曲度編著:細胞死亡通道悖論-Cell death pathway paradox

2009年第2篇:

He C, Klionsky DJ.：Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67-93.

曲 度譯:細胞自噬的調控機制和信號通路

Life Sciences Institute and Departments of Molecular, Cellular and Developmental Biology, and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

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Abstract

Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases.

Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases.

We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis.

We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.

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整理如下:

摘要

自噬是一種細胞成分的自我降解過程；在該過程中，雙膜自噬體隔離細胞器或部分細胞質，並通過所在處的水解酶與溶酶體或液泡融合以便穿透。

在細胞外或細胞應激和某些信號（例如飢餓，生長因子匱乏，內質網應激，和病原體感染）條件的反應中，細胞自噬活動被上調。

缺陷性細胞自噬在人類發病過程中，包括癌症，神經退行性病變和傳染性疾病，起著一種顯著的作用。

我們提出一些自己的最新下列看法：從酵母到真核生物細胞的自噬機關鍵基因構成；對不同類型的應激狀態下的一些信號通路，以及誘導細胞生存及其動態平衡的自噬作用。

我們也對不同水平下調節自噬的分子機制（從轉錄激活作用到翻譯后蛋白修飾作用）方面的最新進展做了文獻複習。

HUABIN 初譯稿

曲 度譯：自食和自殺：自噬與凋亡之間的交談

Maiuri MC, Zalckvar E, Kimchi A,ET AL：Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2007 Sep;8(9):741-52.

INSERM, U848, F-94805 Villejuif, France.

Abstract
The functional relationship between apoptosis ('self-killing') and autophagy ('self-eating') is complex in the sense that, under certain circumstances, autophagy constitutes a stress adaptation that avoids cell death (and suppresses apoptosis), whereas in other cellular settings, it constitutes an alternative cell-death pathway.

Autophagy and apoptosis may be triggered by common upstream signals, and sometimes this results in combined autophagy and apoptosis; in other instances, the cell switches between the two responses in a mutually exclusive manner.

On a molecular level, this means that the apoptotic and autophagic response machineries share common pathways that either link or polarize the cellular responses.

摘要：

細胞凋亡（「自殺」） 自體吞噬（「自食」）的功能之間的關係處於一種複雜的狀況之中；在某些情況下，細胞自噬構成了應激適應，這樣就可避免細胞死亡同時抑制細胞凋亡，而在其他的細胞設置中，它又構成了另一種細胞死亡途徑。

自噬與凋亡可能被一些共同的上游信號所觸發，有時這種混合就導致了自噬與凋亡；在其他情況下，在這兩種反應之間的細胞開關處於一種相互排斥的狀態。在分子水平上，這意味著在細胞凋亡和自噬的反應機制有著共同的通道，要麼鏈接或者要麼分化某些細胞反應。

PMID: 17717517

HUABIN初譯稿

這是Kerr寫的那篇有關"Apoptosis"文章之摘要!現Cited by 9956次!

從這篇文章開始,現用"Apoptosis"可檢索到近19萬次(189,904次)!

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Br J Cancer. 1972 August; 26(4): 239–257. PMCID: PMC2008650


Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics

J. F. R. Kerr, A. H. Wyllie, and A. R. Currie

From the departement of pathology, University of Aberdeen

This article has been cited by other articles in PMC.

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Abstract

The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations.

Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.

The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments.

In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.

Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development.

It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression.

It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.

這是能檢索到的,

比利時學者Christian de Duve在1967年寫的一篇在標題中涉及"AUTOPHAGY"的文獻標題!

截今為止,用"AUTOPHAGY"可檢索的到6131篇英文文獻!  

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J Cell Biol. 1967 November 1; 35(2): C11–C16. PMCID: PMC2107130

Copyright © 1967 by The Rockefeller University Press

Article

PARTICIPATION OF LYSOSOMES IN CELLULAR AUTOPHAGY INDUCED IN RAT LIVER BY GLUCAGON

Russell L. Deter, Pierre Baudhuin, and Christian de Duve

From the Laboratoire de Chimie Physiologique, University of Louvain, Belgium.

Dr. Deter's present address is Department of Anatomy, Baylor University College of Medicine, Houston, Texas

Received July 31, 1967

This article has been cited by other articles in PMC.

曲度編緝:自噬與凋亡在Pubmed所記錄的文獻數量比較I

自噬（autophagy）與凋亡（apoptosis）在Pubmed所記錄的文獻數量比較表1

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.................凋亡（apoptosis） 自噬（autophagy）
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半年之內： 10248篇 ........ 896篇

一年之內： 19378篇 ........ 1616篇

二年之內： 36510篇 ........ 2810篇

三年之內： 53402篇 ........ 3624篇

五年之內： 85123篇 ........ 4551篇

十年之內： 150401篇 ........ 5207篇
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全部文獻： 189904篇（72年始） 6131篇（65年始）
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注:本人聲明保留該貼版權!如引用請註明出處!

D.QU

曲度編緝:自噬與凋亡在Pubmed所記錄的文獻數量比較II

自噬（autophagy）與凋亡（apoptosis），Pubmed所記錄的文獻數量比較表2
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...............................凋亡（apoptosis） 自噬（autophagy）
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1960．1．1—1969。12．31..............0篇...................2篇
1970．1．1—1979。12．31............35篇...............145篇
1980．1．1—1989。12．31..........246篇...............356篇
1990．1．1—1999。12．31......31327篇...............316篇
2000．1．1—2009。12．31....145428篇.............4093篇
最近一年:
2001．1．1—2010。12．31......15327篇..............1351篇
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注1：此表數字系在查Pubmed時得出，為何與上表總數有差異不甚清楚！

注2:本人聲明保留該貼版權!如引用請註明出處!

D.QU

曲 度:凋亡與自嗜兩者論文總數與增長情況:

根據上表I數據,可以得出下面一個比較結果:

一.關於凋亡與自嗜兩者論文增長情況:

1.從近一年到近半年之間:有關凋亡的論文增長了1.89倍; 有關自嗜的論文增長了1.80倍;
2.從近二年到近一年之間:有關凋亡的論文增長了1.88倍; 有關自嗜的論文增長了1.73倍;
3.從近三年到近二年之間:有關凋亡的論文增長了1.46倍; 有關自嗜的論文增長了1.28倍;
4.從近五年到近三年之間:有關調亡的論文增長了1.59倍; 有關自噬的論文增長了1.26倍;

二.關於凋亡與自嗜兩者論文總數情況:

調亡論文189904篇/自嗜論文6131篇=30.81倍

D.QU

下面是可檢索到的最新50篇有關調亡之專業文獻!(截止2010.12)

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1.Diabetic cystopathy is associated with PARP/JNK/ mitochondrial apoptotic pathway-mediated bladder apoptosis.

Li WJ, Oh SJ.

Neurourol Urodyn. 2010 Sep;29(7):1332-7.
PMID: 20879002 [PubMed - in process]

2.Down-regulation of microRNA 106b is involved in p21-mediated cell cycle arrest in response to radiation in prostate cancer cells.

Li B, Shi XB, Nori D, Chao CK, Chen AM, Valicenti R, de Vere White R.

Prostate. 2010 Sep 28. [Epub ahead of print]
PMID: 20878953 [PubMed - as supplied by publisher]
3.Comparison of MTT assay, flow cytometry, and RT-PCR in the evaluation of cytotoxicity of five prosthodontic materials.

Wang X, Xia Y, Liu L, Liu M, Gu N, Guang H, Zhang F.

J Biomed Mater Res B Appl Biomater. 2010 Sep 28. [Epub ahead of print]
PMID: 20878925 [PubMed - as supplied by publisher]
4.DNA-dependent protein kinase catalytic subunit mediates T-cell loss in rheumatoid arthritis.

Shao L, Goronzy JJ, Weyand CM.

EMBO Mol Med. 2010 Sep 28. [Epub ahead of print]
PMID: 20878914 [PubMed - as supplied by publisher]
5.MicroRNA expression changes during zebrafish development induced by perfluorooctane sulfonate.

