s******y
发帖数: 28562 | 1 endosome 和 phagosome主要是来源（膜上的蛋白）不同，导致后面的一
系列处理也不同
endosome 一般是由比较小的单分子结合在受体上引起的，一般需要
clathrin 来协助进入细胞
Phagosome是有比较大的复合体结合在细胞表面上引起的，不需要clathrin。
lysosome 是细胞内部包含着大量蛋白酶的泡泡，可以和其他泡泡融合之后来
降解里面的东西。 |
|
r******k
发帖数: 446 | 2 最近小弟想分离early endosome 但是没找到特别好的protocol。 不知道哪位大神分离
过early endosome？能否转个protocol？
拜谢！！ |
|
r******k
发帖数: 446 | 3 最近小弟想分离early endosome 但是没找到特别好的protocol。 不知道哪位大神分离
过early endosome？能否转个protocol？
拜谢！！ |
|
y**u
发帖数: 7459 | 4 classic endocytosis, for cell surface receptors etc.:
clathrin-mediated endocytois --endosome (pH higher, sorting process here)--
lysosome(low pH, protein lysis)
phagocytosis, for large solid material, by forming larger PM ruffles ---
phagosome |
|
r******k
发帖数: 446 | 5 这两天在extract early endosome。 然后想看看一个蛋白的磷酸化在early endosome
里面的变化。 我用的protocol是用不同梯度浓度的sucrose去extract early endosome
。 然后用超速离心分离。 early endosome在 25%-35%之间的液体中。 但是最后一步
early endosome was then precipitated with methanol/chloroform loaded in SDS-
PAGE for western blot analyses.
我没明白methanol/CHLOROFORM抽提后 我的蛋白在哪里？ 而且难道抽提以后还需要
vaccum dryer把蛋白都弄成干粉 然后加loading buffer煮了以后再上样？ |
|
q******g
发帖数: 3858 | 6 Chloroquine is a lysosomotropic agent that prevents endosomal acidification
[1]. It accumulates inside the acidic parts of the cell, including
endosomes and lysosomes.
This accumulation leads to inhibition of lysosomal enzymes that require an
acidic pH, and prevents fusion of endosomes and lysosomes.
Chloroquine is commonly used to study the role of endosomal acidification in
cellular processes [2, 3], such as the signaling of intracellular TLRs.
Moreover, Chloroquine inhibits autophagy as it ra... 阅读全帖 |
|
B***v
发帖数: 113 | 7 好建议，记下了。
"During vesicle uncoating, primary endocytic vesicles may acquire Rab5, a
small GTPase involved in transport to early endosomes and in early endosome
docking and fusion. Primary endocytic vesicles are targeted to and fuse with
early endosomes expressing phosphatidylinositol(3)-phosphate, PI(3)P, in a
process dependent on Rab5 effector proteins and early endosomal SNAREs."
我觉得抑制endocytosis相对简单。比较困难的是还想找到有哪些靶点，通过对其调节
可以增加endocytosis呢？ |
|
c**g
发帖数: 14 | 8 Unlike TLR3 and TLR7/8/9 which are located and act
exclusively from endosome, TLR4 can signal from both cell surface and
locations within the cell. The difference is that, the activation of TLR4 at
cell surface involves Myd88 and leads to NF-kB pathway activation, while
the activation of TLR4 in endosome involves TRIF and leads to IFN production.
面？ |
|
L****S
发帖数: 89 | 9 Great question! I think it may be all about location. And transfection
reagents alter the distribution of ligands to different subcellular
locations.
For TLR4, it's surface vs. endosome.
For dsRNA, it's endosome vs. cytosolic.
Maybe a cell biologist can tell us how liposome tranfection works? |
|
c**g
发帖数: 14 | 10 I think your explanation of location is logical, however I have read a paper
which said that the DOTAP transfection reagents (liposome based) can
directly deliver DNA/RNA into the endosome, and the TLR3 located mainly in
the endosome,so why is RIG-1/MDA5 activated when dsRNA is transfected
through liposome? |
|
L****S
发帖数: 89 | 11 Thanks for the discussion. I know which papers you are talking about. We
have to think about immunology here.
In the endosomal compartment, for dsRNA, ssRNA, DNA, TLR3/7/9 sense them
accordingly. No big question here.
In the cytosol, dsRNA is a strong "danger" signal showing viral infection
. That's why every cell has PKR, RIG-I/MDA-5, etc. So I speculate, when
liposome delivers ssRNA, RIG-I/MDA-5-mediated cytosolic response dominates
the TLR3 signal, quantitatively or qualitatively.
