関: DEPC、diethylpyrocarbonate

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/03/22 08:55:31」(JST)

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Diethylpyrocarbonate (DEPC), also called diethyl dicarbonate (IUPAC name), is used in the laboratory to inactivate RNase enzymes in water and on laboratory utensils. It does so by the covalent modification of histidine (most strongly), lysine, cysteine, and tyrosine residues.^[1]^[2]^[3]

DEPC-treated (and therefore RNase-free) water is used in handling of RNA in the laboratory to reduce the risk of RNA being degraded by RNases.

Water is usually treated with 0.1% v/v DEPC for at least 2 hours at 37 °C and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC in this manner yields CO₂ and ethanol. Higher concentrations of DEPC are capable of deactivating larger amounts of RNase, but remaining traces or byproducts may inhibit further biochemical reactions such as in vitro translation. Furthermore, chemical modification of RNA such as carboxymethylation is possible when traces of DEPC or its byproducts are present, resulting in impaired recovery of intact RNA even after buffer exchange (after precipitation).

DEPC treated water for use in a laboratory

DEPC is unstable in water and susceptible to hydrolysis to carbon dioxide and ethanol, especially in the presence of a nucleophile. For this reason, DEPC cannot be used with Tris or HEPES buffers. In contrast, it can be used with phosphate-buffered saline or MOPS.^[4] A handy rule is that enzymes or chemicals which have active -O:, -N: or -S: cannot be treated with DEPC to become RNase-free, as DEPC reacts with these species. Furthermore, DEPC degradation products can inhibit in vitro transcription.

DEPC derivatization of histidines is also used to study the importance of histidyl residues in enzymes. Modification of histidine by DEPC results in carbethoxylated derivates at the N-omega-2 nitrogen of the imidazole ring. DEPC modification of histidines can be reversed by treatment with 0.5 M hydroxylamine at neutral pH.

DEPC can also be used for probing the structure of double-stranded DNA.^[4]

References

^ Narumi et al. (1987). Neurochem Res. 12 (4). Missing or empty |title= (help)
^ Chirgwin, John M et al. (1979). "Isolation of biologically active ribonucleic acid from sourcesenriched in ribonuclease". Biochemistry 18 (24): 5294–5299. doi:10.1021/bi00591a005. PMID 518835.
^ Wolf, Barry; Lesnaw, Judith A.; Reichmann, Manfred E. (1970). "A Mechanism of the Irreversible Inactivation of Bovine Pancreatic Ribonuclease by Diethylpyrocarbonate. A General Reaction of Diethylpyrocarbonate with Proteins". European Journal of Biochemistry 13 (3): 519–25. doi:10.1111/j.1432-1033.1970.tb00955.x. PMID 5444158.
^ ^a ^b "FAQ about DEPC". Sigma-Aldrich. Retrieved 12 August 2012.

External links

DEPC at OpenWetWare life scientist wiki
RNase and DEPC Treatment

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English Journal

The second type of transglutaminase regulates immune and stress responses in white shrimp, Litopenaeus vannamei.

Chen YN1, Chen WC1, Cheng W2.Author information 1Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC.2Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC. Electronic address: winton@mail.npust.edu.tw.AbstractThe total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when white shrimp, Litopenaeus vannamei, (7.5 ± 0.5 g) were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or different dsRNA at 3 days of injection. In addition, haemolymph glucose and lactate, and haemocytes crustacean hyperglycemic hormone (CHH), transglutaminase I (TGI), transglutaminase II (TGII) and clottable protein (CP) mRNA expression were determined for the shrimp that received DEPC-H2O and different dsRNA after 3 days, and then transferred to 22 and 28 °C from 28 °C. Results showed that respiratory burst, phagocytic activity and clearance efficiency significantly decreased, but hyaline cells significantly increased in the shrimp received LvTGII dsRNA after 3 days. In hypothermal stress studies, LvTGI and CHH were significantly up-regulated in LvTGII-depleted shrimp following exposure to 28 and 22 °C, and haemolymph glucose and lactate were significantly enhanced in LvTGII-depleted shrimp. The injection of LvTGII dsRNA also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. These results suggest that LvTGII is an important component on the immune resistance of shrimp, and is involved in the regulation of some immune parameters and carbohydrate metabolites, as well as has a complementary effect with LvTGI in immunological and physiological response of shrimp.
Fish & shellfish immunology.Fish Shellfish Immunol.2014 Mar;37(1):30-7. doi: 10.1016/j.fsi.2014.01.010. Epub 2014 Jan 22.
The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when white shrimp, Litopen
PMID 24462912