Zhang L, Li YY, Zeng HC, Wei J, Wan YJ, Chen J, Xu SQ.

J Appl Toxicol. 2010 Sep 28. [Epub ahead of print]
PMID: 20878907 [PubMed - as supplied by publisher]
6.Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrP(Sc), even upon LPS stimulation.

Hughes MM, Field RH, Perry VH, Murray CL, Cunningham C.

Glia. 2010 Sep 27. [Epub ahead of print]
PMID: 20878768 [PubMed - as supplied by publisher]
7.Statin-associated myopathy and its exacerbation with exercise.

Meador BM, Huey KA.

Muscle Nerve. 2010 Oct;42(4):469-79.
PMID: 20878737 [PubMed - in process]
8.[Impact of fragile histidine triad gene transfection on the proliferation and apoptosis of human colorectal cancer cell.]

Cao J, Liang LY, Yang P, Qian YJ, Wang H, Sun Z, Li WL, Tan MH.

Zhonghua Wei Chang Wai Ke Za Zhi. 2010 Sep;13(9):691-4. Chinese.
PMID: 20878579 [PubMed - in process]
9.Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca(2+) fluxes in oxidative cell death.

Blenn C, Wyrsch P, Bader J, Bollhalder M, Althaus FR.

Cell Mol Life Sci. 2010 Sep 29. [Epub ahead of print]
PMID: 20878536 [PubMed - as supplied by publisher]
10.Combined treatment with TRAIL and PPARγ ligands overcomes chemoresistance of ovarian cancer cell lines.

Bräutigam K, Biernath-Wüpping J, Bauerschlag DO, von Kaisenberg CS, Jonat W, Maass N, Arnold N, Meinhold-Heerlein I.

J Cancer Res Clin Oncol. 2010 Sep 29. [Epub ahead of print]
PMID: 20878528 [PubMed - as supplied by publisher]
11.1p36.32 rearrangements and the role of PI-PLC η2 in nervous tumours.

Lo Vasco VR.

J Neurooncol. 2010 Sep 29. [Epub ahead of print]
PMID: 20878447 [PubMed - as supplied by publisher]
12.Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines.

Lundh M, Christensen DP, Rasmussen DN, Mascagni P, Dinarello CA, Billestrup N, Grunnet LG, Mandrup-Poulsen T.

Diabetologia. 2010 Sep 28. [Epub ahead of print]
PMID: 20878317 [PubMed - as supplied by publisher]
13.Subcellular TSC22D4 Localization in Cerebellum Granule Neurons of the Mouse Depends on Development and Differentiation.

Canterini S, Bosco A, Carletti V, Fuso A, Curci A, Mangia F, Fiorenza MT.

Cerebellum. 2010 Sep 28. [Epub ahead of print]
PMID: 20878296 [PubMed - as supplied by publisher]
14.Functional properties of synthetic N-acyl-L-homoserine lactone analogs of quorum-sensing gram-negative bacteria on the growth of human oral squamous carcinoma cells.

Chai H, Hazawa M, Shirai N, Igarashi J, Takahashi K, Hosokawa Y, Suga H, Kashiwakura I.

Invest New Drugs. 2010 Sep 28. [Epub ahead of print]
PMID: 20878204 [PubMed - as supplied by publisher]
15.Suppressed protein expression of the death-associated protein kinase enhances 5-fluorouracil-sensitivity but not etoposide-sensitivity in human endometrial adenocarcinoma cells.

Tanaka T, Bai T, Yukawa K.

Oncol Rep. 2010 Nov;24(5):1401-5.
PMID: 20878137 [PubMed - in process]
16.microRNA-143, down-regulated in osteosarcoma, promotes apoptosis and suppresses tumorigenicity by targeting Bcl-2.

Zhang H, Cai X, Wang Y, Tang H, Tong D, Ji F.

Oncol Rep. 2010 Nov;24(5):1363-9.
PMID: 20878132 [PubMed - in process]
17.Knockdown of the c-Jun-N-terminal kinase expression by siRNA inhibits MCF-7 breast carcinoma cell line growth.

Parra E, Ferreira J.

Oncol Rep. 2010 Nov;24(5):1339-45.
PMID: 20878129 [PubMed - in process]
18.Replication-dependent γ-H2AX formation is involved in docetaxel-induced apoptosis in NSCLC A549 cells.

Zhang F, Zhang T, Qu Y, Jiang T, Cao YX, Li C, Fan L, Mei QB.

Oncol Rep. 2010 Nov;24(5):1297-305.
PMID: 20878124 [PubMed - in process]
19.Curcumin promotes apoptosis in human lung adenocarcinoma cells through miR-186* signaling pathway.

Zhang J, Du Y, Wu C, Ren X, Ti X, Shi J, Zhao F, Yin H.

Oncol Rep. 2010 Nov;24(5):1217-23.
PMID: 20878113 [PubMed - in process]
20.Regulation of apoptosis by p53-inducible transmembrane protein containing sushi domain.

Cui H, Kamino H, Nakamura Y, Kitamura N, Miyamoto T, Shinogi D, Goda O, Arakara H, Futamura M.

Oncol Rep. 2010 Nov;24(5):1193-200.
PMID: 20878110 [PubMed - in process]
21.Discrepancy of biologic behavior influenced by bone marrow derived cells in lung cancer.

Zhang J, Niu XM, Liao ML, Liu Y, Sha HF, Zhao Y, Yu YF, Tan Q, Xiang JQ, Fang J, Lv DD, Li XB, Lu S, Chen HQ.

Oncol Rep. 2010 Nov;24(5):1185-92.
PMID: 20878109 [PubMed - in process]
22.Induction of apoptosis by HAC-Y6, a novel microtubule inhibitor, through activation of the death receptor 4 signaling pathway in human hepatocellular carcinoma cells.

Tsai JY, Hung CM, Bai ST, Huang CH, Chen WC, Chung JG, Kuo SC, Way TD, Huang LJ.

Oncol Rep. 2010 Nov;24(5):1169-78.
PMID: 20878107 [PubMed - in process]
23.Schedule-dependent antitumor activity of the combination with erlotinib and docetaxel in human non-small cell lung cancer cells with EGFR mutation, KRAS mutation or both wild-type EGFR and KRAS.

Furugaki K, Iwai T, Shirane M, Kondoh K, Moriya Y, Mori K.

Oncol Rep. 2010 Nov;24(5):1141-6.
PMID: 20878103 [PubMed - in process]
24.Differential regulation of proliferation, cell cycle control and gene expression in cultured human aortic and pulmonary artery endothelial cells by resveratrol.

Hsieh TC, Lu X, Guo J, Wu JM.

Int J Mol Med. 2010 Nov;26(5):743-9.
PMID: 20878097 [PubMed - in process]
25.Simvastatin inhibits cell growth and induces apoptosis and G0/G1 cell cycle arrest in hepatic cancer cells.

Relja B, Meder F, Wilhelm K, Henrich D, Marzi I, Lehnert M.

Int J Mol Med. 2010 Nov;26(5):735-41.
PMID: 20878096 [PubMed - in process]
26.Induction of apoptosis by 5,7-dihydroxy-8-nitrochrysin in breast cancer cells: The role of reactive oxygen species and Akt.

Zhao XC, Tian L, Cao JG, Liu F.

Int J Oncol. 2010 Nov;37(5):1345-52.
PMID: 20878083 [PubMed - in process]
27.Hedyotis Diffusa Willd extract induces apoptosis via activation of the mitochondrion-dependent pathway in human colon carcinoma cells.

Lin J, Chen Y, Wei L, Chen X, Xu W, Hong Z, Sferra TJ, Peng J.

Int J Oncol. 2010 Nov;37(5):1331-8.
PMID: 20878081 [PubMed - in process]
28.Calpain is involved in cisplatin-induced endothelial injury in an in vitro three-dimensional blood vessel model.

Eguchi R, Fujimori Y, Ohta T, Kunimasa K, Nakano T.

Int J Oncol. 2010 Nov;37(5):1289-96.
PMID: 20878076 [PubMed - in process]
29.Cantharidin induces apoptosis in human bladder cancer TSGH 8301 cells through mitochondria-dependent signal pathways.

