatewe mu cyumba cyo gukuramo imyaka kandi umubare w'imbuto zavutsemo warabazwe nyuma y'iminsi 14. Ku isuzuma ry’imikurire y’uturemangingo, intanga 9 zavuye kuri buri sahani zashyizwe ku gipimo cya agar kirimo urugero rwa KAND cyangwa DMSO rwagaragajwe hanyuma zishyirwa mu cyumba cy’imikurire mu gihe cy’iminsi 14.
Koresha ingemwe zasizweho ibara rya 5 mg/ml rya propidium iodide (PI) kugira ngo urebe imiterere y'imizi ya meristem. Ibimenyetso bya PI byagaragaye hakoreshejwe mikorosikopi ya fluorescence hakoreshejwe mikorosikopi ya TCS SPE confocal laser scanning (Leica Microsystems).
Gusiga irangi ry’imizi histochemical hakoreshejwe β-glucuronidase (GUS) byakozwe hakurikijwe amabwiriza yavuzwe na Malami na Benfey36. Ingemwe zashyizwe muri 90% ya acetone mu ijoro ryose, zishyirwaho 0.5 mg/ml ya 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acide muri GUS buffer mu gihe cy’isaha 1 hanyuma zishyirwa mu gikoresho cya chloraldehyde gifite amazi. (8 g ya chloral hydrate, 2 ml y’amazi na 1 ml ya glycerol) kandi bireberwa hakoreshejwe mikorosikopi ya differential interference contrast hakoreshejwe mikorosikopi ya Axio Imager M1 (Carl Zeiss).
Inguni z'imizi zapimwe ku mbuto z'iminsi 7 zatewe ku masahani ahagaze. Pima inguni y'imizi ukurikije icyerekezo cy'umuvuduko nk'uko byasobanuwe mu ntambwe ya 6.
Imiterere ya mikorotubule za cortical yagaragaye nk'uko byasobanuwe, hamwe n'impinduka nto kuri protocole 37. Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) na Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) byakoreshejwe nk'ingufu z'ibanze n'iz'inyongera kuri dilutions ya 1:1000 na 1:100, uko bikurikirana. Amashusho ya Fluorescence yabonetse hakoreshejwe mikorosikopi ya TCS SPE confocal laser scanning (Leica Microsystems). Shaka amashusho ya Z-stack kandi ukore projections y'ubukana ntarengwa hakurikijwe amabwiriza y'uwakoze.
Isuzuma ry’ubwiyongere bw’uturemangingo twa HeLa ryakozwe hakoreshejwe uburyo bwa 8 bwo kubara uturemangingo (Dojindo) hakurikijwe amabwiriza y’uwakoze.
Ikura rya E. coli DH5α ryasesenguwe hapimwa ubucucike bw'uturemangingo mu mikurire hakoreshejwe spectrophotometer kuri 600 nm (OD600).
Imiterere ya cytoskeletal mu turemangingo twa BY-2 twahinduwe mu ngirabuzimafatizo yabonetse hakoreshejwe mikorosikopi y’urumuri ifite igikoresho cya CSU-X1 cyo gupima confocal (Yokogawa) na kamera ya sCMOS (Zyla, Andor Technology). Ubucucike bwa cytoskeletal bwapimwe hakoreshejwe isesengura ry’amashusho, ryapimye ijanisha rya pixels za cytoskeletal muri pixels za cytoplasmic mu mashusho ya confocal hakoreshejwe porogaramu ya ImageJ nk’uko byasobanuwe38,39.
Kugira ngo hamenyekane urupfu rw'uturemangingo mu turemangingo twa BY-2, igice kimwe cy'uturemangingo cyashyizwemo ubururu bwa Evans bwa 0.05% mu gihe cy'iminota 10 ku bushyuhe bw'icyumba. Gusiga irangi ry'ubururu rya Evans ku turemangingo twapfuye biterwa no gusohora irangi mu turemangingo tuzima hakoreshejwe membrane ya plasma idahinduka40. Uturemangingo twagaragaye hakoreshejwe mikorosikopi y'ikirere cyiza (BX53, Olympus).
Uturemangingo twa HeLa twakuriye muri DMEM twongerewemo 10% FBS mu cyuma gishyushye gifite ubushyuhe kuri 37°C na 5% CO2. Uturemangingo twavuwe na 100 μM KAND 11, aside kumamonamic 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), cyangwa 100 ng/ml Nocodmaze (Sigma) mu gihe cy'amasaha 6 kuri 37°C. Uturemangingo twakosowe na MetOH mu gihe cy'iminota 10 hanyuma dushyirwamo acetate mu gihe cy'iminota 5 ku bushyuhe bw'icyumba. Uturemangingo twakosowe twashyizwemo antibody y'ibanze ya β-tubulin (1D4A4, Proteintech: 66240-1) yakuwemo 0.5% BSA/PBS mu gihe cy'amasaha 2, twozwa inshuro 3 na TBST, hanyuma dushyirwamo antibody y'ihene ya Alexa Fluor. 488 Isaha 1. – Imbeba IgG (Thermo Fisher Scientific: A11001) na 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) yakuwemo 0.5% BSA/PBS. Nyuma yo gukaraba na TBST inshuro eshatu, uturemangingo twanduye twagaragaye kuri mikorosikopi ya Nikon Eclipse Ti-E. Amashusho yafashwe hifashishijwe kamera ikonje ya Hamamatsu ORCA-R2 CCD hakoreshejwe porogaramu ya MetaMorph (Molecular Devices).
Igihe cyo kohereza: Kamena-17-2024