In the pape... 阅读全帖 |
|
g*****p
发帖数: 451 | 12 哦，这样的话就很合情合理了
你开始的CHO分泌型表达，蛋白在培养基里面的浓度是很低的
一般表达良好的外源蛋白在CHO中最多也就1~10 mg/L，如果你的蛋白分子量在一般范围
20~50 kD, 那蛋白酶浓度也就0.02uM或者多点，况且pH比较高，酶的活性也低，所以你
能看到很多full length的酶，E.coli表达的东西都是包涵体，活性全无，自然也就大
都是
full length了
合成多肽切割实验不是证明有其他蛋白酶能切，而是证明它自己能识别这个位点，而且
可以
把动力学参数算出来，不过这个是反式剪切实验，至于你的自切是反式切割还是顺式切割
就没办法证明了
你说的pH induced 酶自切活性是个很有趣的发现
我觉得你得查查文献把这个酶的细胞定位搞清楚
因为在endosome pH5-6,如果酶定位在这里，那跟其生理功能是非常相关的
至少我知道的一个例子是流感病毒HA，它发挥膜融合功能的时候就是可以在endosome通过
pH induced构象变化，然后进行病毒－细胞膜融合
如果你的pH导致构象变化是有意义的
那做药就至少有3种途径了
1.catalytic site... 阅读全帖 |
|
t*d
发帖数: 1290 | 13 IMHO, to find novel drug delivering methods might be better ways to conquer
cancer comparing to studying the mechanisms of cancer initiation and
progression.
Nature 491, S58–S60 (22 November 2012)
Nanotechnology: Carrying drugs
When Joseph DeSimone makes nanomedicines, he compares himself to a baker. He
mixes drugs with different chemical 'batters', puts them in tiny moulds,
cures them and then turns them out. He can mould almost any shape: discs,
cubes, long sticks, roughened doughn... 阅读全帖 |
|
b***2
发帖数: 348 | 14 endosomal escape主要不通过binding partner，出不了endosome蛋白就是死路一条，
而且相对来说gfp更容易一些。如果gfp都搞不定，其他蛋白更悬。 |
|
s****9
发帖数: 932 | 15 如果你可以让抗体efficiently进入细胞内，我可以非常负责的说，明天就专利这个技
术（想法），10年之内你就可以获得noble prize，20年之内你就是世界首富。
如果抗体可以进入细胞内，你可以target无数的transcription factor，
intracellular protein，调节protein－protein interaction，整个生物医药将发生
革命，创造无数的就业机会。
抗体size太大，要跨膜几乎不可能。当然你说，可以通过endocytosis，但是但是进入
endosome就出不来了（这个也是SiRNA最大的问题）。你如果能，在不toxic细胞的情况
下，控制让抗体（甚至siRNA）出endosome就足够赚很多很多钱了。 |
|
l******a
发帖数: 3339 | 16 进endosome, lysosome，golgi和ER跟cytosol是两个完全不同的概念。你能link一篇
reference吗？antibody也可以进endosome，并且被recycle出来，但是进不了cytosol。 |
|
a*****n
发帖数: 2835 | 17 蛋白沉淀最后应该在管底，用loading buffer溶解煮了上样
endosome
endosome
SDS- |
|
t*d
发帖数: 1290 | 18 【 以下文字转载自 Biology 讨论区 】
发信人: ttd (oldcat), 信区: Biology
标 题: better way to conquer cancer
发信站: BBS 未名空间站 (Wed Jan 16 14:57:14 2013, 美东)
IMHO, to find novel drug delivering methods might be better ways to conquer
cancer comparing to studying the mechanisms of cancer initiation and
progression.