Kuo JH, Chu YL, Yang JS, Lin JP, Lai KC, Kuo HM, Hsia TC, Chung JG.

Int J Oncol. 2010 Nov;37(5):1243-50.
PMID: 20878071 [PubMed - in process]
30.Triptolide inactivates Akt and induces caspase-dependent death in cervical cancer cells via the mitochondrial pathway.

Kim MJ, Lee TH, Kim SH, Choi YJ, Heo J, Kim YH.

Int J Oncol. 2010 Nov;37(5):1177-85.
PMID: 20878065 [PubMed - in process]
31.Mechanisms behind COX-1 and COX-2 inhibition of tumor growth in vivo.

Axelsson H, Lönnroth C, Andersson M, Lundholm K.

Int J Oncol. 2010 Nov;37(5):1143-52.
PMID: 20878062 [PubMed - in process]
32.Identification of NEEP21, encoding neuron-enriched endosomal protein of 21 kDa, as a transcriptional target of tumor suppressor p53.

Ohnishi S, Futamura M, Kamino H, Nakamura Y, Kitamura N, Miyamoto Y, Miyamoto T, Shinogi D, Goda O, Arakawa H.

Int J Oncol. 2010 Nov;37(5):1133-41.
PMID: 20878061 [PubMed - in process]
33.Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration.

Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, Mittal M, Barit D, Schwarz T, Geis C, Kraft P, Barthel K, Schuhmann MK, Herrmann AM, Meuth SG, Stoll G, Meurer S, Schrewe A, Becker L, Gailus-Durner V, Fuchs H, Klopstock T, de Angelis MH, Jandeleit-Dahm K, Shah AM, Weissmann N, Schmidt HH.

PLoS Biol. 2010 Sep 21;8(9). pii: e1000479.
PMID: 20877715 [PubMed - in process]
34.CXCL12 Chemokine Expression and Secretion Regulates Colorectal Carcinoma Cell Anoikis through Bim-Mediated Intrinsic Apoptosis.

Drury LJ, Wendt MK, Dwinell MB.

PLoS One. 2010 Sep 22;5(9). pii: e12895.
PMID: 20877573 [PubMed - in process]
35.Gene expression profiles of colonic mucosa in healthy young adult and senior dogs.

Kil DY, Vester Boler BM, Apanavicius CJ, Schook LB, Swanson KS.

PLoS One. 2010 Sep 22;5(9). pii: e12882.
PMID: 20877568 [PubMed - in process]
36.siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model.

Liang Y, Gao H, Lin SY, Goss JA, Brunicardi FC, Li K.

PLoS One. 2010 Sep 20;5(9). pii: e12860.
PMID: 20877462 [PubMed - in process]
37.Improved mitochondrial function in brain aging and Alzheimer disease - the new mechanism of action of the old metabolic enhancer piracetam.

Leuner K, Kurz C, Guidetti G, Orgogozo JM, Müller WE.

Front Neurosci. 2010 Sep 7;4. pii: 44.
PMID: 20877425 [PubMed - in process]
38.Fenretinide-dependent upregulation of death receptors through ASK1 and p38α enhances death receptor ligand-induced cell death in Ewing's sarcoma family of tumours.

White DE, Burchill SA.

Br J Cancer. 2010 Sep 28. [Epub ahead of print]
PMID: 20877355 [PubMed - as supplied by publisher]
39.A Therapeutic Approach to Nasopharyngeal Carcinomas by DNAzymes Targeting EBV LMP-1 Gene.

Yang L, Lu Z, Ma X, Cao Y, Sun LQ.

Molecules. 2010 Sep 1;15(9):6127-39.
PMID: 20877211 [PubMed - in process]
40.Geno-transcriptomic dissection of proteinuria in the uninephrectomized rat uncovers a molecular complexity with sexual dimorphism.

Yagil Y, Hessner MJ, Schulz H, Gosele C, Lebdev L, Barkalifa R, Sapojnikov M, Hubner N, Yagil C.

Physiol Genomics. 2010 Sep 28. [Epub ahead of print]
PMID: 20876844 [PubMed - as supplied by publisher]
41.Inhibition of Autophagy Enhances Anticancer Effects of Atorvastatin in Digestive Malignancies.

Yang PM, Liu YL, Lin YC, Shun CT, Wu MS, Chen CC.

Cancer Res. 2010 Sep 28. [Epub ahead of print]
PMID: 20876807 [PubMed - as supplied by publisher]
42.Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia.

Chiarini F, Grimaldi C, Ricci F, Tazzari PL, Evangelisti C, Ognibene A, Battistelli M, Falcieri E, Melchionda F, Pession A, Pagliaro P, McCubrey JA, Martelli AM.

Cancer Res. 2010 Sep 28. [Epub ahead of print]
PMID: 20876803 [PubMed - as supplied by publisher]
43.RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype.

Yang D, Kedei N, Li L, Tao J, Velasquez JF, Michalowski AM, Tóth BI, Marincsák R, Varga A, Bíró T, Yuspa SH, Blumberg PM.

Cancer Res. 2010 Sep 28. [Epub ahead of print]
PMID: 20876802 [PubMed - as supplied by publisher]
44.Aerobic Glycolysis Suppresses p53 Activity to Provide Selective Protection from Apoptosis upon Loss of Growth Signals or Inhibition of BCR-Abl.

Mason EF, Zhao Y, Goraksha-Hicks P, Coloff JL, Gannon H, Jones SN, Rathmell JC.

Cancer Res. 2010 Sep 28. [Epub ahead of print]
PMID: 20876800 [PubMed - as supplied by publisher]
45.Preclinical Characterization of Mitochondria-Targeted Small Molecule Hsp90 Inhibitors, Gamitrinibs, in Advanced Prostate Cancer.

Kang BH, Siegelin MD, Plescia J, Raskett CM, Garlick DS, Dohi T, Lian JB, Stein GS, Languino LR, Altieri DC.

Clin Cancer Res. 2010 Sep 28. [Epub ahead of print]
PMID: 20876793 [PubMed - as supplied by publisher]
46.Identification of c-FLIPL and c-FLIPS as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells.

Haag C, Stadel D, Zhou S, Bachem MG, Möller P, Debatin KM, Fulda S.

Gut. 2010 Sep 28. [Epub ahead of print]
PMID: 20876774 [PubMed - as supplied by publisher]
47.Different effects of oleate versus palmitate on mitochondrial function, apoptosis and insulin signaling in L6 skeletal muscle cells: role of oxidative stress.

Yuzefovych L, Wilson G, Rachek L.

Am J Physiol Endocrinol Metab. 2010 Sep 28. [Epub ahead of print]
PMID: 20876761 [PubMed - as supplied by publisher]
48.Colon Tumor Cell Growth-Inhibitory Activity of Sulindac Sulfide and Other Nonsteroidal Anti-Inflammatory Drugs Is Associated with Phosphodiesterase 5 Inhibition.

Tinsley HN, Gary BD, Thaiparambil J, Li N, Lu W, Li Y, Maxuitenko YY, Keeton AB, Piazza GA.

Cancer Prev Res (Phila). 2010 Sep 28. [Epub ahead of print]
PMID: 20876730 [PubMed - as supplied by publisher]
49.Potential cytotoxic effect of hydroxypyruvate produced from D-serine by astroglial D-amino acid oxidase.

Chung SP, Sogabe K, Park HK, Song Y, Ono K, Abou El-Magd RM, Shishido Y, Yorita K, Sakai T, Fukui K.

J Biochem. 2010 Sep 27. [Epub ahead of print]
PMID: 20876609 [PubMed - as supplied by publisher]
50.TGF-{beta}1 protects against mesangial cell apoptosis via induction of autophagy.

Ding Y, Kim JK, Kim SI, Na HJ, Jun SY, Lee SJ, Choi ME.

