Mahalo no kou kipa ʻana iā Nature.com.ʻO ka mana o ka polokalamu kele pūnaewele āu e hoʻohana nei he kākoʻo CSS palena ʻole.No nā hualoaʻa maikaʻi loa, paipai mākou e hoʻohana ʻoe i kahi mana hou o kāu polokalamu kele pūnaewele (a i ʻole e hoʻopau i ke ʻano Compatibility Mode ma Internet Explorer).I kēia manawa, e hōʻoia i ke kākoʻo mau ʻana, ke hōʻike nei mākou i ka pūnaewele me ka ʻole o ka styling a i ʻole JavaScript.
ʻO ka ʻike a me ka hoʻohana pono ʻana i nā huahana kūlohelohe hiki ke kōkua i ka hoʻomaikaʻi ʻana i ke ola kanaka.Hoʻohana nui ʻia nā kemika pale ulu ulu e like me nā herbicides e hoʻopaʻa ai i ka mauʻu.Ma muli o ka pono e hoʻohana i nā ʻano herbicides like ʻole, pono e ʻike i nā pūhui me nā hana hou o ka hana.Ma kēia haʻawina, ua ʻike mākou i kahi pūhui N -alkoxypyrrole hou, coumamonamide, mai Streptomyces werraensis MK493-CF1 a hoʻokumu i ke kaʻina hana synthesis piha.Ma o ka ho'āʻo ʻana o ka hana biological, ua ʻike mākou he urs-monoamic acid kahi mea hoʻohuihui synthetic o urs-monoamide a me kahi mea hiki.mea hoʻohiolo ulu.Eia hou, ua hoʻomohala mākou i nā ʻano urbenonic acid derivatives, me ka urbenyloxy derivative (UDA), nona ka hana herbicidal kiʻekiʻe me ka ʻole o ka hopena maikaʻi ʻole i ka ulu ʻana o nā cell HeLa.Ua ʻike pū mākou ua hoʻopau nā mea hoʻoheheʻe urmotonic acid i nā microtubules mea kanu;Eia kekahi, pili ka KAND i nā filament actin a hoʻoulu i ka make cell;ʻOkoʻa kēia mau hopena multifaceted mai nā mea hoʻopiʻi microtubule i ʻike ʻia a hōʻike i kahi hana hou o ka hana no ka waika ursonic, ʻo ia ka mea e hōʻike ai i kahi pōmaikaʻi nui i ka hoʻomohala ʻana i nā herbicides hou.
ʻO ka ʻike ʻana a me ka hoʻohana pono ʻana i nā huahana kūlohelohe maikaʻi a me kā lākou derivatives kahi ala e hoʻomaikaʻi ai i ka maikaʻi o ke ola kanaka.ʻO nā metabolites lua i hana ʻia e nā microorganisms, nā mea kanu a me nā inike i alakaʻi i ka holomua nui i ka lāʻau lapaʻau a me ka mahiʻai.Nui nā lāʻau antibiotic a me nā lāʻau anti-leukemia i kūkulu ʻia mai nā huahana kūlohelohe.Eia kekahi, nā ʻano like ʻole opesticides, Wehe ʻia nā fungicides a me nā herbicides mai kēia mau huahana kūlohelohe no ka hoʻohana ʻana i ka mahiʻai.ʻO ka mea nui, he mau mea hana koʻikoʻi ka weed control herbicides no ka hoʻonui ʻana i nā hua i ka mahiʻai hou, a ua hoʻohana ʻia nā ʻano mea like ʻole ma ke kālepa.ʻO kekahi mau kaʻina kelepona i loko o nā mea kanu, e like me photosynthesis, amino acid metabolism, cell wall synthesis, regulation of mitosis, phytohormone signaling, a i ʻole synthesis protein, i manaʻo ʻia he mau pahuhopu maʻamau o nā herbicides.ʻO nā pūhui e kaohi ana i ka hana microtubule he ʻano lāʻau lapaʻau maʻamau e pili ana i ka ulu ʻana o ka mea kanu ma o ka hoʻopili ʻana i ka mitotic regulation2.
ʻO nā microtubules nā ʻāpana o ka cytoskeleton a mālama nui ʻia i nā cell eukaryotic.ʻO ka heterodimer tubulin he α-tubulin a me ka β-tubulin e hana ana i nā protofilaments microtubule linear, me 13 protofilaments e hana ana i kahi ʻano cylindrical.Hoʻokani nā microtubules i nā hana he nui i nā mea kanu, me ka hoʻoholo ʻana i ke ʻano o ka cell, ka mahele cell, a me ka transport intracellular3,4.Loaʻa i nā mea kanu nā microtubules ma lalo o ka membrane plasma interphase, a ua manaʻo ʻia kēia mau mea i kapa ʻia ʻo cortical microtubules e hoʻomalu i ka hoʻonohonoho ʻana o nā microfibrils cellulose ma o ka hoʻoponopono ʻana o ka cellulose synthase complexes4,5.ʻO nā microtubules cortical o nā pūnaewele epidermal aʻa, aia i loko o ka ʻāpana o ka elongation wikiwiki o ke kumu aʻa, aia ma ka ʻaoʻao, a me nā microfibers cellulose e hahai i kēia mau microtubules a kaupalena i ke kuhikuhi o ka hoʻonui ʻana o ke kelepona, a laila e hoʻoikaika ana i ka elongation cell anisotropic.No laila, pili pili ka hana microtubule i ka morphology mea kanu.ʻO ka hoʻololi ʻana i ka waikawa amino i loko o nā genes encoding tubulin ke kumu skew o cortical microtubule arrays a me ka ulu hema-a ʻaoʻao ʻākau paha ma Arabidopsis 6,7.Pēlā nō, hiki i ka hoʻololi ʻana i nā protein pili i ka microtubule e hoʻoponopono i ka dynamics microtubule hiki ke alakaʻi i ka ulu ʻana o ke aʻa 8,9,10,11,12,13.Eia kekahi, ʻo ka mālama ʻana me nā microtubule-disrupting herbicides e like me disopyramide, ʻike ʻia hoʻi ʻo pretilachlor, ke kumu hoʻi i ka ulu ʻana o ke aʻa oblique ʻaoʻao hema14.Hōʻike kēia mau ʻikepili he mea koʻikoʻi ka hoʻoponopono pololei o ka hana microtubule no ka hoʻoholo ʻana i ke kuhikuhi o ka ulu ʻana o ka mea kanu.
Ua ʻike ʻia nā ʻano like ʻole o ka microtubule inhibitors, a ua hāʻawi nui kēia mau lāʻau i ka noiʻi cytoskeletal, a me ka mahiʻai a me ka lāʻau lapaʻau2.ʻO ka mea nui, oryzalin, dinitroaniline pūhui, disopyramide, benzamide-pili pūhui, a me kā lākou mau analogs hiki ke kāohi i ka hana microtubule a ma laila e pale ai i ka ulu ʻana o ka mea kanu.No laila, hoʻohana nui ʻia lākou e like me nā herbicides.Eia naʻe, no ka mea he mea nui nā microtubules o nā mea kanu a me nā mea holoholona, ʻo ka hapa nui o nā microtubule inhibitors he cytotoxic i nā ʻano cell ʻelua.No laila, ʻoiai ʻo kā lākou pono i ʻike ʻia he herbicides, ua hoʻohana ʻia kahi helu liʻiliʻi o nā mea hana antimicrotubule no nā kumu hana.