Efficient siRNA delivery and tumor accumulation mediated by ionically cross-linked folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate nanoparticles: for the potential targeted ovarian cancer gene therapy.

Li TS1, Yawata T2, Honke K3.Author information 1Department of Biochemistry, Kochi Medical School, Kochi University, Oko-Cho, Nankoku, Kochi 783-8505, Japan.2Department of Neurosurgery, Kochi Medical School, Kochi University, Oko-Cho, Nankoku, Kochi 783-8505, Japan; Center for Innovative and Translational Medicine, Kochi Medical School, Kochi University, Oko-Cho, Nankoku, Kochi 783-8505, Japan.3Department of Biochemistry, Kochi Medical School, Kochi University, Oko-Cho, Nankoku, Kochi 783-8505, Japan; Center for Innovative and Translational Medicine, Kochi Medical School, Kochi University, Oko-Cho, Nankoku, Kochi 783-8505, Japan. Electronic address: khonke@kochi-u.ac.jp.AbstractFor effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA-PEG-COL conjugate was characterized by MALDI-TOF-MS, FT-IR and (1)H NMR. The average size of siRNA/FA-PEG-COL nanoparticles was approximately 200 nm, and the surface charge was +8.4 mV compared to +30.5 mV with siRNA/COL nanoparticles. FA-PEG-COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76-62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA-PEG-COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA-PEG-COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA-PEG-COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy.
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.Eur J Pharm Sci.2014 Feb 14;52:48-61. doi: 10.1016/j.ejps.2013.10.011. Epub 2013 Oct 29.
For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective F
PMID 24178005

Antioxidative capacity in the fat body of Bombyx mori is increased following oral administration of 4-methylumbelliferone.

Fang Y1, Wang H, Zhu W, Wang L, Liu H, He Y, Xu X, Yin W, Sima Y, Xu S.Author information 1Department of Applied Biology, School of Biology and Basic Medical Sciences, Medical College, Soochow University, Suzhou 215123, China; National Engineering Laboratory for Modern Silk, Institute of Agricultural Biotechnology & Ecology, Soochow University, Suzhou 215123, China.AbstractPlant sources of umbelliferones have tumor-inhibitory effects at the cellular level. However, their physiological functions in animals are largely unresolved. In this study, we provide evidence to show that 4-methylumbelliferone (4-MU) participates in the regulation of antioxidative capacity in the fat body of Bombyx mori, a tissue similar to mammalian liver in this model invertebrate. Larvae (3rd day of the 5th instar) were orally exposed to 4 mM 4-MU, an umbelliferone, which swiftly induced the generation of a large number of ROS (e.g. H2O2 increased 6 to 8-fold), and 4-MU was detected in the fat body 8 min after administration. In addition, the activities of CAT and GPx were up-regulated 4 to 11-fold and 2 to 16-fold, respectively, and were helpful in defending fat body cells against oxidative injury in combination with NADPH. Furthermore, significant increases in the contents of T-AOC (up to approx. 2-fold), antioxidants of ASAFR (by 2 to 4-fold) and GSH were detected.
Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.Comp Biochem Physiol C Toxicol Pharmacol.2014 Jan;159:31-7. doi: 10.1016/j.cbpc.2013.09.003. Epub 2013 Sep 28.
Plant sources of umbelliferones have tumor-inhibitory effects at the cellular level. However, their physiological functions in animals are largely unresolved. In this study, we provide evidence to show that 4-methylumbelliferone (4-MU) participates in the regulation of antioxidative capacity in the
PMID 24080584