J Biol Chem. 2010 Sep 28. [Epub ahead of print]
PMID: 20876581 [PubMed - as supplied by publisher]

曲度:"細胞死亡通道"的問題對人類來說至今它是一個黑洞"

"細胞死亡通道"的問題,至今它是一個人類認識領域的"黑洞",本人預計在至少100年內,不可能會有根本性的突破!

原因在於:"細胞"本身就相當與一個"小宇宙"! 人類研究"細胞"其困難程度一點都不會小於研究"宇宙"!能說不困難?

此外,研究"細胞死亡通道"問題,就好似研究臨床機體整體層次的"多臟器衰竭死亡"問題一樣!

如果后一個問題那麼容易攻克,臨床還會死人嗎! 您說有可能嗎?前者也同此理!只是層次不同!

D.QU

廣譜抗病毒物質"天龍,DRACO"與細胞凋亡:

鏈接:http://news.dxy.cn/bbs/topic/21562857

曲 度譯:"天龍,DRACO"到底是什麼?DRACO廣譜抗病毒之機制!

引自原文如下:

In its simplest form, a DRACO is a chimeric protein with one domain that binds to viral dsRNA and a second domain (e.g., a procaspase-binding domain or a procaspase) that induces apoptosis when two or more DRACOs crosslink on the same dsRNA.

DRACO其最簡單形式就是一種嵌合蛋白;其一個區域與病毒dsRNA結合,第二個區域（例如:一個前半胱天冬酶"procaspase"結合區域,或者一個前半胱天冬酶"procaspase"結合區域）則誘導細胞凋亡(當兩個或兩個以上的DRACOs交聯相同的dsRNA之時。)

If viral dsRNA is present inside a cell, DRACOs will bind to the dsRNA and induce apoptosis of that cell. If viral dsRNA is not present inside the cell, DRACOs will not crosslink and apoptosis will not occur.

如果病毒雙鏈RNA存在細胞內，DRACOs將結合雙鏈RNA,並誘導該細胞凋亡。如果病毒dsRNA在細胞內不存在，DRACOs將不會發生交聯，細胞凋亡也就不會發生。

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"天龍,DRACO"是什麼?

"天龍,DRACO"是下列一組專業英文詞的縮寫:

Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO)
即是一種"活化Caspase Oligomerizer的雙鏈RNA" !
Oligomerizer這個詞不好譯,先放在這裡;有誰能譯嗎?

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HUABIN註釋:

1.Caspase 是細胞死亡酶系之中的一個家族!其在1996年被命此名,前面的"C"指的是半胱氨酸,後面的"aspase "指的是天門冬氨酸.可以簡稱為"半胱天冬酶"!Caspase 現被認為是程序性細胞死亡(PCD)之關鍵,任何引起細胞凋亡的基因(例如Fas死亡因子, P53,TNFR1等)均須激活Caspase 方能引起PCD;故上述因子也被稱為促細胞凋亡基因,反之則為抗細胞凋亡基因(例如,BCL-2,P35等).

2.Oligomerizer 其前面部分的"Oligo"是一個前綴詞,表示"過少,不足,微,寡"等義之字干;Oligomerizer其後面部分的"merizer"查不到,僅在新英漢醫學大辭典中查到"merisis"(裂生,分裂性增大)與"merism"(裂殖)兩個相近的詞.

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Introduction

A serious threat is posed by viral pathogens, including clinical viruses (HIV, hepatitis viruses, etc.), natural emerging viruses (avian and swine influenza strains, SARS, etc.), and viruses relevant to potential bioterrorism (Ebola, smallpox, etc.). Unfortunately, there are relatively few prophylactics or therapeutics for these viruses, and most which do exist can be divided into three broad categories [1]–[3]:

(1) Specific inhibitors of a virus associated target (e.g., HIV protease inhibitors, RNAi) generally must be developed for each virus or viral strain, are prone to resistance if a virus mutates the drug target, are not immediately available for emerging or engineered viral threats, and can have unforeseen adverse effects.

(2) Vaccines also require a new vaccine to be developed for each virus or viral strain, must be administered before or in some cases soon after exposure to be effective, are not immediately available for emerging or engineered viral threats, can have unforeseen adverse effects, and are difficult to produce for certain pathogens (e.g., HIV).

(3) Interferons and other pro- or anti-inflammatories are less virus specific, but still are only useful against certain viruses, and they can have serious adverse effects through their interactions with the immune and endocrine systems.

To overcome these shortcomings of existing approaches, we have developed and demonstrated a novel antiviral approach that is effective against a very broad spectrum of viruses, nontoxic in vitro and in vivo, and potentially suitable for either prophylactic or therapeutic administration. Our approach, which we call a Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO), is designed to selectively and rapidly kill virus-infected cells while not harming uninfected cells.

Our DRACO approach combines two natural cellular processes. The first process involves dsRNA detection in the interferon pathway. Most viruses have double- or single-stranded RNA (ssRNA) genomes and produce long dsRNA helices during transcription and replication; the remainder of viruses have DNA genomes and typically produce long dsRNA via symmetrical transcription [4]–[5]. In contrast, uninfected mammalian cells generally do not produce long dsRNA (greater than ~21–23 base pairs) [4]–[5]. Natural cellular defenses exploit this difference in order to detect and to attempt to counter viral infections [6]–[7]. For example, protein kinase R (PKR) contains an N-terminal domain with two dsRNA binding motifs (dsRBM 1 and 2) and a C-terminal kinase domain [8]–[9]. Binding of multiple PKR proteins to dsRNA with a length of at least 30–50 base pairs [5] activates the PKRs via trans-autophosphorylation; activated PKR then phosphorylates eIF-2α, thereby inhibiting translation of viral (and cellular) proteins. Other examples of proteins that detect viral dsRNA include 2′,5′-oligoadenylate (2–5A) synthetases [10], RNase L (activated via dimerization by 2–5A produced by 2–5A synthetases in response to dsRNA [11]), TLR 3 [12], interferon-inducible ADAR1 [13], and RIG-I and Mda-5 [6]–[7].

The second natural process used by our approach is one of the last steps in the apoptosis pathway [14], in which complexes containing intracellular apoptosis signaling molecules, such as apoptotic protease activating factor 1 (Apaf-1) [15]–[16] or FLICE-activated death domain (FADD) [17]–[18], simultaneously bind multiple procaspases. The procaspases transactivate via cleavage, activate additional caspases in the cascade, and cleave a variety of cellular proteins [14], thereby killing the cell.

Many viruses attempt to counter these defenses. A wide variety of viruses target dsRNA-induced signaling proteins, including IPS-1, interferon response factors (IRFs), interferons and interferon receptors, JAK/STAT proteins, and eIF-2α [19]–[20]. Some viral products attempt to sequester dsRNA (e.g., poxvirus E3L [21]) or to directly interfere with cellular dsRNA binding domains (e.g., HIV TAR RNA [19]–[20]). Virtually all viruses that inhibit apoptosis do so by targeting early steps in the pathway, for example by inhibiting p53, mimicking anti-apoptotic Bcl-2, or interfering with death receptor signaling [22]–[23]. Among the few viral proteins that directly inhibit one or more caspases are African swine fever virus A224L (which inhibits caspase 3) [24], poxvirus CrmA (which inhibits caspases 1, 8, and 10 but not others) [25], and baculovirus p35 (which inhibits several caspases but is relatively ineffective against caspase 9) [25].

Because PKR activation and caspase activation function in similar ways and involve proteins that have separate domains with well-defined functions, these two processes can be combined to circumvent most viral blockades [26]–[27]. In its simplest form, a DRACO is a chimeric protein with one domain that binds to viral dsRNA and a second domain (e.g., a procaspase-binding domain or a procaspase) that induces apoptosis when two or more DRACOs crosslink on the same dsRNA. If viral dsRNA is present inside a cell, DRACOs will bind to the dsRNA and induce apoptosis of that cell. If viral dsRNA is not present inside the cell, DRACOs will not crosslink and apoptosis will not occur.

For delivery into cells in vitro or in vivo, DRACOs can be fused with proven protein transduction tags, including a sequence from the HIV TAT protein [28], the related protein transduction domain 4 (PTD) [29], and polyarginine (ARG) [30]. These tags have been shown to carry large cargo molecules into both the cytoplasm and the nucleus of all cell types in vitro and in vivo, even across the blood-brain barrier.