ʻO Streptomyces kahi ʻano o ka ʻohana Streptomyces, e komo pū ana me ka aerobic, gram-positive, filamentous bacteria a ua ʻike nui ʻia no kona hiki ke hana i nā ʻano metabolites lua.No laila, ua manaʻo ʻia ʻo ia kekahi o nā kumu koʻikoʻi o nā huahana kūlohelohe kūlohelohe hou.Ma ka noiʻi o kēia manawa, ua ʻike mākou i kahi hui hou i kapa ʻia ʻo coumamonamide, i hoʻokaʻawale ʻia mai Streptomyces werraensis MK493-CF1 a me S. werraensis ISP 5486. Ma ka hoʻohana ʻana i ka spectral analysis a me ka spectral analysis piha, ua hōʻike ʻia ke ʻano o ka coumamonamide a me kāna iwi N-alkoxypyrrole kūikawā. ua hooholoia.hoʻohuihui.ʻO ka waika Ursmonic, kahi kikowaena synthetic o ursmonoamide a me kāna mau derivatives, ua ʻike ʻia e kāohi i ka ulu a me ka germination o ka mea kanu kumu hoʻohālike kaulana ʻo Arabidopsis thaliana.I loko o kahi haʻawina pili ka hana-hana, ua ʻike mākou ua hoʻololi ʻia kahi hui me C9 i ka waika ursonic, i kapa ʻia nonyloxy derivative o ka ursonic acid (KAND), hoʻonui nui i ka hopena pale i ka ulu a me ka germination.ʻO ka mea nui, ua hoʻopilikia ʻia ka ulu ʻana o ka paka a me ka liverwort a ʻaʻole ia he cytotoxic i ka bacteria a i ʻole nā pūnaewele HeLa.Eia kekahi, ʻo kekahi mau mea urmotonic acid derivatives e hoʻoulu ai i kahi phenotype aʻa i hoʻoheheʻe ʻia, e hōʻike ana i kēia mau derivatives e hoʻopili pololei a ʻole ʻole i nā microtubules.E like me kēia manaʻo, ʻo kā mākou ʻike ʻana i nā microtubules i kapa ʻia i ka immunohistochemically a i ʻole me nā protein fluorescent e hōʻike ana i ka mālama ʻana i ka KAND depolymerizes microtubules.Eia kekahi, ʻo ka mālama ʻana me nā derivatives ʻakika kumamotonic i hoʻopau i nā microfilaments actin.No laila, ua ʻike mākou i kahi mea hoʻoulu ulu mea kanu hou nona ka hana ʻokoʻa o ka hana e pili ana i ka luku ʻana i ka cytoskeleton.
Ua hoʻokaʻawale ʻia ʻo Strain MK493-CF1 mai ka lepo ma Shinagawa-ku, Tokyo.Hoʻokumu ʻo Strain MK493-CF1 i ka mycelium stromal lālā maikaʻi.Ua hoʻoholo ʻia ka ʻāpana ʻāpana o ka 16S ribosomal RNA gene (1422 bp).Ua like loa kēia ʻano me S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: ʻano maʻamau, 99.93%).Ma muli o kēia hopena, ua hoʻoholo ʻia ua pili pili kēia ʻano i ke ʻano o ke ʻano o S. werraensis.No laila, ua kapa mākou i kēia ʻano S. werraensis MK493-CF1.Hoʻopuka pū ʻo S. werraensis ISP 5486T i nā pūhui bioactive like.Ma muli o ka liʻiliʻi o ka noiʻi mua ʻana i ka loaʻa ʻana o nā huahana kūlohelohe mai kēia microorganism, ua hana hou ʻia ka noiʻi kemika.Ma hope o ka mahi ʻana i ka S. werraensis MK493-CF1 ma ka pā bale ma ka fermentation solid-state ma 30 ° C no 14 mau lā, ua unuhi ʻia ka mea me 50% EtOH.60 ml o ka laʻana ua maloʻo e loaʻa 59.5 mg o ka crude extract.Ua hoʻokomo ʻia ka ʻāpana palaka i ka HPLC hoʻohuli e hāʻawi iā N-methoxy-1H-pyrrole-2-carboxamide (1, kapa ʻia coumamonamide, 36.0 mg).ʻO ka nui o ka 1 ma kahi o 60% o ka extract crude.No laila, ua hoʻoholo mākou e aʻo kikoʻī i nā waiwai o kumamotoamide 1.
ʻO ka Coumamonamide 1 he pauka amorphous keʻokeʻo a me ka spectrometry nuipaʻa kiʻekiʻe (HRESIMS) e hōʻoia ana iā C6H8N2O2 (Fig. 1).Hōʻike ʻia ka ʻāpana pyrrole C2-substituted o kēia pūhui e δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH ma 1H NMR spectrum: 4.5 Hz. , H-5) a me δH 6.78 (1H, d, J = 2.5 Hz, H-6), a hōʻike ka spectrum 13C NMR i ka loaʻa ʻana o ʻehā sp2 carbon atoms.ʻO ka loaʻa ʻana o kahi pūʻulu amide ma ke kūlana C2 i loiloi ʻia e ka HMBC correlation mai ka C-3 proton i ka amide carbonyl carbon ma δC 161.1.Eia hou, 1 H a me 13 C NMR peaks ma δH 4.10 (3H, S) a me δC 68.3 hōʻike i ka hiki mai o nā pūʻulu N-methoxy i loko o ka mole.ʻOiai ʻaʻole i hoʻoholo ʻia ke kūlana kūpono o ka pūʻulu methoxy me ka hoʻohana ʻana i ka nānā ʻana spectroscopic e like me ka hoʻonui ʻokoʻa spectroscopy a me ka pōkole Overhauser abbreviation (NOEDF), ua lilo ʻo N-methoxy-1H-pyrrole-2-carboxamide i hui moho mua.
No ka hoʻoholoʻana i ka hoʻolālā kūpono o 1, ua hanaʻia kahi synthesis holoʻokoʻa (Fig. 2a).ʻO ka mālama ʻana i ka 2-aminopyridine 2 i loaʻa i ke kūʻai aku me ka m-CPBA i loaʻa i ka N-oxide 3 e like me ka hua nui.Ma hope o ka 2-aminoazidation o 2, ua hoʻokō ʻia ka hopena cyclocondensation i wehewehe ʻia e Abramovich i benzene ma 90 ° C e loaʻa ai ka 1-hydroxy-1H-pyrrole-2-carbonitrile 5 i makemake ʻia i ka grams.Ka wikiwiki 60% (ʻelua pae).15,16.Hāʻawi ka methylation a me ka hydrolysis o 4 i ka 1-methoxy-1H-pyrrole-2-carboxylic acid (i kapa ʻia ʻo "cumotonic acid", 6) i ka hua maikaʻi (70%, ʻelua mau ʻanuʻu).ʻO ka mea hope loa, hāʻawi ʻo Kumamoto amide 1 i ka 98% o ka hua o ka amidation ma o ka acid chloride intermediate 6 me ka ammonia wai.ʻO nā ʻikepili spectral a pau o ka synthesized 1 ua like me ka 1 i hoʻokaʻawale ʻia, no laila ua hoʻoholo ʻia ke ʻano o ka 1;
ʻO ka synthesis maʻamau a me ka nānā ʻana o ka hana biological o ka urbenamide a me ka urbenic acid.(a) Huina huina o Kumamoto amide.(b) ʻEhiku mau lā ʻano ʻano ʻāhiu ʻano Arabidopsis Columbia (Col) i ulu ʻia ma nā pā Murashige a me Skoog (MS) i loaʻa ka coumamonamide 6 a i ʻole coumamonamide 1 ma nā ʻano i hōʻike ʻia.ʻO ka pā unahi = 1 knm.