Nature 491, S58–S60 (22 November 2012)
Nanotechnology: Carrying drugs
When Joseph DeSimone makes nanomedicines, he compares himself to a baker. He
mixes drugs with different chemical 'batters', ... 阅读全帖 |
|
c******n
发帖数: 5697 | 19 Chloroquine, a widely-used anti-malarial and autoimmune disease drug, has
recently been reported as a potential broad-spectrum antiviral drug.8,9
Chloroquine is known to block virus infection by increasing endosomal pH
required for virus/cell fusion, as well as interfering with the
glycosylation of cellular receptors of SARS-CoV.10 Our time-of-addition
assay demonstrated that chloroquine functioned at both entry, and at post-
entry stages of the 2019-nCoV infection in Vero E6 cells (Fig. 1c, d).... 阅读全帖 |
|
k**********4
发帖数: 16092 | 20 1. We know what it is
The first cases of AIDS were described in June 1981 and it took more than
two years to identify the virus (HIV) causing the disease. With COVID-19,
the first cases of severe pneumonia were reported in China on December 31,
2019 and by January 7 the virus had already been identified. The genome was
available on day 10. We already know that it is a new coronavirus from group
2B, of the same family as the SARS, which we have called SARSCoV2. The
disease is called COVID-19. It ... 阅读全帖 |
|
d*********o
发帖数: 6388 | 21 http://www.weibo.com/1251560221/IBpp2ontJ
子陵在听歌
今天 07:01 来自 iPhone 11 Pro Max
武汉大学人民医院报道了第一个羟氯喹（hydroxychloroquine, HCQ）治疗COVID-19的
随机对照RCT临床试验（ChiCTR2000029559），preprint上传到了medRxiv上。研究入组
了62名COVID-19患者，31人接受5天HCQ（400 mg/d）；另外31人进入对照组。研究主要
评价治疗5天后，临床康复时间（TTCR）、临床特征、影像学表现。
研究发现5天后羟氯喹组患者临床康复时间明显缩短，发热、咳嗽症状缓解更快（p<0.
05）。羟氯喹组81%患者肺炎表现明显改善，而对照组为55%（p<0.05）。所有进展成为
重症患者均在对照组。这项RCT研究非常重要，肯定了羟氯喹的治疗效果。
同时，Nature Nanotech的一篇Comment评述了羟氯喹的潜在作用机制，似乎羟氯喹主要
是作用于宿主细胞，而非病毒本身，羟氯喹可能抑制PICALM蛋白，从而抑制clathrin介
导的SARS-Co... 阅读全帖 |
|
B*********a
发帖数: 6244 | 22 武汉大学人民医院报道了第一个羟氯喹（hydroxychloroquine, HCQ）治疗COVID-19的
随机对照RCT临床试验（ChiCTR2000029559），preprint上传到了medRxiv上。研究入组
了62名COVID-19患者，31人接受5天HCQ（400 mg/d）；另外31人进入对照组。研究主
要 评价治疗5天后，临床康复时间（TTCR）、临床特征、影像学表现。
研究发现5天后羟氯喹组患者临床康复时间明显缩短，发热、咳嗽症状缓解更快（p<
;0. 05）。羟氯喹组81%患者肺炎表现明显改善，而对照组为55%（p<0.05）。所有
进展成为 重症患者均在对照组。这项RCT研究非常重要，肯定了羟氯喹的治疗效果。
同时，Nature Nanotech的一篇Comment评述了羟氯喹的潜在作用机制，似乎羟氯喹主要
是作用于宿主细胞，而非病毒本身，羟氯喹可能抑制PICALM蛋白，从而抑制clathrin
介 导的SARS-CoV-2被细胞内吞；羟氯喹还可以抑制endosome-溶酶解介导的S蛋白剪切
和进 入细胞。 |
|
f******g
发帖数: 1003 | 23 肖俊宇
1. Tagliabracci VS#, Xiao J#, Dixon JE. Phosphorylation of Substrates
Destined for Secretion by the Fam20 Kinases. Biochem Soc Trans. 2013 Aug 1;
41(4):1061-5. (# Equal contribution).
2. Xiao J, Tagliabracci VS, Wen J, Kim SA, Dixon JE. Crystal Structure of
the Golgi Casein Kinase. Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):
10574-9.
6. Xiao J, Engel JL, Zhang J, Chen MJ, Manning G, and Dixon JE.
Structural and functional analysis of PTPMT1, a phosphatase required for
cardiolipin... 阅读全帖 |
|
T**7
发帖数: 264 | 24 杂志是皇家化学学会的杂志，印象因子是6。
想审的同学请发给我：
1.名字
2.email
3.expertise 关键词（5个左右）
4.发表的文章
谢谢关注，希望大家好运
题目：Redox-triggered intracellular dePEGylation based on diselenide-linked
polycations for DNA delivery
ABSTRACT: Extracellular stability to protect DNA against nucleases and
stimuli-triggered intracellular DNA release are key factors in designing non
-viral gene vectors. In this study, diselenide-linked polycation mPEG-SeSe-
PEI was developed as a new type of PEG-detachable gene vector for redox-
responsive gen... 阅读全帖 |
|
v*******e
发帖数: 961 | 25 关键词：Solid Lipid Nanoparticles, nanomedicine / nanocarriers, DNA release,
endosomal acidification, HEK293T cells
请把你的姓名，单位email及简介（一两句话，第三人称）发我站内信箱。谢谢。 |
|
d*****r
发帖数: 2583 | 26 ☆─────────────────────────────────────☆
nmda (SingingMouse) 于 (Mon Feb 2 16:26:06 2009) 提到:
表达一个和膜有关的GFP fusion protein然后看他的localization,同时用各种oganell
marker去染co-localization,看这个fusion protein定位或者聚集在哪个oganell,这
个方法和结果有多大的可信度? 有人说完全不可信,任选一个oganell marker,总能找到
一个细胞co-localized;有人说所有这个protein到过的地方都应该能找到co-
localization,所以ER,Golgi,endosome,lysosome都应该有co-localization. 如果这样
,那到底应该如何胞内定位这个protein?