Results and Discussion

We produced DRACOs with different dsRNA detection domains, apoptosis induction domains, and transduction tags (Figure 1). The dsRNA detection domains included PKR1–181, PKR1–181 with dsRBM 1 (NTE3L), dsRBM 2 (CTE3L), or dsRBM 1 and 2 (2×E3L) replaced by the dsRNA binding motif from poxvirus E3L, and RNaseL1–335 (which binds to 2–5A produced by endogenous cellular 2–5A synthetases in response to viral dsRNA). The apoptosis induction domains included FADD1–90 Death Effector Domain (DED, which binds to procaspase 8), Apaf-11–97 caspase recruitment domain (CARD, which binds to procaspase 9), and murine Apaf-11–97 (mApaf) CARD. Except for mApaf, all domains refer to the human sequence. Isolated dsRNA detection domains and apoptosis induction domains were produced as negative controls. Mutant DRACOs with deleterious K64E [9] and homologous K154E mutations in the PKR domain were also produced as negative controls. Proteins were produced with TAT, PTD, or ARG tags on the N terminus, C terminus, or both termini. Proteins were expressed in BL21(DE3)pLysS Rosetta E. coli. An empty expression vector was transformed into the E. coli and the same purification protocol was followed, resulting in control extract without DRACOs.  

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Figure 2.DRACOs penetrated cells and persisted for days.

(A) DRACOs with PTD or TAT tags entered H1-HeLa cells more readily than DRACO without a transduction tag. 400 nM PKR-Apaf
DRACO was added to medium for 1 hour, and then cells were trypsinized and washed to remove any DRACO on the cell surface.
Cells were lysed and analyzed for DRACO by westerns using anti-His6 antibodies. Lysate from approximately 105 cells was loaded
in each lane. A known amount of purified PKR-Apaf DRACO was used as a standard as indicated. (B) DRACOs entered HeLa cells
within 10 minutes and reached a maximum after 1.5 hours. 400 nM TAT-PKR-Apaf DRACO was added to medium for the specified
time, and then cells were analyzed as in (A). (C) DRACOs persisted within HeLa cells for at least 8 days. 500 nM PTD-PKR-Apaf
DRACO was added to cell medium for 1 hour, and then cells were put into DRACO-free medium. After the specified number of days,
cells were analyzed as in (A).

doi:10.1371/journal.pone.0022572.g002



Figure 3.DRACOs mediated apoptosis in cells containing dsRNA.

L929 cells transfected with both DRACO and poly(I):poly(C) dsRNA exhibited apoptosis within 24 hours, whereas cells that received
only DRACO did not. Caspase inhibitors eliminated DRACO-mediated apoptosis in the presence of dsRNA.

doi:10.1371/journal.pone.0022572.g003

We measured the viability of normal human lung fibroblast (NHLF) cells that had been treated with PKR-Apaf DRACOs or negative

controls and then challenged with 130 plaque forming units (pfu) per well rhinovirus 1B (Figures 4, S2, S3).
Untreated cell populations succumbed to virus within days, indicating that any innate cellular responses were ineffective against the
virus or blocked by the virus. DRACOs with PTD, TAT, and ARG tags prevented significant cytopathic effects (CPE) in virus-challenged
cell populations by rapidly killing any initially infected cells, thereby terminating the infection in its earliest stages. DRACOs had no
apparent toxicity in unchallenged cells. Isolated PKR1–181 and Apaf-11–97 domains were nontoxic but not antiviral, even when added
simultaneously (but not covalently linked). DRACO with deleterious amino acid changes also had little efficacy. Likewise, an amount
of purified bacterial extract (without DRACOs) approximately 10-fold greater than the average volume of DRACOs added to cells was
nontoxic and not efficacious, demonstrating that any remaining　bacterial contaminants such as lipopolysaccharide did not affect
the cells or produce antiviral activity. Thus the antiviral efficacy
appears to require intact functional DRACOs. Tests using DRACOs with protein transduction tags on the N terminus, C terminus,
or both termini indicated that N-terminal tags generally worked the best (data not shown). DRACOs with transduction tags penetrated cells and were antiviral when
administered by themselves (Figures 2, S2A), but efficacy was enhanced by co-administration with Roche FuGene 6 to maximize uptake (Figure S2B), so FuGene was used in experiments unless otherwise noted. Cell viability measured 7 days post infection (dpi) showed little difference if DRACO-containing medium was removed 3 dpi after untreated cells had widespread CPE; there was no relapse of viral CPE in treated cells
after DRACOs were withdrawn (Figure 4B).




Figure 4.DRACOs were effective against rhinovirus 1B in NHLF cells.

(A) 100 nM DRACO was effective against 130 pfu/well rhinovirus, whereas 100 nM negative controls were not (12 dpi). (B) Cell
viability measured 7 dpi showed little difference if 100 nM DRACO-containing medium was removed 3 dpi when untreated cells
had widespread CPE from 130 pfu/well rhinovirus 1B; there was no relapse of viral CPE in treated cells after DRACOs were
withdrawn. (C) 1 dose of 25 nM PTD-PKR-Apaf DRACO was effective against rhinovirus 1B in NHLF cells when it was added from
6 days before infection to 3 days after infection. (Complete viral CPE in untreated cell populations required 3–4 days in our
experiments, and for these experiments a significant fraction of cells were still uninfected 3 dpi.) Cell viability was measured 14 dpi.

doi:10.1371/journal.pone.0022572.g004

DRACOs were added approximately 24 hours before virus unless otherwise noted, but other dosing times were tested (Figure 4C).
One dose of PTD-PKR-Apaf DRACO was efficacious against rhinovirus 1B in NHLF cells when added up to 6 days before infection,
supporting the western data (Figure 2C) that DRACO persisted inside cells for at least 8 days. Up to 3 days after infection, one
DRACO dose could still rescue a significant percentage of the cell population. After 3 days, virtually all of the cells had already been
killed or at least infected by the virus.

Additional DRACO designs exhibited efficacy against rhinovirus (Figure 5A). Other effective dsRNA detection domains included
NTE3L, CTE3L, 2×E3L, and RNaseL1–335. Other effective apoptotic domains included FADD1–90, mApaf11–97, and procaspases
[26]–[27]. Although the initial performance of these alternate DRACOs was generally inferior to that of PKR-Apaf human DRACO
in these experiments, better performance might be achieved with further optimization. These results demonstrate that the alternate
DRACO designs are nontoxic and efficacious against virus, and they support the DRACO mechanism of action.

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Figure 5.DRACOs were effective against rhinovirus 1B and other viruses.

(A) Multiple 100 nM DRACOs were effective against 130 pfu/well rhinovirus (4 dpi). Even better performance of these alternate DRACOs might be achieved with further optimization. (B) PKR-Apaf DRACOs reduced the viral titer in supernatant from NHLF cells challenged with 300 pfu/well rhinovirus 1B to undetectable levels. PKR and Apaf-1 domains not covalently linked increased viral titers somewhat, possibly by interfering with the antiviral activity of endogenous wild-type PKR and Apaf-1. Cells were treated with 100 nM DRACO or controls. Supernatants were collected 4 dpi and their viral titers determined by serial dilution onto fresh 96-well NHLF plates. (C) The EC50 for PTD-PKR-Apaf DRACO was 2–3 nM against 130 pfu/well rhinovirus 1B in NHLF cells (measured 3 dpi), and 50 pfu/well murine encephalomyelitis (3 dpi) and 50 pfu/well murine adenovirus (11 dpi) in L929 cells.

doi:10.1371/journal.pone.0022572.g005

In addition to improving survival of the cell population, DRACOs reduced viral titers from virus-challenged cells (Figures 5B, S4). One dose of PKR-Apaf DRACO administered to NHLF cells 24 hours before 300 pfu/well rhinovirus 1B eliminated any measurable viral titer in cell supernatant samples collected 4 dpi.