ʻO ka mea mua, ua loiloi mākou i nā hana olaola o urbenamide a me kāna mau kikowaena no ko lākou hiki ke hoʻololi i ka ulu ʻana o nā mea kanu.Ua hoʻohui mākou i nā ʻano ʻokoʻa like ʻole o ka ursmonamide 1 a i ʻole ka ursmonic acid 6 i ka medium MS agar a me nā mea kanu Arabidopsis thaliana moʻomeheu ma kēia ʻano.Ua hōʻike kēia mau ho'āʻo i nā kiʻekiʻe kiʻekiʻe (500 μM) o 6 i kāohi i ka ulu aʻa (Fig. 2b).Ma hope aʻe, hana mākou i nā derivatives like ʻole ma ka hoʻololi ʻana i ke kūlana N1 o 6 a hana i nā haʻawina pili pili i ka hoʻolālā-hana ma luna o lākou (ua wehewehe ʻia ke kaʻina hana analogue ma ka Supporting Information (SI)).Ua ulu ʻia nā mea kanu Arabidopsis ma kahi ʻano me 50 μM ursonic acid derivatives, a ua ana ʻia ka lōʻihi o ke aʻa.e like me ka mea i hoikeia ma ke kii.E like me ka hōʻike ʻana ma nā Kiʻi 3a, b, a me S1, ʻokoʻa ka lōʻihi o nā ʻakika coumamo o nā kaulahao alkoxy linear (9, 10, 11, 12, a me 13) a i ʻole nā kaulahao alkoxy nui (15, 16, a me 17) ma ke kūlana N1.Ua hōʻike nā derivatives i ka pale nui o ka ulu aʻa.Eia kekahi, ua ʻike mākou i ka hoʻohana ʻana o 200 μM 10, 11, a i ʻole 17 i kāohi i ka germination (Fig. 3c a me S2).
Ke aʻo ʻana i ka pilina hana-hana o Kumamoto amide a me nā pūhui pili.(a) Ka hoʻolālā a me ke ʻano hoʻohālikelike o nā analogues.(b) Ka helu o ka lōʻihi o ke aʻa o nā mea kanu he 7 lā i ulu ma luna o MS me ka 50 μM coumamonamide derivatives a i ʻole.Hōʻike nā asterisk i nā ʻokoʻa koʻikoʻi me ka mālama hoʻopunipuni (t test, p<0.05).n>18. Hōʻike ʻia nā ʻikepili ma ke ʻano he ± SD.nt 'o ia ho'i "'a'ole i ho'ā'o 'ia" no ka mea, 'oi aku ma mua o 50% o nā 'anoano 'a'ole i ulu.(c) Ka helu ʻana o ka germination rate o nā ʻanoʻano i mālama ʻia i hoʻomoʻa ʻia no 7 mau lā ma MS medium me ka 200 μM coumamonamide a me nā pūhui pili ʻole.Hōʻike nā asterisk i nā ʻokoʻa koʻikoʻi me ka hoʻomaʻamaʻa sham (hoʻāʻo chi-square).n=96.
ʻO ka mea hoihoi, ʻo ka hoʻohui ʻana o nā kaulahao ʻaoʻao alkyl ʻoi aku ka lōʻihi ma mua o C9 i hōʻemi i ka hana hoʻopā, me ka manaʻo e koi ʻia nā pūhui pili i ka waikona kumamotoic i nā kaulahao ʻaoʻao o kekahi nui e hōʻike i kā lākou hana olaola.
Ma muli o ka hōʻike ʻana i ka pilina pili i ka hoʻolālā-hana ua hoʻololi ʻia ʻo C9 i ka waika ursonic a me ka derivative nonyloxy o ka ursonic acid (i kapa ʻia ʻo KAND 11 ma hope) ʻo ia ka mea hoʻopiʻi ulu ulu maikaʻi loa, ua hana mākou i kahi kikoʻī kikoʻī o KAND 11. me 50 μM KAND 11 aneane pale loa i ka germination, akā naʻe nā haʻahaʻa haʻahaʻa (40, 30, 20, a i ʻole 10 μM) o KAND 11 i keakea i ka ulu ʻana o ke aʻa ma ke ʻano o ka hopena (Fig. 4a, b).No ka hoʻāʻo ʻana inā pili ʻo KAND 11 i ke ola meristem aʻa, ua nānā mākou i nā meristem aʻa i hoʻopaʻa ʻia me ka propidium iodide (PI) a ana i ka nui o ka meristem.ʻO ka nui o ka meristem o nā mea kanu i ulu ʻia ma luna o kahi mea i loaʻa iā 25 μM KAND-11 he 151.1 ± 32.5 μm, aʻo ka nui o ka meristem o nā mea kanu i ulu ma luna o kahi mea hoʻomalu i loaʻa DMSO he 264.7 ± 30.8 μm (Fig. 4c, d) , e hōʻike ana e hoʻihoʻi ka KAND-11 i ka hana kelepona.pahola ana.Meristem kumu.E like me keia, ua hoemi ka lapaau KAND 11 i ka nui o ka marker division cell CDKB2;1p::CDKB2;1-GUS hoailona ma ke kumu meristem (Fig. 4e) 17 .Hōʻike kēia mau hopena i ka KAND 11 ke kāohi i ka ulu ʻana o ke aʻa ma o ka hōʻemi ʻana i ka hana hoʻonui cell.
Ka nānā 'ana i ka hopena o ka urbenonic acid derivatives (urbenyloxy derivatives) i ka ulu.(a) 7 mau lā-ʻano ʻano ʻāhiu Col seedlings i ulu ʻia ma nā papa MS me ka ʻike ʻia ʻana o ka KAND 11. Paʻa pālākiō = 1 knm.(b) Ka helu o ka lōʻihi o ke aʻa.Hōʻike nā leka i nā ʻokoʻa koʻikoʻi (Tukey HSD test, p<0.05).n>16. Hōʻike ʻia nā ʻikepili ma ke ʻano he ± SD.(c) Confocal microscopy o propidium iodide-stained wild-type Col aʻa i ulu ma nā papa MS me ka 25 μM KAND 11 a i ʻole.ʻO ka pā unahi = 100 µm.(d) Ka helu o ka nui meristem kumu (n = 10 a hiki i ka 11).Hoʻoholo ʻia nā ʻokoʻa helu me ka hoʻohana ʻana i ka hoʻāʻo-t (p<0.05).Hōʻike nā pā i ka nui meristem maʻamau.(e) Microscopy hoʻokolo like ʻole (DIC) o kahi meristem kumu i loaʻa ka hana CDKB2;1pro: CDKB2;ʻO 1-GUS i hoʻopaʻa ʻia a hoʻopaʻa ʻia ma nā mea kanu he 5 mau lā i ulu ʻia ma nā papa MS me ka 25 µM KAND assay a i ʻole.
Ua hoʻāʻo hou ʻia ka phytotoxicity o KAND 11 me ka hoʻohana ʻana i kahi mea kanu dicotyledonous ʻē aʻe, ka paka (Nicotiana tabacum), a me kahi ʻano mea kanu ʻāina nui, liverwort (Marchantia polymorpha).E like me ka Arabidopsis, ua ulu ka paka SR-1 mea kanu ma ka waena me 25 μM KAND 11 i hua mai i nā aʻa pōkole (Fig. 5a).Hoʻohui hou, 40 o 48 mau hua i hoʻoulu ʻia ma nā papa i loaʻa ka 200 μM KAND 11, aʻo nā hua 48 a pau i hoʻoulu ʻia ma ka media i hoʻomāʻewaʻewa ʻia, e hōʻike ana he koʻikoʻi ke kiʻekiʻe o ka KAND (p< 0.05;chi test -square) keʻakeʻa i ka germination o ka paka.(Fig. 5b).Eia kekahi, ua like ka hoʻopaʻa ʻana o KAND 11 i kāohi i ka ulu ʻana o ka maʻi bacteria i loko o ka liverwort me ke ʻano kūpono o ka Arabidopsis (Fig. 5c).Hōʻike kēia mau hualoaʻa he hiki i ka KAND 11 ke kāohi i ka ulu ʻana o nā mea kanu like ʻole.A laila ua noiʻi mākou i ka cytotoxicity o ka bear monoamide-pili pūhui i loko o nā mea ola ʻē aʻe, ʻo ia hoʻi nā cell HeLa kanaka a me ka Escherichia coli strain DH5α, ma ke ʻano he mau ʻelele o nā holoholona kiʻekiʻe a me nā cell bacteria.I loko o ke kaʻina o ka hoʻonui ʻana o ka cell proliferation assays, ua ʻike mākou i ka coumamonamide 1, coumamonamidic acid 6, a me KAND 11 ʻaʻole i hoʻopilikia i ka ulu ʻana o HeLa a i ʻole E. coli cell ma nā ʻano o 100 μM (Fig. 5d, e).
