英语翻译Since the major target of [Pt(L3)Cl]+ is the nucleolus,which is a major site of RNA synthesis,[20] we tested theeffect of [Pt(L3)Cl]+ on cellular transcription.Nascent RNAtranscripts can be labelled by fluorouridine,which can beimmunostai

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英语翻译Sincethemajortargetof[Pt(L3)Cl]+isthenucleolus,whichisamajorsiteofRNAsynthesis,[20]wetestedtheef

英语翻译Since the major target of [Pt(L3)Cl]+ is the nucleolus,which is a major site of RNA synthesis,[20] we tested theeffect of [Pt(L3)Cl]+ on cellular transcription.Nascent RNAtranscripts can be labelled by fluorouridine,which can beimmunostai
英语翻译
Since the major target of [Pt(L3)Cl]+ is the nucleolus,
which is a major site of RNA synthesis,[20] we tested the
effect of [Pt(L3)Cl]+ on cellular transcription.Nascent RNA
transcripts can be labelled by fluorouridine,which can be
immunostained by an antibody against halogen-containing
nucleosides.[21] In the absence of [Pt(L3)Cl]+,newly synthesised
RNA,labelled by fluorouridine,was detected in a punctate
pattern in cell nuclei (Figure 5D).After 15 min of incubation
with [Pt(L3)Cl]+ at 0.1 mm,however,newly synthesised
RNA was dramatically reduced in most cells (Figure
5E).At 0.5 mm,all RNA synthesis was abolished (Figure
5F).Hence,[Pt(L3)Cl]+ is a potent inhibitor of
transcription.
The mechanism by which [Pt(L3)Cl]+ mediates its transcription
inhibition is not clear.It is likely that on binding to
endogenous structures in cells,the cyclometalated PtII complex
blocks transcription-related reactions.It is therefore
important to identify the biological molecules interacting
with [Pt(L3)Cl]+.Since the majority of the cellular RNA accumulates
in the nucleolus,it is possible that [Pt(L3)Cl]+
can bind to RNA.To test this hypothesis,we incubated
methanol-fixed HeLa cells with RNase at a concentration
that is sufficient for removing the cellular RNA content.[22]
The RNase-treated cells were then stained with [Pt(L3)Cl]+.
As shown in Figure 6A and B,RNase-digested cells (Figure
6B) were still labelled brightly by [Pt(L3)Cl]+,and the
staining intensity and patterns were indistinguishable from
those in untreated cells (Figure 6A).It is therefore unlikely
that the interaction of [Pt(L3)Cl]+ with cellular structures is
RNA dependent.To confirm this data,we incubated total
cell lysate,purified DNA,RNA and proteins spotted on nitrocellulose
membrane with [Pt(L3)Cl]+.As shown in Fig-
ACHTUNGTRENUNGure 6C,[Pt(L3)Cl]+ effectively stained total cell lysate on
the blot,reproducing its cell binding properties observed by
microscopy.Interestingly,[Pt(L3)Cl]+ did not bind to DNA
or RNA but bound strongly to proteins,suggesting the staining
of [Pt(L3)Cl]+ may be mediated by its specific interaction
with some nuclear or nucleolar proteins.