The median effective concentration for DRACOs with PTD, TAT, and ARG tags against a variety of viruses was 2–3 nM, as illustrated for PTD-PKR-Apaf DRACO against rhinovirus 1B, murine encephalomyelitis, and murine adenovirus (Figures 5C).

DRACOs were effective against a broad spectrum of other viruses in a variety of cell types (Tables 1–2). DRACOs were effective against rhinoviruses 2 and 30 in NHLF cells (data not shown) and rhinovirus 14 in HeLa cells (Figure S4). DRACOs were effective against murine adenovirus in L929 cells if added before or up to at least 72 hours after virus (Figures 6, S5), demonstrating efficacy against a DNA virus (Figures 6A, S5), in murine cells (using human apoptotic DRACO domains to recruit endogenous murine procaspases), when treatment is delayed until significantly after infection (Figure 6B), and with a variety of DRACO designs (Figure 6C). DRACOs were effective against murine encephalomyelitis in L929 cells regardless of whether the DRACO-containing medium was removed 3 dpi (Figure 7A), whether DRACOs were added before or after infection (Figure 7B), and which DRACOs were used (Figures 7C, S6). DRACOs were effective in Vero E6 cells against Amapari and Tacaribe, arenaviruses that are closely related to lymphocytic choriomeningitis virus (LCMV), Lassa, and Junin viruses (Figures 8A, S7, S8). Likewise, DRACOs were effective against Guama strain Be An 277 (Figures 8B, S9); comparable results were obtained for Guama strain Be Ar 12590 (data not shown). Guama virus is a significant human pathogen and is closely related to other bunyaviruses such as Rift Valley fever, hantavirus, and Crimean-Congo virus. DRACOs were similarly effective against dengue type 2 (New Guinea C) hemorrhagic fever virus, a major human pathogen that is very closely related to other flaviviruses such as West Nile virus, Yellow fever virus, and Omsk virus (Figures 8C, S10, S11). DRACOs were also effective against H1N1 influenza A/PR/8/34 in normal human hepatocytes (Figure S12 left), reovirus 3 in BALB/3T3 murine cells (Figure S12 center), and adenovirus 5 in AD293 cells (Figure S12 right).


Figure 6.DRACOs were effective against murine adenovirus in L929 cells.

(A) 100 nM DRACOs were effective against 50 pfu/well murine adenovirus, whereas all negative controls were not (16 dpi). (B) 100 nM PTD-PKR-Apaf DRACO was effective if added before or up to at least 72 hours after adenovirus (16 dpi). (C) Multiple 100 nM DRACOs were effective against 50 pfu/well murine adenovirus (11 dpi). Even better performance of these alternate DRACOs might be achieved with further optimization.

doi:10.1371/journal.pone.0022572.g006



Figure 7.DRACOs were effective against murine encephalomyelitis in L929 cells.

(A) 100 nM DRACOs were effective against 50 pfu/well encephalomyelitis. Cell viability measured 6 dpi showed little difference if DRACO-containing medium was removed 3 dpi when untreated cells had widespread CPE; there was no relapse of viral CPE in treated cells after DRACOs were withdrawn. (B) 100 nM PTD-PKR-Apaf DRACO was effective if added before, simultaneously with, or up to at least 6 hours after encephalomyelitis. (C) Multiple 100 nM DRACOs were effective against 50 pfu/well murine encephalomyelitis (4 dpi). Even better performance of these alternate DRACOs might be achieved with further optimization.

doi:10.1371/journal.pone.0022572.g007


Figure 9.DRACOs appeared promising when administered via intraperitoneal (i.p.) injection in proof-of-concept trials with adult BALB/c mice.

(A) 2.5 mg PTD-PKR-Apaf DRACO administered i.p. penetrated the liver, kidney, and lungs and persisted for at least 48 hours. Averages of 3 mice per data point are plotted, and error bars show s.e.m. (B) PTD-PKR-Apaf and TAT-PKR-Apaf DRACOs administered i.p. from day -1 through day 3 greatly reduced the morbidity and day-2 lung viral titers in mice challenged intranasally (i.n.) with 1.3 LD50 influenza H1N1 A/PR/8/34. (C) PTD-RNaseL-Apaf, TAT-RNaseL-Apaf, and ARG-RNaseL-Apaf DRACOs administered i.p. from day -1 through day 3 greatly reduced the morbidity and day-2 lung viral titers in mice challenged i.n. with 0.3 LD50 influenza H1N1 A/PR/8/34.

doi:10.1371/journal.pone.0022572.g009


Figure 10.DRACOs appeared promising when administered via intranasal (i.n.) injection in proof-of-concept trials with adult BALB/c mice.

(A) 0.5 mg PKR-Apaf DRACO administered i.n. to adult BALB/c mice penetrated the lungs and persisted over 24 hours. Averages of 3 mice per data point are plotted, and error bars show s.e.m. (B) PTD-PKR-Apaf, TAT-PKR-Apaf, and ARG-PKR-Apaf DRACOs administered i.n. on day 0 reduced the morbidity in mice challenged i.n. with 1 LD50 influenza H1N1 A/PR/8/34.

doi:10.1371/journal.pone.0022572.g010

Based on these encouraging initial animal trials, future work should be done to test and optimize antiviral efficacy, pharmacokinetics, and absence of toxicity in vitro and in vivo. Future experiments can further characterize and optimize dsRNA binding, apoptosis induction, cellular transduction, and other DRACO properties. More extensive trials are also needed to determine how long after infection DRACOs can be used successfully, or if DRACOs are useful against chronic viral infections without producing unacceptable levels of cell death in vivo.

DRACOs should be effective against numerous clinical and NIAID priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered. We have demonstrated that DRACOs are effective against viruses with DNA, dsRNA, positive-sense ssRNA, and negative-sense ssRNA genomes; enveloped and non-enveloped viruses; viruses that replicate in the cytoplasm and viruses that replicate in the nucleus; human, bat, and rodent viruses; and viruses that use a variety of cellular receptors (Table 1).

Materials and Methods

Ethics statement for mouse trials

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on Animal Care of MIT (Assurance Number: A-3125-01). Guidelines to minimize suffering were followed, and avertin anesthetic was used for intranasal procedures.

Cloning

E.F. Meurs provided PKR cDNA and Y. Shi donated human Apaf-1 cDNA. RNaseL1–335 sequence was cloned from HeLa cells. PKR-E3L, FADD1–90 (with L75A and L76A to prevent spontaneous self-association [18]) and murine Apaf-11–97 sequences came from BioBasic. Genes for DRACOs and controls were constructed using PCR and restriction cloning. TAT (YGRKKRRQRRR), PTD-4 (YARAAARQARA), and ARG (R9) tags were incorporated at N- and/or C-termini. Genes were inserted into pET100/D-TOPO (Invitrogen).

Protein production

Each vector was transformed into Rosetta BL21(DE3)pLysS E. coli (EMD Biosciences), bacteria were plated on Luria broth (LB) agar with 100 ?g ml?1 ampicillin and 34 ?g ml?1 chloramphenicol, and plates were incubated overnight at 37°C. One colony was inoculated into ampicillin-chloramphenicol LB and grown overnight (37°C, 225 r.p.m.), then diluted 1:30 into ampicillin-chloramphenicol LB and incubated (30°C, 225 r.p.m.) until OD600 reached 1.0. 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added and flasks were incubated overnight. E. coli were recovered by centrifugation (5,000 r.p.m., 30 min., 4°C) and lysed by sonication, and His6-tagged proteins were purified using Ni-NTA agarose (Invitrogen) following the manufacturer's protocols. Proteins were eluted into 1.5× PBS with 300 mM imidazole and 10% (vol/vol) glycerol, concentrated with Amicon-15 (10 kDa cutoff, 3,000 g) to >5 mg/ml, and filter-sterilized. Protein concentrations were measured relative to BSA standards by Bradford assay (BioRad) and Gel Doc densitometry.