ʻO ka pale ʻana i ka ulu ʻana o KAND 11 i nā mea ola ʻole Arabidopsis.(a) Ua ulu ʻia nā mea kanu paka SR-1 ʻelua pule ma luna o nā papa MS i hoʻonohonoho pono ʻia i loaʻa ka 25 μM KAND 11. (b) Ua ulu ʻia nā mea kanu paka SR-1 ʻelua pule ma luna o ka pae ʻaoʻao. ʻO nā papa MS he 200 μM KAND 11. (c) ʻElua mau pule he ʻano ʻāhiu ʻano ʻē aʻe ʻo Tak-1 i ulu ʻia ma nā pā Gamborg B5 me nā manaʻo i hōʻike ʻia o KAND 11. Hōʻike nā pua ʻulaʻula i nā spores i pau ka ulu ʻana i loko o ka incubation ʻelua pule. manawa.(d) Ka ho'āʻo hoʻomāhuahua ʻana o nā cell HeLa.Ua ana ʻia ka heluna o nā pūnaewele ola i nā manawa paʻa me ka hoʻohana ʻana i kahi pahu helu kelepona 8 (Dojindo).Ma keʻano he mana, ua mālamaʻia nā pūnaewele HeLa me ka 5 μg / ml actinomycin D (Act D), ka mea e kāohi ai i ka RNA polymerase transcription a ke kumu i ka make cell.Ua hana ʻia nā loiloi ma ka ʻekolu.(e) E. coli cell proliferation assay.Ua kālailai ʻia ka ulu ʻana o E. coli ma ke ana ʻana iā OD600.Ma ke ʻano he mana, ua mālama ʻia nā cell me 50 μg/ml ampicillin (Amp), ka mea e pale ai i ka synthesis o ka paia cell bacteria.Ua hana ʻia nā loiloi ma ka ʻekolu.
No ka wehewehe ʻana i ke ʻano o ka hana o ka cytotoxicity i hoʻokumu ʻia e nā pūhui pili i ka uramide, ua loiloi hou mākou i nā derivatives urbenic acid me nā hopena inhibitory haʻahaʻa.e like me ka mea i hoikeia ma ke kii.E like me ka mea i hōʻike ʻia ma nā Kiʻi 2b, 6a, ʻo nā mea kanu i ulu ʻia ma nā papa agar i loaʻa nā kiʻekiʻe kiʻekiʻe (200 μM) o ka urmotonic acid 6 i hoʻopuka i nā aʻa pōkole a me ka ʻāʻī hema (θ = - 23.7 ± 6.1), akā ʻo nā mea kanu i ulu ʻia ma ke ʻano mana, ua hoʻopuka nā mea kanu i nā aʻa pololei (θ = – 3.8 ± 7.1).ʻIke ʻia kēia ʻano ulu oblique ma muli o ka hana ʻino ʻana o nā microtubules cortical14,18.E like me kēia ʻike, ʻo ka microtubule-destabilizing drugs disopyramide a me ka oryzalin i hoʻoulu i ka tilting aʻa like ma lalo o kā mākou ulu ulu (Fig. 2b, 6a).I ka manawa like, ua hoʻāʻo mākou i nā derivatives urmotonic acid a koho i kekahi o lākou i hoʻoulu ʻia i ka ulu aʻa oblique.Hoʻololi nā pūhui 8, 9, a me 15 i ke kuhikuhi o ka ulu ʻana o ke aʻa ma 75 μM, 50 μM, a me 40 μM, e hōʻike ana e hiki i kēia mau pūhui ke hoʻopau pono i nā microtubules (Fig. 2b, 6a).Ua hoʻāʻo pū mākou i ka derivative ursolic acid ʻoi loa, KAND 11, ma kahi haʻahaʻa haʻahaʻa (15 µM) a ʻike ʻia ʻo ka hoʻohana ʻana o KAND 11 i keʻakeʻa i ka ulu ʻana o ke aʻa a ʻaʻole pololei ke kuhikuhi o ka ulu ʻana o ke aʻa, ʻoiai ua maʻa lākou i ka piʻi ʻana i ka hema ( Kiʻi C3)..No ka mea, ʻoi aku ka kiʻekiʻe o nā lāʻau lapaʻau microtubule-destabilizing i kekahi manawa ke keʻakeʻa nei i ka ulu ʻana o ka mea kanu ma mua o ke kumu hili ʻana o ke aʻa, a laila ua loiloi mākou i ka hiki ke hoʻopili ʻia ka KAND 11 i nā microtubules ma ka nānā ʻana i nā microtubules cortical i loko o nā pūnaewele epidermal aʻa.ʻO ka Immunohistochemistry me ka hoʻohana ʻana i nā anti-β-tubulin antibodies i loko o nā epidermal cell of seedling roots i mālama ʻia me 25 μM KAND 11 i hōʻike i ka nalowale ʻana o nā microtubules cortical a pau i loko o nā cell epidermal i ka elongation zone (Fig. 6b).Hōʻike kēia mau hopena i ka hana pololei ʻana a i ʻole ke ʻano ʻole o ka waikawa kumamotonic a me kāna mau mea hoʻohui i nā microtubules e hoʻopilikia iā lākou a ʻo kēia mau pūhui he mea hoʻopaneʻe microtubule hou.
Hoʻololi ka ʻakika Ursonic a me kāna mau mea i nā microtubules cortical ma Arabidopsis thaliana.(a) Ua ana ʻia ka huina ʻaʻa ma ke alo o nā mea hoʻoheheʻe waika urmotonic like ʻole ma nā ʻāpana i hōʻike ʻia.ʻO nā hopena o nā pūhui ʻelua i ʻike ʻia e kaohi i nā microtubules: disopyramide a me oryzalin ua nānā pū ʻia.Hōʻike ka inset i ka maʻamau i hoʻohana ʻia no ke ana ʻana i ke kihi ulu aʻa.Hōʻike nā asterisk i nā ʻokoʻa koʻikoʻi me ka mālama hoʻopunipuni (t test, p<0.05).n>19. Paʻa unahi = 1 knm.(b) Cortical microtubules i loko o ka epidermal cell i ka elongation zone.ʻO nā microtubules i loko o nā aʻa Arabidopsis Col i ulu ʻia ma nā papa MS me ka 25 μM KAND 11 a i ʻole ka ʻike ʻia e ka immunohistochemical staining me ka hoʻohana ʻana i nā antibodies mua β-tubulin a me Alexa Fluor-conjugated second antibodies.ʻO ka pā unahi = 10 µm.(c) ʻO ke ʻano mitotic o nā microtubules i ke kumu meristem.ʻIke ʻia nā microtubules me ka hoʻohana ʻana i ka immunohistochemical staining.Ua helu ʻia nā hale mitotic, me nā ʻāpana prophase, spindles, a me nā phragmoplasts mai nā kiʻi confocal.Hōʻike nā pua i nā hanana microtubule mitotic.Hōʻike nā asterisk i nā ʻokoʻa koʻikoʻi me ka mālama hoʻopunipuni (t test, p<0.05).n>9. Paʻa unahi = 50 µm.