☆─────────────────────────────────────☆
n158 (土人) 于 (Mon Feb 2 17:12:41 2009) 提到:
I think it i |
|
i******k
发帖数: 4625 | 27 nice drawing
还有recycling endosome呢？ |
|
w******e
发帖数: 1187 | 28 I hope I knew this earlier...:)
I guess you are talking about endosomal escape? or chemical conjugation
simply
abrogate the drug property?
I'm hooked with the simplicity of conjugating aptamer with siRNA. both can
achieve certain level of specificity; both can be synthesized in a single
run; the toxicity profile of ONs are widely available due to the gold
rush in anti-sense field... Yet it's a shame that you only have that few
aptamers to choose from (well, only 1 or 2, according to my knowledge |
|
g****0
发帖数: 425 | 29 至少要共染DNA，才好猜吧。
我猜：
fused mitochondria or endosome;
or
microtubule bundle after failed mitosis; |
|
w******e
发帖数: 1187 | 30 hope to see some fellow researchers working on aptamers/targeted
delivery/endosome escape:)
also looking for roommate! |
|
b******r
发帖数: 111 | 31 Note: NHEs is membrane proteins that transmembrane 12 times. NHE5 is
localized to the plasma membrane;NHE6-9 reside on organellar membranes(
endosome,Golgi);
1. pcDNA transfects NHE5-9 through Fugene 6(ratio is 3 uL of Fugene/2 ug of
DNA) in 293 cells. After three days,collect cells.
2. Cold PBS washes cells. Add 150 uL of lysis buffer(final concentration:
50mM Tris pH7.4, 150mM NaCl, 1% triton, 0.1% sds, cocktail inhibitor)
into each well of 6-well plate. Scrape cells.
3. The lysate gets st... 阅读全帖 |
|
s****9
发帖数: 932 | 32 My two cents:
1. Not sure what kind of particle it is. But for lyposome, MPLA can bind
lyposome particles in the outside domains.
2. Still in debate whether TLR4 can be internalized and remain active in the
endosome. But it can be a possibility.
面？ |
|
S****r
发帖数: 982 | 33 Thanks! endosome vs cytosolic, it nicely addressed the question tangled me
long time. |
|
S****r
发帖数: 982 | 34 Does DOTAP bring DNA/RNA contents only to endosome?
paper |
|
v***a
发帖数: 1242 | 35 谢谢哈。ubiquitin方面的我不是太懂。有没有可能ubiquitin导致结合的蛋白一块
internalized，然后也进入endosome了？ |
|
p*****m
发帖数: 7030 | 36 AA又不能自己穿过细胞膜 那个只能算mTOR上游一个很重要的分子罢了。AA本身怎么从循
环系统进入细胞调节细胞内活动还是个未知问题，而且是个更有意思的问题，因为吧wh
ole organism和细胞联系起来了。
endosome上好像有AA transporter有报道，但是这个和细胞膜上的是不是一样的还不知
道，八成不是一样的 |
|
p*****m
发帖数: 7030 | 37 AA又不能自己穿过细胞膜 那个只能算mTOR上游一个很重要的分子罢了。AA本身怎么从循
环系统进入细胞调节细胞内活动还是个未知问题，而且是个更有意思的问题，因为吧wh
ole organism和细胞联系起来了。
endosome上好像有AA transporter有报道，但是这个和细胞膜上的是不是一样的还不知
道，八成不是一样的 |
|
a*****n
发帖数: 2835 | 38 看看是不是endosome/lysosome
染EEA1或者LAMP试试
转到别的细胞有同样的现象吗？ |
|
k***r
发帖数: 100 | 39 I agree with Ankyrin, looks like endosomes and lysosomes (try EEA1 and some
lysozyme markers). Some times, truncated protein after translation it
misfolds and directly sent to lysosome for degradation. |
|
M**a
发帖数: 4816 | 40 26 January, 2012 Volume 1, Issue 1EditorialMore than the Sum of Its Parts p1
Boyana Konforti
Full Text | PDF (62 kb)
More than the Sum of Its Parts
Boyana Konforti
Editor, Cell Reports
Welcome to the first issue of Cell Reports, a new open-access journal that
covers all of biology with a focus on high-quality short papers. There are,
of course, other open-access journals—in fact, quite a number have launched
just in recent years (and there will almost certainly be more to follow)—
though few hav... 阅读全帖 |
|
M**a
发帖数: 4816 | 41 26 January, 2012 Volume 1, Issue 1EditorialMore than the Sum of Its Parts p1
Boyana Konforti
Full Text | PDF (62 kb)
More than the Sum of Its Parts
Boyana Konforti
Editor, Cell Reports
Welcome to the first issue of Cell Reports, a new open-access journal that
covers all of biology with a focus on high-quality short papers. There are,
of course, other open-access journals—in fact, quite a number have launched
just in recent years (and there will almost certainly be more to follow)—
though few hav... 阅读全帖 |
|
l**********1
发帖数: 5204 | 42 Exactly your opinion is right.