Cells

L929 (CCL-1), NIH/3T3 (CRL-1658), BALB/3T3 (CCL-163), H1-HeLa (CRL-1958), MDCK (CCL-34), and Vero E6 (CRL-1586) (ATCC) and AD293 (Stratagene) were cultured in complete DMEM (Gibco). Normal human lung fibroblasts, small airway epithelial cells, osteoblasts, hepatocytes, and aortic smooth muscle cells (Lonza) were cultured in cell-specific media (Lonza).

Viruses

Dengue type 2 (New Guinea C, VR-1584), Amapari (VR-477), Tacaribe (VR-1272), Guama (Be An 277, VR-407; Be Ar 12590, VR-420), murine adenovirus (VR-550), Theiler's murine encephalomyelitis (VR-57), reovirus 3 (VR-824), influenza H1N1 A/PR/8/34 (ATCC VR-1469), influenza H1N1 A/WS/33 (ATCC VR-1520), rhinovirus 1B (VR-481), rhinovirus 2 (VR-482), rhinovirus 14 (VR-284), and rhinovirus 30 (VR-505) were obtained from ATCC. Adenovirus 5 was obtained from Stratagene. Influenza A/PR/8/34 for animal trials was donated by P. Palese.

Cell assays

Contact-inhibited cells were grown to 50–80% confluence and non-contact inhibited cells to 20–50% confluence in 96-well plates with 100 ?l/well medium. DRACOs or controls were added to columns of wells, 8 wells/column. Except in Figs. 2 and S2, 0.4–1% (vol/vol) Roche FuGene 6 was co-administered with DRACOs and controls to optimize cellular uptake. Wells received virus approximately 24 hours after DRACO unless otherwise noted. On selected days, cell viability in each plate was measured using CellTiter 96 (Promega). Assay schedules, viral doses, and other parameters were optimized for different cell/virus systems. Micrographs were taken in 24-well plates under similar conditions.

DRACO cell penetration assays

Cells in 24-well plates were incubated with DRACOs for varying lengths of time, then trypsinized, washed thoroughly in PBS, and lysed. Lysate from approximately 105 cells was loaded in each lane. DRACOs were detected via westerns using mouse anti-His6 (Invitrogen) and goat anti-mouse IgG HRP (Jackson).

Apoptosis assays

70% confluent 96-well L929 plates were treated with 10 ?M Z-VAD-FMK pan-caspase inhibitor or 20 ?M Z-LEHD-FMK caspase-9 inhibitor (R&D Systems), then 75 ?M camptothecin (Calbiochem) or 100 nM DRACOs with or without 25 ng/well poly(I):poly(C) dsRNA (Sigma) transfected using FuGene (Roche) following manufacturers' protocols. After 24 hours, apoptosis was determined using Caspase-Glo 3/7 (Promega).

Viral titers

Titers were determined by serial dilutions onto 96-well NHLF (for rhinovirus 1B) or H1-HeLa (for rhinovirus 14) plates, with 8 wells per 10-fold dilution and with the number of wells exhibiting CPE measured 5 dpi. Reed-Muench titers were calculated from the results (1 TCID50≈0.7 pfu). Error bars indicate s.e.m. from 3 trials.

Statistical analysis

CellTiter 96 cell viabilities were normalized to 100% for untreated uninfected and 0% for untreated virus-killed cells. Graphs indicate averages (n = 8) with s.e.m. Experiments were repeated at least 3 times with similar results.

Mouse trials

7-week-old female BALB/c mice (Charles River) received DRACO i.n. (~0.5 mg in 50 ?l) or i.p. (0.8–2.5 mg in 200 ?l). Mice were challenged i.n. with 0.3–1.3 LD50 influenza H1N1 A/PR/8/34. Mice received DRACO i.p. once daily on days -1 and 1–3 and twice on day 0, or just one i.n. DRACO dose simultaneously with virus. Lungs were harvested on day 2 and viral titers determined by serial dilutions onto 96-well MDCK plates. For pharmacokinetics, organs were harvested at designated times, then sonicated into 1 ml PBS with 1% Triton X-100. 1 mg organ solution was mixed with 2× Laemmeli buffer, boiled 5 min., and run on a 10–20% SDS PAGE gel with a standard curve of purified DRACO, followed by western blots with anti-Apaf (Millipore) and HEP-labeled anti-rabbit IgG (Jackson Immunoresearch). Blots were developed with Pierce luminescent reagent and exposed to film. DRACO bands were quantitated by Gel Doc densitometry vs. the standards.


Supporting Information

Figure S1.

DRACOs entered normal human lung fibroblasts. NHLF cells were incubated overnight with 500 nM PTD-PKR-Apaf DRACO labeled with Lumio (Invitrogen), washed with Hank's balanced salt solution, and photographed with a fluorescent microscope to compare (A) untreated and (B) DRACO-treated cells. DRACOs appeared to be distributed throughout each cell in both the cytoplasm and the nucleus.

(TIF)

Figure S2.

FuGene co-administration with DRACOs improved cellular uptake and antiviral efficacy. (A) 100 nM DRACOs with PTD, TAT, and ARG protein transduction tags were effective against rhinovirus 1B in NHLF cells without FuGene co-administration. Cell viability was measured 3 days after infection with 130 pfu/well. (B) Co-administration of FuGene with DRACOs lowered the EC50 of DRACOs, as shown here for PTD-PKR Apaf DRACO against 130 pfu/well rhinovirus 1B in NHLFs.

(TIF)

Figure S3.

200 nM PTD-PKR-Apaf DRACO was effective against rhinovirus 1B in NHLF cells. Representative photographs were taken 20 days after challenge with 300 pfu/well. Scale bar = 50 ?m.

(TIF)

Figure S4.

DRACOs decreased the viral titer of rhinovirus 14 in H1-HeLa cells. One 120 nM dose of PTD-PKR-Apaf DRACO administered to cells 24 hours before or simultaneously with 10 pfu/well rhinovirus 14 eliminated any measurable titer 3 dpi. One DRACO dose administered 24 or 30 hours after infection halved the 3-dpi viral titer.

(TIF)

Figure S5.

200 nM PTD-PKR-Apaf DRACO was effective against murine adenovirus in L929 cells. Representative photographs were taken 15 days after challenge with 30 pfu/well. Scale bar = 25 ?m.

(TIF)

Figure S6.

200 nM PTD-PKR-Apaf DRACO was effective against murine encephalomyelitis in L929 cells. Representative photographs were taken 21 days after challenge with 50 pfu/well. Scale bar = 25 ?m.

(TIF)

Figure S7.

100 nM PTD-PKR-Apaf DRACO was effective against Amapari arenavirus in Vero E6 cells. Representative photographs were taken 11 days after challenge with 300 pfu/well. Scale bar = 100 ?m.

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Figure S8.

100 nM PTD-PKR-Apaf DRACO was effective against Tacaribe arenavirus in Vero E6 cells. Photographs were taken 8 days after challenge with 140 pfu/well. Scale bar = 100 ?m.

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Figure S9.

200 nM PTD-PKR-Apaf DRACO was effective against Guama Be An 277 bunyavirus in Vero E6 cells. Photographs were taken 4 days after challenge with 30 pfu/well. Scale bar = 100 ?m.

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Figure S10.

200 nM PKR-Apaf DRACO was effective against dengue flavivirus in Vero E6 cells. Cell viability was measured 18 days after challenge with 16 pfu/well.

(TIF)

Figure S11.

100 nM PTD-PKR-Apaf DRACO was effective against dengue flavivirus in Vero E6 cells. Photographs were taken 7 days after challenge with 160 pfu/well. Scale bar = 100 ?m.

(TIF)

Figure S12.

DRACOs were effective against a broad spectrum of other viruses in a variety of cell types. Left four photos: 100 nM PKR-Apaf DRACO was effective against H1N1 influenza A/PR/8/34 in normal human hepatocytes. Untreated cells challenged with 105 pfu/well died within 3 days, whereas treated challenged cells were cultured for 72 days with no sign of viral CPE. Center four photos: 100 nM PTD-PKR-Apaf DRACO was effective against reovirus 3 in BALB/3T3 murine cells. Photographs were taken 11 days after challenge with 30 pfu/well reovirus 3. Right four photos: 200 nM PTD-2×E3L-Apaf DRACO was effective against adenovirus 5 in human embryonic kidney AD293 cells. Fluorescent microscope photographs were taken 4 days after challenge with 25 pfu/well adenovirus 5 expressing enhanced green fluorescent protein (EGFP). Scale bars = 50 ?m.