ʻOiai ua hiki iā Ursa ke hoʻopau i ka hana microtubule, ua manaʻo ʻia he ʻokoʻa kona ʻano hana mai nā mea hana depolymerizing microtubule maʻamau.No ka laʻana, ʻoi aku ka kiʻekiʻe o ka microtubule depolymerizing agents e like me ka disopyramide a me ka oryzalin e hoʻoulu i ka hoʻonui anisotropic o nā cell epidermal, akā ʻo KAND 11 ʻaʻole.Eia hou, ua hoʻohana pū ʻia ka KAND 11 a me ka disopyramide i ka hopena i hoʻohui ʻia i ka disopyramide-induced root growth response a me ka KAND 11-induced growth inhibition (Fig. S4).Ua nānā pū mākou i ka pane o ka hypersensitive disopyramide 1-1 (phs1-1) mutant i KAND 11. phs1-1 he non-canonical tubulin kinase point mutation a hoʻopuka i nā aʻa pōkole ke mālama ʻia me ka disopyramide9,20.ʻO nā kumulāʻau mutant phs1-1 i ulu ma luna o ka media agar me KAND 11 he mau aʻa pōkole e like me nā aʻa i ulu ma ka disopyramid (fig. S5).
Eia kekahi, ua ʻike mākou i nā ʻōnaehana microtubule mitotic, e like me nā prophase zones, spindles, a me phragmoplasts, ma ke kumu meristem o nā mea kanu i mālama ʻia me KAND 11. Kūlike me nā ʻike no CDKB2;1p::CDKB2;1-GUS, he emi nui o ka. uaʻikeʻia ka helu o nā microtubules mitotic (Fig. 6c).
No ka ʻike ʻana i ka cytotoxicity o KAND 11 ma ka hoʻonā subcellular, ua mālama mākou i ka paka BY-2 suspension cell me KAND 11 a nānā i kā lākou pane.Hoʻohui mua mākou i ka KAND 11 i nā pūnaewele BY-2 e hōʻike ana i ka TagRFP-TUA6, ka mea e hoʻopaʻa inoa i nā microtubules, e loiloi i ka hopena o KAND 11 ma nā microtubules cortical.Ua loiloi ʻia ka nui o ka microtubule cortical me ka hoʻohana ʻana i ka nānā ʻana i ke kiʻi, ka mea i helu i ka pakeneka o nā cytoskeletal pixels ma waena o nā cytoplasmic pixels.Ua hōʻike ʻia nā hopena assay ma hope o ka mālama ʻana me 50 μM a i ʻole 100 μM KAND 11 no ka hola 1, ua emi nui ka density i 0.94 ± 0.74% a i ʻole 0.23 ± 0.28%, ʻoiai ʻo ka nui o nā cell i mālama ʻia me DMSO, ʻo 1.61 ± 0.34. % (Fig. 7a).Ua kūlike kēia mau hopena me ka nānā ʻana ma Arabidopsis i ka mālama ʻana i ka KAND 11 e hoʻoulu i ka depolymerization o nā microtubules cortical (Fig. 6b).Ua nānā pū mākou i ka laina BY-2 me nā filament actin-labeled GFP-ABD ma hope o ka mālama ʻana me ka hoʻohālikelike like o KAND 11 a ʻike ʻia ua hoʻopau ka lāʻau KAND 11 i nā filament actin.Hoʻomaʻamaʻa me 50 μM a i ʻole 100 μM KAND 11 no ka 1 h ua hoʻemi nui ʻia ka actin filament density i 1.20 ± 0.62% a i ʻole 0.61 ± 0.26%, ʻoiai ʻo ka nui o nā cell i mālama ʻia DMSO he 1.69 ± 0.51% (Fig.7b).Hoʻohālikelike kēia mau hopena me nā hopena o propyzamide, ʻaʻole pili i nā filament actin, a me latrunculin B, he actin depolymerizer ʻaʻole pili i nā microtubules (SI Figure S6).Eia kekahi, ʻaʻole i hoʻopilikia ka lāʻau me ka coumamonamide 1, ka coumamonamide acid 6, a i ʻole KAND 11 i nā microtubules i nā cell HeLa (SI Figure S7).No laila, ʻokoʻa ke ʻano o ka hana o KAND 11 mai nā mea hoʻopilikia cytoskeleton i ʻike ʻia.Eia kekahi, ʻo kā mākou ʻike microscopic o nā cell BY-2 i mālama ʻia me KAND 11 i hōʻike i ka hoʻomaka ʻana o ka make cell i ka wā o ka mālama ʻana o KAND 11 a hōʻike ʻia ʻaʻole i hoʻonui nui ʻia ka nui o nā mea make polū polū Evans ma hope o 30 min o ka mālama ʻana KAND 11, ʻoiai. ma hope o 90 mau minuke o ka mālama ʻana me 50 μM a i ʻole 100 μM KAND, ua hoʻonui ʻia ka nui o nā mea make i 43,7% a i ʻole 80,1% (Fig 7c).Hoʻopili pū ʻia, hōʻike kēia mau ʻikepili i ka moʻolelo ursolic acid derivative KAND 11 he mea kanu-specific cytoskeletal inhibitor me kahi ʻano hana i ʻike ʻole ʻia.
Hoʻopilikia ʻo KAND i nā microtubules cortical, nā filament actin, a me ka hiki ʻana o ka paka BY-2 cell.(a) Ka ʻike ʻana i nā microtubules cortical i loko o nā pūnaewele BY-2 ma ke alo o TagRFP-TUA6.Ua nānā ʻia nā cell BY-2 me KAND 11 (50 μM a i ʻole 100 μM) a i ʻole DMSO e ka microscopy confocal.Ua helu ʻia ka nui o ka microtubule cortical mai nā micrographs o 25 mau keena kūʻokoʻa.Hōʻike nā leka i nā ʻokoʻa koʻikoʻi (Tukey HSD test, p<0.05).ʻO ka pā unahi = 10 µm.(b) ʻO nā filament cortical actin i loko o nā pūnaewele BY-2 i ʻike ʻia ma ke alo o GFP-ABD2.Ua nānā ʻia nā cell BY-2 me KAND 11 (50 μM a i ʻole 100 μM) a i ʻole DMSO e ka microscopy confocal.Ua helu ʻia ka nui o nā filament cortical actin mai nā micrographs o 25 mau cell kūʻokoʻa.Hōʻike nā leka i nā ʻokoʻa koʻikoʻi (Tukey HSD test, p<0.05).ʻO ka pā unahi = 10 µm.(c) Ka nānā ʻana i nā pūnaewele BY-2 make e ka ʻoni polū ʻo Evans.Ua nānā ʻia nā cell BY-2 i mālama ʻia me KAND 11 (50 μM a i ʻole 100 μM) a i ʻole DMSO e ka microscopy māmā.n=3.ʻO ka pā unahi = 100 µm.