英语翻译Since the major target of [Pt(L3)Cl]+ is the nucleolus,which is a major site of RNA synthesis,[20] we tested theeffect of [Pt(L3)Cl]+ on cellular transcription.Nascent RNAtranscripts can be labelled by fluorouridine,which can beimmunostai
由于[Pt(L3)Cl]+的主要目标是核仁,这是RNA合成的主要场所,[20] 所以我们试验了[Pt(L3)Cl]+对细胞转录的影响.初期发生的RNA转录产物可以用氟尿苷来标志,它可用一种针对含卤核苷的抗体免疫染色.[21] 在不存在[Pt(L3)Cl]+时,由氟尿标志的新合成的RNA在细胞核的点状图形中被检测到（图5D）.可是,在用[Pt(L3)Cl]+在0.1mM下培养15min后,新合成的RNA在大多数细胞中大大减少（图5E）.在0.5mM下,所有RNA的合成都被废止（图5F）.因此,[Pt(L3)Cl]+是一种转录的有效抑制剂.
[Pt(L3)Cl]+调定自己转录抑制作用的机理还不清楚.它很可能是在与细胞的内生结构结合时,环金属铂II络合物阻断了转录相关反应.因此很重要的是识别与[Pt(L3)Cl]+相互作用的生物分子.因为大多数细胞的RNA累积在细胞核内,所以有可能[Pt(L3)Cl]+能与RNA结合.为了实验这种假设,我们用RNase培养了甲醇固定的HeLa细胞,RNase的浓度足以去除细胞的RNA含量.[22] 经RNase处理的细胞随后用[Pt(L3)Cl]+染色.如图6A和B所示的那样,经RNase消化细胞（图6B）仍然被[Pt(L3)Cl]+所明亮地标志,而染色的强度和图形与未处理过的细胞中的强度和图形不能区分（图6A）.因此,[Pt(L3)Cl]+与细胞结构的相互作用不太可能是取决于RNA的.为了确认这一数据,我们培养了整个细胞的溶胞产物,净化了由[Pt(L3)Cl]+玷污在硝化纤维薄膜上的DNA、RNA和蛋白质.如在图6C中所示的那样,[Pt(L3)Cl]+有效地染色了在污斑上的整个细胞的溶胞产物,再现了其有显微镜术观察到的细胞结合性质.令人感兴趣的是,[Pt(L3)Cl]+不与DNA或RNA结合,但被强烈结合到蛋白质,表明了[Pt(L3)Cl]+的染色可以通过其与某些核蛋白质或核仁蛋白质的特异互作用来调定.

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自从主要目标的[Pt(L3)Cl)+的核仁,
它是一个主要的RNA合成、[20]我们测试了吗
[Pt(L3效果)Cl)+于细胞的方法。初生的RNA
文本可以被fluorouridine,即可
通过对halogen-containing immunostained抗体
核苷。(21)在缺乏[Pt(L3)。Cl)+、新合成的
fluorouridine RNA,他被发现,在一个斑点状的
细胞核内的模式(图5D)。15分钟后的孵化
[Pt(L3)。Cl)+在0.1毫米,然而,新合成的
在大多数RNA是大大减少细胞(如图)
5E)。在0.5毫米,废除了RNA合成(见图)
五)。因此,[Pt(L3)。Cl)+是一种强有力的抑制剂
抄写。
这个机制[Pt(L3)Cl)+调和它的方法
抑制还不清楚。它是可能的,在结合
在细胞内结构cyclometalated PtII复杂
transcription-related块时的反应。因此
重要的生物分子识别的交互
[Pt(L3)Cl)+。因为绝大多数的细胞RNA积累
在核仁,很可能是[Pt(L3)Cl)+
能与RNA。为了检验该假说,我们孵化
methanol-fixed RNase HeLa细胞浓度
这足以消除细胞RNA内容(22)。
这个RNase-treated细胞然后沾[Pt(L3)Cl)+。
如图里,RNase-digested和B细胞(如图)
译林出版社)仍被贴上鲜艳[Pt(L3)。Cl)+、
染色的强度和模式
那些在未经处理的细胞(图6安培)。这是不太可能的
那间的相互作用[Pt(L3)Cl)+和细胞结构
RNA依赖。确认这些数据,我们孵化的总数
细胞lysate,净化了DNA、RNA和蛋白质的纸上
[Pt(膜)Cl)+。L3如图-
ACHTUNGTRENUNGure 6摄氏度,[Pt(L3)Cl)+有效lysate总细胞染色
这个污点,再现其手机绑定属性观测到
显微镜下。有趣的是,[Pt(L3)。Cl)+没有绑定的DNA
但强烈或RNA的蛋白质,暗示了染色
[Pt(L3)。Cl)+可以通过其特有的互动
有些核或核仁蛋白质。

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