(TIF)

Acknowledgments

We thank

E.F. Meurs (Institut Pasteur) for PKR cDNA,
Y. Shi (Princeton University) for human Apaf-1 cDNA,
P. Palese (Mount Sinai School of Medicine) for A/PR/8/34 influenza,
and MIT Division of Comparative Medicine for mouse facilities and advice.

We are grateful to

G. Johnson for assistance in the lab,
B. Lemus for producing the EGFP adenovirus,
and E. Schwoebel, C. Cabrera, G. Beltz, and S. Chiang for helpful discussions.

Author Contributions

Conceived and designed the experiments:
THR. Performed the experiments: THR. Wrote the paper: THR. Designed DRACOs: THR. Conducted cell assays: CZ TB. Lead for animal trials: TB. Lead for protein production and purification: SW. Assisted with experiments: JP BZ.
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References

•Kr?usslich HG, Bartenschlager R, editors. (2009) Handbook of Experimental Pharmacology 189: Antiviral Strategies. Berlin: Springer.
•Demberg T, Robert-Guroff M (2009) Mucosal immunity and protection against HIV/SIV infection: strategies and challenges for vaccine design. Int Rev Immunol 28: 20–48. Find this article online
•Boomker JM, de Leij LF, The TH, Harmsen MC (2005) Viral chemokine-modulatory proteins: tools and targets. Cytokine Growth Factor Rev 16: 91–103. Find this article online
•Knipe DM, Howley PM, editors. (2006) Fields Virology, 5th ed. Philadelphia: Lippincott Williams & Wilkins.
•Samuel CE (2004) Knockdown by RNAi—proceed with caution. Nat Biotechnol 22: 280–282. Find this article online
•Sadler AJ, Williams BRG (2008) Interferon-inducible antiviral effectors. Nat Rev Immunol 8: 559–568. Find this article online
•Yoneyama M, Fujita T (2010) Recognition of viral nucleic acids in innate immunity. Rev Med Virol 20: 4–22. Find this article online
•Sadler AJ, Williams BR (2007) Structure and function of the protein kinase R. Curr Top Microbiol Immunol 316: 253–292. Find this article online
•Wu S, Kaufman RJ (1996) Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR). J Biol Chem 271: 1756–1763. Find this article online
•Hovanessian AG, Justesen J (2007) The human 2′-5′oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2′-5′ instead of 3′-5′ phosphodiester bond formation. Biochimie 89: 779–788. Find this article online
•Bisbal C, Silverman RH (2007) Diverse functions of RNase L and implications in pathology. Biochimie 89: 789–798. Find this article online
•Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11: 373–84. Find this article online
•Placido D, Brown BA, Lowenhaupt K, Rich A, Athanasiadis A (2007) A left-handed RNA double helix bound by the Zα domain of the RNA-editing enzyme ADAR1. Structure 15: 395–404. Find this article online
•Pop C, Salvesen GS (2009) Human caspases: activation, specificity, and regulation. J Biol Chem 284: 21777–21781. Find this article online
•Qin H, Srinivasula SM, Wu G, Fernandes-Alnemri T, Alnemri ES, et al. (1999) Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature 399: 549–557. Find this article online
•Bao Q, Shi Y (2007) Apoptosome: a platform for the activation of initiator caspases. Cell Death Differ 14: 56–65. Find this article online
•Eberstadt M, Huang B, Chen Z, Meadows RP, Ng SC, et al. (1998) NMR structure and mutagenesis of the FADD (Mort1) death-effector domain. Nature 392: 941–945. Find this article online
•Muppidi JR, Lobito AA, Ramaswamy M, Yang JK, Wang L, et al. (2006) Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis. Cell Death Differ 13: 1641–50. Find this article online
•Randall RE, Goodbourn S (2008) Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 89: 1–47. Find this article online
•Weber F, Haller O (2007) Viral suppression of the interferon system. Biochimie 89: 836–842. Find this article online
•Langland JO, Kash JC, Carter V, Thomas MJ, Katze MG, et al. (2006) Suppression of proinflammatory signal transduction and gene expression by the dual nucleic acid binding domains of the vaccinia virus E3L proteins. J Virol 80: 10083–10095. Find this article online
•Galluzzi L, Brenner C, Morselli E, Touat Z, Kroemer G (2008) Viral control of mitochondrial apoptosis. PLoS Pathogens 4: e1000018. Find this article online
•Postigo A, Ferrer PE (2009) Viral inhibitors reveal overlapping themes in regulation of cell death and innate immunity. Microbes Infect 11: 1071–1078. Find this article online
•Nogal ML, González de Buitrago G, Rodríguez C, Cubelos B, Carrascosa AL, et al. (2001) African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells. J Virol 75: 2535–43. Find this article online
•Callus BA, Vaux DL (2007) Caspase inhibitors: viral, cellular and chemical. Cell Death Differ 14: 73–78. Find this article online
•Rider TH (2006) Anti-pathogen treatments. U.S. Patent 7,125,839.
•Rider TH (2009) Anti-pathogen treatments. U.S. Patent 7,566,694.
•Gump JM, Dowdy SF (2007) TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med 13: 443–448. Find this article online
•Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF (2001) Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 61: 474–477. Find this article online
•Goun EA, Pillow TH, Jones LR, Rothbard JB, Wender PA (2006) Molecular transporters: synthesis of oligoguanidinium transporters and their application to drug delivery and real-time imaging. ChemBioChem 7: 1497–1515. Find this article online

Jerry E. Chipuk, Gavin P. McStay, Archana Bharti,ET AL:Sphingolipid Metabolism Cooperates with BAK and BAX to Promote the Mitochondrial Pathway of Apoptosis,Cell, Volume 148, Issue 5, 988-1000, 2 March 2012

•Highlights
•Mitochondria devoid of heterotypic membranes are resistant to cytochrome c release
•Mitochondrial sphingolipids promote BAK/BAX activation and cytochrome c release
•Sphingosine-1-PO4 and hexadecenal coordinate BAK and BAX activation, respectively
•Inhibition of sphingolipid metabolism blocks cytochrome c release and apoptosis

Summary

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO4 and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.

曲度: 哈默羅夫的調諧客觀還原理論(Orch-OR)--靈魂出竅理論

1.靈魂之物質基礎:腦細胞內微管的量子態.
2.意識之本質是大腦內一台量子計算機的程序.意識是宇宙的一個組成部分.
3.人類靈魂之本質:是大腦內神經元細胞之間的交互作用.
4.Orch-OR理論概要:
在瀕死經歷中，微管失去了它們的量子態，但裡面的信息並沒有遭到破壞。也就是說，靈魂離開肉體，重回宇宙。哈默羅夫在紀錄片《科學頻道 -穿越蟲洞》中表示：「心臟停止跳動，血液停止流動，微管失去了它們的量子態，但微管內的量子信息並沒有遭到破壞，也無法被破壞，離開肉體后重新回到宇宙。如果患者蘇醒過來，這種量子信息又會重新回到微管，患者會說『我體驗了一次瀕死經歷』。如果沒有蘇醒過來，患者便會死亡，這種量子信息將存在於肉體外，以靈魂的形式。」

D.QU

鏈接:
http://news.dxy.cn/bbs/topic/18124558?ppg=4

跋:
細胞像一個小宇宙,裡面有很多種細胞器與外界有管道相通,結構異常複雜.它們的功能非常複雜且相互關聯,
細胞死亡通道(機制),人類研究了很多年,目前認識仍不很深入(包括當代研究者在內).
細胞死亡通道悖論,是我們提出的一個新概念.
本貼彙編了該領域研究大致進展情況(截止2010年底),對欲了解它來龍去脈的初學者或許有所幫助.是為跋.
D.QU
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