ʻO ka loaʻa ʻana a me ka hoʻohana ʻana i nā huahana kūlohelohe hou i alakaʻi i nā holomua nui i nā ʻano like ʻole o ke ola kanaka, me ka lāʻau lapaʻau a me ka mahiʻai.Ua hana ʻia ka noiʻi mōʻaukala no ka loaʻa ʻana o nā pūhui pono mai nā kumuwaiwai kūlohelohe.ʻO ka mea nui, ʻike ʻia nā actinomycetes he mea pono e like me nā antiparasitic antibiotics no nā nematodes ma muli o ko lākou hiki ke hana i nā metabolites lua like ʻole e like me avermectin, ka hui alakaʻi o ka ivermectin a me ka bleomycin a me kāna mau mea i hoʻohana ʻia i ka lāʻau lapaʻau ma ke ʻano he anticancer agent21,22.Pēlā nō, ua ʻike ʻia nā ʻano pūhui herbicidal mai actinomycetes, a ua hoʻohana mua ʻia kekahi o ia mau mea ma ke kālepa1,23.No laila, ʻo ka nānā ʻana i nā metabolites actinomycete e hoʻokaʻawale i nā huahana kūlohelohe me nā hana olaola i makemake ʻia he hoʻolālā kūpono.I loko o kēia noiʻi ʻana, ua ʻike mākou i kahi pūhui hou, coumamonamide, mai S. werraensis a hoʻohui maikaʻi ʻia.ʻO ka waika Ursonic kahi kikowaena synthetic o ka urbenamide a me kāna mau derivatives.Hiki iā ia ke hoʻoulu i ke aʻa wili, hōʻike i ka haʻahaʻa haʻahaʻa i ka ikaika herbicidal, a hōʻino pololei a ʻaʻole pololei paha i nā microtubules mea kanu.Eia naʻe, ʻokoʻa paha ke ʻano o ka hana ʻana o ka waika urmotonic mai nā mea hoʻopaneʻe microtubule e kū nei, ʻoiai ʻo KAND 11 ke hoʻopau nei i nā filament actin a make i ka cell cell, e hōʻike ana i kahi ʻano hoʻoponopono e hoʻoikaika ai ka waika urmotonic a me nā mea i loaʻa i kahi ākea o nā hale cytoskeletal..
ʻO ka wehewehe kikoʻī hou aku o ka waika urbenonic e kōkua i ka hoʻomaopopo maikaʻi ʻana i ke ʻano o ka hana o ka waika urbenonic.ʻO ka mea nui, ʻo ka pahuhopu aʻe e loiloi i ka hiki o ka ursonic acid e hoʻopaʻa i nā microtubules i hoʻemi ʻia e hoʻoholo ai inā hana pololei ka ursonic acid a me kāna mau derivatives i nā microtubules a depolymerize iā lākou, a i ʻole ka hopena o kā lākou hana i ka microtubule destabilization.Eia kekahi, i ka hihia inā ʻaʻole ʻo microtubules kahi pahuhopu pololei, ʻo ka ʻike ʻana i ka pae o ka hana a me nā molecular targets o ka ursonic acid ma nā mea kanu e kōkua hou i ka hoʻomaopopo ʻana i nā waiwai o nā pūhui pili a me nā ala hiki ke hoʻomaikaʻi i ka hana herbicidal.Ua hōʻike kā mākou bioactivity assay i ka mana cytotoxic kūʻokoʻa o ka ursonic acid i ka ulu ʻana o nā mea kanu e like me Arabidopsis thaliana, ka paka a me ka liverwort, ʻoiai ʻaʻole i hoʻopilikia ʻia nā cell E. coli a me HeLa.ʻO ka liʻiliʻi a ʻaʻohe mea ʻawaʻawa i nā pūnae holoholona he mea maikaʻi ia o ka ursonic acid derivatives inā hoʻomohala ʻia lākou e like me nā herbicides no ka hoʻohana ʻana i nā māla mahiʻai ākea.ʻOiaʻiʻo, no ka mea he mau hale maʻamau nā microtubules i nā eukaryotes, ʻo kā lākou koho koho ʻana i nā mea kanu he mea nui ia no ka herbicides.No ka laʻana, propyzamide, he microtubule depolymerizing agent i hoʻopaʻa pololei i ka tubulin a keʻakeʻa i ka polymerization, hoʻohana ʻia ma ke ʻano he herbicide ma muli o kona ʻona haʻahaʻa i nā cell holoholona24.ʻAʻole like me ka disopyramide, loaʻa nā benzamides pili i nā kikoʻī kikoʻī.Ma waho aʻe o nā microtubules kanu, ʻo RH-4032 a i ʻole benzoxamide ke kāohi nei i nā microtubules o nā pūnae holoholona a i ʻole nā oomycetes, kēlā me kēia, a hoʻohana ʻia ka zalilamide ma ke ʻano he fungicide ma muli o kona haʻahaʻa phytotoxicity25,26,27.ʻO ka bea i ʻike hou ʻia a me kāna mau mea hoʻohui e hōʻike i ka cytotoxicity koho e kūʻē i nā mea kanu, akā pono e hoʻomaopopo ʻia hiki i nā hoʻololi hou ke hoʻololi i kā lākou kiko kikoʻī, hiki ke hāʻawi i nā derivatives hou no ka mālama ʻana i nā fungi pathogenic a i ʻole nā oomycetes.
ʻO nā waiwai kūʻokoʻa o ka waika urbenonic a me kāna mau mea e pono ai no ko lākou ulu ʻana ma ke ʻano he herbicides a hoʻohana ʻia ma ke ʻano he mea noiʻi.ʻIke nui ʻia ke koʻikoʻi o ka cytoskeleton i ka hoʻomalu ʻana i ke ʻano o nā mea kanu.Ua hōʻike ʻia nā haʻawina mua ua hoʻomohala nā mea kanu i nā mīkini paʻakikī o ka hui cortical microtubule ma o ka hoʻomalu ʻana i ka microtubule dynamics e hoʻomalu pono i ka morphogenesis.Ua ʻike ʻia ka heluna nui o nā moleke no ka hoʻoponopono ʻana i ka hana microtubule, a ke hoʻomau nei ka noiʻi e pili ana3,4,28.ʻO kā mākou ʻike i kēia manawa o ka microtubule dynamics i loko o nā mea kanu ʻaʻole wehewehe piha i nā hana o ka hui cortical microtubule.No ka laʻana, ʻoiai hiki i ka disopyramide a me ka oryzalin ke depolymerize i nā microtubules, ʻo ka disopyramide ke kumu o ka distortion aʻa koʻikoʻi aʻo ka oryzalin ka hopena maʻalahi.Eia kekahi, ʻo ka hoʻololi ʻana i ka tubulin, ka mea e hoʻopaʻa ai i nā microtubule, ke kumu hoʻi i ka dextrorotation i nā aʻa, ʻoiai ʻo paclitaxel, ka mea e hoʻopaʻa i ka microtubule dynamics, ʻaʻole ia.No laila, ʻo ke aʻo ʻana a me ka ʻike ʻana i nā pahuhopu molekala o ka ursolic acid e hāʻawi i nā ʻike hou i ka hoʻoponopono ʻana i nā microtubules cortical kanu.Pēlā nō, ʻo ka hoʻohālikelike ʻana o nā kinikini e hiki mai ana i ka hoʻoikaika ʻana i ka ulu ʻino, e like me ka disopyramide, a me nā kemika ʻoi aku ka maikaʻi, e like me ka oryzalin a i ʻole ka waikawa kumamotoric, e hāʻawi i nā hōʻailona i ke ʻano o ka ulu ʻana.