plus even without CryoEM below group still can do simulation with modeling for their most possbile
mechanism discovery
please refer the figure G its scale bar 200 nm it is below florescent microscopy resolution limits,
so they cannot resolve the fusion of the individual FIP3-endosomes.
cited from
//www.ncbi.nlm.nih.gov/pubmed/21486954
and
//www.ncbi.nlm.nih.gov/pubmed/16818889
will |
|
j******i
发帖数: 939 | 43 现在没有试剂盒吧。把蛋白直接导入细胞很难 现在比较成熟的是在目的蛋白一端加上
cell penetrating domain, 但是提存蛋白需要专业设备。使用这种方法，蛋白折叠可
能会出现问题且多数蛋白困在endosome，最后被降解。使用病毒包装蛋白的方法还不成
熟。另外，很好奇楼主做什么方向的啊。 |
|
h****k
发帖数: 182 | 44 google也没查到什么权威的解释，有人知道吗？ |
|
h****k
发帖数: 182 | 45 really clear now. thank you! |
|
l**********1
发帖数: 5204 | 46 楼主 你能发现特例 就是 既可以 endo 一移动又可以lyso or phago 的话
draft 上CNS 吧 |
|
u**********d
发帖数: 573 | 47 赞！
很好奇两点：
Fab是怎么进细胞的？如果是endocytosis干的，又是怎么从endosome释放到细胞质里的呢
Fab能不能和GFP做成嵌合蛋白？Fab的糖基化对其特异性影响大不大 |
|
i***0
发帖数: 160 | 48 For GPCR internalization, you can try beta-galactosidase complement assay.
search if DiscoverRx has already made the corresponding cell line. That
assay can give you sig/background=10. Internalized GPCR (tagged small
subunit of beta-gal) collocalized with endosome anchored marker protein (
tagged with large subunit of beta-gal) will boost beta-Gal activity. I have
tried this assay. once you have the stable cell line, the protocol is very
simple and the result is very potent. |
|
l**********1
发帖数: 5204 | 49 咳 那是泛指瑞士的人均论文引用次数是连续五年 世界第一的话
以下的名单里边 位于瑞士的PIs
今后 有成生物顶级大牛的至少有一到二位吧
更不要说 S. Tonegawa 的博士后 包括炸药奖的那篇免疫论文可是在巴塞尔做的
Ps:
EMBO Conference Series
Dynamic Endosomes:
Mechanisms Controlling Endocytosis
24 - 29 September, 2011 | Chania | Greece
Speakers
from web site:
http://events.embo.org/11-endocytosis/speakers.html
only now in Switzerland as shown as below:
a)
Marcos Gonzalez-Gaitan
University of Geneva
Switzerland
Wartlick, O. at al. and González-Gaitán, M. (2011)
Science, 331: 1154-1159
link:
http:/... 阅读全帖 |
|
C********4
发帖数: 308 | 50 1）DT注射到体内的量很少，一个细胞分不到多少DT; 2) DT有两个亚基A和B，B负责跟
受体结合进细胞，并在endosome/lysosome里降解，A进cytosol里搞破坏。即使A流出来
，没有B也进不了别的细胞。3）现在打靶载体上都带DTA做阴性筛选，从来没有见到DTA
从被杀死细胞流出来影响别的细胞。
再有就是这个方法已经应用了超过10年，没有遇到过问题。做这个改进主要是两个目的
： 1）提高细胞剔除效率: A)用强启动子，使DTR表达水平提高；2）加EGFP使细胞可以
被追踪又可以随时被清除，比如我们想观察某群细胞被杀死之后的再生情况。 |
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