Ma ka ʻaoʻao ʻē aʻe, ʻo ka hoʻoponopono hou ʻana i ka cytoskeletal e pili ana i ka pale kekahi mea hiki ke wehewehe i ka cytotoxicity o ka ursonic acid.ʻO ka maʻi o kahi pathogen a i ʻole ka hoʻokomo ʻana o kahi elicitor i loko o nā mea kanu i kekahi manawa ke kumu i ka luku ʻana i ka cytoskeleton a me ka make ʻana o ka cell29.No ka laʻana, ua hōʻike ʻia ka cryptoxanthin i loaʻa i ka oomycete e hoʻopau i nā microtubules a me nā filament actin ma mua o ka make ʻana o ka paka, e like me ka mea i hana ʻia me ka mālama KAND30,31.ʻO nā mea like ma waena o nā pane pale a me nā pane cellular i hoʻokomo ʻia e ka ursonic acid i alakaʻi iā mākou i ka hypothesize e hoʻomaka lākou i nā kaʻina kelepona maʻamau, ʻoiai ʻoi aku ka wikiwiki a me ka ikaika o ka ursonic acid ma mua o ka cryptoxanthin.Eia naʻe, ua hōʻike ʻia nā haʻawina ʻo ka hoʻopau ʻana i nā filament actin e hoʻoikaika i ka make cell spontaneous, ʻaʻole i hui pū ʻia me ka microtubule disruption29.Eia hou, e ʻike ʻia inā ʻo ka pathogen a i ʻole elicitor ke kumu o ka ulu ʻana o ke aʻa, e like me ka ursonic acid derivatives.No laila, ʻo ka ʻike molekala e pili ana i nā pane pale a me ka cytoskeleton he pilikia hoihoi e hoʻoponopono ʻia.Ma ka hoʻohana ʻana i ka loaʻa ʻana o nā pūhui paona haʻahaʻa e pili ana i ka waika ursonic, a me kahi ʻano o nā derivatives me nā mana like ʻole, hiki iā lākou ke hāʻawi i nā manawa e huli ai i nā mīkini kelepona ʻike ʻole.
Hoʻohui pū ʻia, ʻo ka loaʻa ʻana a me ka hoʻohana ʻana i nā pūhui hou e hoʻololi ai i ka microtubule dynamics e hāʻawi i nā ala ikaika e hoʻoponopono ai i nā mīkini molekala paʻakikī e pili ana i ka hoʻoholo ʻana i ke ʻano o nā mea kanu.Ma kēia pōʻaiapili, hiki i ka urmotonic acid i kūkulu hou ʻia, e hoʻopilikia i nā microtubules a me nā filament actin a hoʻoulu i ka make cell, hiki ke hāʻawi i kahi manawa e wehewehe ai i ka pilina ma waena o ka mana microtubule a me kēia mau hana ʻē aʻe.No laila, ʻo ka loiloi kemika a me ka biological me ka hoʻohana ʻana i ka urbenonic acid e kōkua iā mākou e hoʻomaopopo i nā ʻōnaehana hoʻoponopono molekala e hoʻomalu i ka cytoskeleton mea kanu.
Inoculate S. werraensis MK493-CF1 i loko o ka pahu Erlenmeyer 500 mL pohihihi i loaʻa he 110 mL o ka mea kanu me 2% (w/v) galactose, 2% (w/v) Essence paste, 1% ( w/v) Bacto haku. .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) kānana extract (KOGOSTCH Co., Ltd., Iapana), 0.2% (w / v) (NH4) 2SO4 a me 0.2% CaCO3 i ka wai deionized.(pH 7.4 ma mua o ka sterilization).Hoʻomoʻa ʻia nā ʻanoʻano moʻomeheu i kahi mea hoʻoheheʻe rotary (180 rpm) ma 27 ° C no 2 mau lā.Hoʻoulu ʻia ma o ka fermentation mokuʻāina paʻa.Ua hoʻoili ʻia ka moʻomeheu hua (7 ml) i loko o kahi pahu 500 ml K-1 i loaʻa he 40 g o ka mea hana hana me ka 15 g o ka bale i kaomi ʻia (MUSO Co., Ltd., Iapana) a me 25 g o ka wai deionized (pH ʻaʻole i hoʻoponopono ʻia. ma mua o ka sterilization).).Lawe ʻia ka fermentation ma 30 ° C i ka pōʻeleʻele no 14 mau lā.Ua unuhiʻia ka mea fermentation me ka 40 ml /'ōmole EtOH a me ka centrifuged (1500 g, 4 ° C, 10 min).Ua unuhi ʻia ka supernatant moʻomeheu (60 ml) me ka hui ʻana o 10% MeOH/EtOAc.Ua hoʻokuʻu ʻia ka papa organik ma lalo o ke kaomi hoʻemi ʻia e loaʻa ai kahi koena (59.5 mg), i hoʻopaʻa ʻia i ka HPLC me ka elution gradient (0-10 mau minuke: 90%) ma kahi kolamu hoʻohuli (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID. 10 mm × lōʻihi 250 mm) H2O/CH3CN, 10–35 minuke: 90% H2O/CH3CN i 70% H2O/CH3CN (gradient), 35–45 minuke: 90% H2O/EtOH, 45–155 minuke: 90% H2O / EtOH i 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) ma ke kahe kahe o 1.5 ml / min, coumamonamide (1, 36.0 mg) i hoʻokaʻawale ʻia e like me ka pauka amorphous keʻokeʻo.
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]+ helu helu: 141.0659, ana waiwai: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1533, 1593.
Ua loaʻa nā hua Columbia (Col-0) mai ka Arabidopsis Biological Resource Center (ABRC) me ka ʻae ʻia no ka hoʻohana noiʻi.Ua hoʻolaha ʻia a mālama ʻia nā hua Col-0 ma lalo o kā mākou mau kūlana hoʻokolohua a hoʻohana ʻia e like me nā mea kanu Arabidopsis ʻano hihiu.Ua hoʻomaʻemaʻe ʻia nā hua Arabidopsis a hoʻomaʻamaʻa ʻia i ka hapalua ikaika Murashige a me Skoog medium i loaʻa ka 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) ( Fujifilm Wako Pure Chemical. ).) a me 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, ma 23 °C a me ka malamalama mau.Ua hoʻolako ʻia nā hua o ka mutant phs1-1 e T. Hashimoto (Nara Institute of Science and Technology).
Na T. Hashimoto (Nara Institute of Science and Technology) na hua o ka strain SR-1 a ua hoohana ia ma ke ano he mau mea kanu paka.Hoʻopili ʻia nā ʻanoʻano paka i ka ʻili a hoʻomoʻi ʻia i loko o ka wai maʻemaʻe no ʻekolu pō e hoʻoikaika ai i ka ulu ʻana, a laila waiho ʻia i loko o kahi hopena hapalua ikaika me 2% sucrose, 0.05% (w/v) MES, a me 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige.a me Skoog medium) me ka pH 5.7 a hoʻokomo ʻia ma 23 ° C ma lalo o ka mālamalama mau.
Ua hoʻolako ʻia ʻo Strain Tak-1 e T. Kohchi (Kyoto University) a ua hoʻohana ʻia ʻo ia ka ʻāpana hoʻokolohua maʻamau no ka haʻawina liverwort.Loaʻa ʻia ʻo Gemma mai nā mea kanu i hoʻomaʻemaʻe ʻia a laila hoʻopaʻa ʻia ma luna o Gamborg B5 medium (Fujifilm Wako Pure Chemical) i loaʻa ka 1% sucrose a me 0.3% gellan gum a hoʻomoʻa ʻia ma 23°C ma lalo o ka mālamalama mau.
ʻO ka paka BY-2 pūnaewele (Nicotiana tabacum L. cv. Bright Yellow 2) na S. Hasezawa (Ke Kulanui o Tokyo).Ua hoʻoheheʻe ʻia nā pūnaewele BY-2 i 95-fold ma Linsmeier a me Skoog medium i hoʻololi ʻia a hoʻohui ʻia i kēlā me kēia pule me 2,4-dichlorophenoxyacetic acid 32.Ua hui pū ʻia ka hoʻokuʻu ʻana o ke kelepona ma kahi haʻalulu rotary ma 130 rpm ma 27 ° C i ka pōʻeleʻele.E holoi i nā pūnaewele me 10 mau manawa ka nui o ka mea hou a hoʻomaha hou i ka mea like.BY-2 transgenic cell lines stably expressing the microtubule marker TagRFP-TUA6 or the actin filament marker GFP-ABD2 under the cauliflower mosaic virus 35S promoter was generated as describe33,34,35.Hiki ke mālama ʻia kēia mau laina kelepona me ka hoʻohana ʻana i nā kaʻina hana e like me nā mea i hoʻohana ʻia no ka laina kelepona mua BY-2.
Ua kanu ʻia nā pūnaewele HeLa ma Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) i hoʻohui ʻia me 10% fetal bovine serum, 1.2 U/ml penicillin, a me 1.2 μg/ml streptomycin i loko o kahi incubator 37°C me 5% CO2.
Ua hana ʻia nā hoʻokolohua āpau i wehewehe ʻia i loko o kēia manuscript e like me nā lula a me nā alakaʻi biosafety Iapana.
Ua hoʻoheheʻe ʻia nā mea hoʻohui i ka dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) ma ke ʻano he hoʻonā waiwai a hoʻoheheʻe ʻia i loko o ka medium MS no Arabidopsis a me ka paka a i ʻole Gamborg B5 medium no ka liverwort.No ka hoʻāʻo ʻana i ka ulu ʻana o ke aʻa, ʻoi aku ma mua o 10 mau ʻanoʻano no ka pā i lulu ʻia ma luna o ka media agar i loaʻa nā pūhui i hōʻike ʻia a i ʻole DMSO.Hoʻokomo ʻia nā hua i loko o kahi keʻena ulu no 7 mau lā.Ua kiʻi ʻia nā mea kanu a ua ana ʻia ka lōʻihi o nā aʻa.No ka Arabidopsis germination assay, 48 mau hua no ka pā i lulu ʻia ma luna o ka media agar i loaʻa ka 200 μM pūhui a i ʻole DMSO.Hoʻoulu ʻia nā hua Arabidopsis i loko o kahi keʻena ulu a helu ʻia ka helu o nā mea kanu i hoʻoulu ʻia i nā lā 7 ma hope o ka ulu ʻana (dag).No ka hoʻāʻo ʻana i ka ulu ʻana o ka paka, ua lūlū ʻia nā hua he 24 i kēlā me kēia pā ma luna o ka media agar me 200 μM KAND a i ʻole DMSO.Ua ulu ʻia nā hua paka i loko o kahi keʻena ulu a helu ʻia ka nui o nā mea kanu i hoʻoulu ʻia ma hope o 14 mau lā.No ka hoʻāʻo ʻana i ka ulu ʻana o ka liverwort inhibition assay, 9 embryos mai kēlā me kēia pā i hoʻopaʻa ʻia ma luna o ka media agar e loaʻa ana nā manaʻo i hōʻike ʻia o KAND a i ʻole DMSO a hoʻokomo ʻia i loko o kahi keʻena ulu no nā lā 14.
E hoʻohana i nā mea kanu i hoʻopaʻa ʻia me 5 mg/ml propidium iodide (PI) e ʻike i ka hui meristem aʻa.Ua ʻike ʻia nā hōʻailona PI e ka microscopy fluorescence me ka hoʻohana ʻana i kahi microscope scanning laser confocal TCS SPE (Leica Microsystems).
Hana ʻia ka histochemical staining o nā aʻa me β-glucuronidase (GUS) e like me ka protocol i wehewehe ʻia e Malami a me Benfey36.Ua hoʻopaʻa ʻia nā mea kanu i ka 90% acetone i ka pō, i hoʻopaʻa ʻia me ka 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid i loko o ka GUS buffer no 1 hola a waiho ʻia i loko o kahi solution hydrated chloraldehyde.(8 g chloral hydrate, 2 ml wai a me 1 ml glycerol) a nānā ʻia e ka microscopy hoʻohālikelike ʻokoʻa me ka hoʻohana ʻana i kahi microscope Axio Imager M1 (Carl Zeiss).
Ua ana ʻia nā kihi aʻa ma luna o nā mea kanu he 7 mau lā i ulu ʻia ma luna o nā papa i kau ʻia.E ana i ka huina o ke kumu mai ke kuhikuhi o ka umekaumaha e like me ia i hoakakaia ma ka papa 6.
Ua nānāʻia ka hoʻonohonohoʻana o nā microtubules cortical e like me ka mea i ho'ākākaʻia, me nā hoʻololi liʻiliʻi i ka protocol 37.ʻO ka anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) a me Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) ua hoʻohana ʻia e like me nā antibodies mua a me ka lua ma 1:1000 a me 1:100 dilutions, pakahi.Ua loaʻa nā kiʻi Fluorescence me ka hoʻohana ʻana i kahi microscope scanning laser confocal TCS SPE (Leica Microsystems).E kiʻi i nā kiʻi Z-stack a hana i nā manaʻo ʻoi loa e like me nā ʻōlelo a ka mea hana.
Ua hana ʻia ʻo HeLa cell proliferation assay me ka hoʻohana ʻana i ka Cell Counting Kit 8 (Dojindo) e like me nā ʻōlelo a ka mea hana.
Hoʻopili ʻia ka ulu ʻana o E. coli DH5α ma ke ana ʻana i ka nui o nā cell i ka moʻomeheu me ka hoʻohana ʻana i kahi spectrophotometer ma 600 nm (OD600).
Ua ʻike ʻia ka hui cytoskeletal i nā cell transgenic BY-2 me ka microscope fluorescence i lako me kahi CSU-X1 confocal scanning device (Yokogawa) a me kahi kāmela sCMOS (Zyla, Andor Technology).Ua helu ʻia ka nui o ka cytoskeletal e ka nānā ʻana i ke kiʻi, ka mea i helu i ka pākēneka o nā pika cytoskeletal ma waena o nā cytoplasmic pixels i nā kiʻi confocal me ka hoʻohana ʻana i ka polokalamu ImageJ e like me ka wehewehe ʻana38,39.
No ka ʻike ʻana i ka make ʻana o ke kelepona i loko o nā pūnaewele BY-2, ua hoʻokomo ʻia kahi aliquot o ka hoʻokuʻu ʻia o ka cell me 0.05% Evans blue no 10 mau minuke ma ke ana wela.ʻO ke kala ʻulaʻula o Evans i koho ʻia ma muli o ka hoʻokuʻu ʻia ʻana o ke kala mai nā cell ola e ka membrane plasma intact40.Ua ʻike ʻia nā cell stained me ka microscope bright-field (BX53, Olympus).
Ua ulu ʻia nā pūnaewele HeLa i DMEM i hoʻohui ʻia me 10% FBS i loko o kahi incubator humidified ma 37 ° C a me 5% CO2.Ua mālama ʻia nā kelepona me 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng / ml colcemid (Gibco), a i ʻole 100 ng / ml Nocodmaze (Sigma) no 6 mau hola ma 37 ° C.Ua hoʻopaʻa ʻia nā kelepona me MetOH no 10 min a laila me ka acetate no 5 min i ka lumi wela.Ua hoʻopili ʻia nā pūnaewele paʻa me ka antibody β-tubulin mua (1D4A4, Proteintech: 66240-1) i hoʻoheheʻe ʻia ma 0.5% BSA/PBS no 2 mau hola, holoi ʻia ʻo 3 mau manawa me TBST, a laila hoʻomoʻi ʻia me ka Alexa Fluor kao antibody.488 1 hola.- Mouse IgG (Thermo Fisher Scientific: A11001) a me 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) i hoʻoheheʻe ʻia ma 0.5% BSA/PBS.Ma hope o ka holoi ʻana me TBST ʻekolu manawa, ʻike ʻia nā cell stained ma kahi Nikon Eclipse Ti-E inverted microscope.Ua kiʻi ʻia nā kiʻi me kahi kāmeʻa Hamamatsu ORCA-R2 CCD me ka hoʻohana ʻana i ka polokalamu MetaMorph (Molecular Devices).
Ka manawa hoʻouna: Jun-17-2024