Japanese Journal

ホールマウントin situハイブリダイゼーション法(3. 遺伝子をみる, III. 遺伝子・分子をみる)

本間俊作
組織細胞化学 2005, 127-138, 2005-07-10
NAID 110004028632

Chemical modification of contractile 3 nm diameter filaments in Vorticellidae spasmoneme by diethyl-pyrocarbonate and its reversible renaturation by hydroxylamine

FANG J.
Biochem Biophys Res Commun 310, 1067-1072, 2003
NAID 80016247432

Molecular and Biochemical Characterization of a Novel Hydroxycinnamoyl-CoA : Anthocyanin 3-O-Glucoside-6"-O-Acyltransferase from Perilla frutescens

Yonekura-Sakakibara Keiko,Tanaka Yoshikazu,Fukuchi-Mizutani Masako [他],Fujiwara Hiroyuki,Fukui Yuko,Ashikari Toshihiko,Murakami Yasuyuki,Yamaguchi Masaatsu,Kusumi Takaaki
Plant and cell physiology 41(4), 495-502, 2000-04
… The inhibitory effects of diethyl pyrocarbonate and N-ethylmaleimide on the recombinant 3AT activities suggest that histidine and cysteine residues are important for their catalytic function. …
NAID 110003722795

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★リンクテーブル★

リンク元	「DEPC」「二炭酸ジエチル」「ジエチルピロカーボネート」「diethylpyrocarbonate」

「DEPC」

Diethylpyrocarbonate
Names
	IUPAC name Diethyl dicarbonate
	Other names Diethyl oxydiformate; Ethoxyformic anhydride; Pyrocarbonic acid diethyl ester; DEPC
Identifiers
CAS Registry Number	1609-47-8
ChEBI	CHEBI:59051
ChEMBL	ChEMBL55517
ChemSpider	2943
	InChI InChI=1S/C6H10O5/c1-3-9-5(7)11-6(8)10-4-2/h3-4H2,1-2H3 ; Key: FFYPMLJYZAEMQB-UHFFFAOYSA-N ; InChI=1/C6H10O5/c1-3-9-5(7)11-6(8)10-4-2/h3-4H2,1-2H3; Key: FFYPMLJYZAEMQB-UHFFFAOYAU ;
Jmol-3D images	Image
KEGG	C11592
MeSH	Diethylpyrocarbonate
PubChem	3051
	SMILES O=C(OCC)OC(=O)OCC ;
Properties
Molecular formula	C6H10O5
Molar mass	162.14 g·mol−1
Appearance	Clear, colorless liquid
Density	1.101 g/mL at 25 °C; 1.121 g/mL at 20 °C
Boiling point	93 to 94 °C (199 to 201 °F; 366 to 367 K) at 24 hPa
Hazards
Main hazards	Harmful
R-phrases	R22 R36/37/38
Flash point	69 °C (156 °F; 342 K) closed cup
LD50 (Lethal dose)	Oral - rat - 850 mg/kg
Related compounds
Related compounds	Di-tert-butyl dicarbonate; Dimethyl dicarbonate
	Except where noted otherwise, data is given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
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	Infobox references

　　[★]

ジエチルピロカーボネート、ジエチルピロカルボネート

関: diethyl pyrocarbonate、diethylpyrocarbonate

「二炭酸ジエチル」

　　[★]

英: diethyl pyrocarbonate
関: ジエチルピロカルボネート、ジエチルピロカーボネート

「ジエチルピロカーボネート」

　　[★]

英: diethyl pyrocarbonate、DEPC
関: ジエチルピロカルボネート、二炭酸ジエチル

「diethylpyrocarbonate」

　　[★]

ジエチルピロカルボネート

関: DEPC、diethyl pyrocarbonate

[narumi-1] Narumi et al. (1987). Neurochem Res. 12 (4). Missing or empty |title= (help)

[2] Chirgwin, John M et al. (1979). "Isolation of biologically active ribonucleic acid from sourcesenriched in ribonuclease". Biochemistry 18 (24): 5294–5299. doi:10.1021/bi00591a005. PMID 518835.

[3] Wolf, Barry; Lesnaw, Judith A.; Reichmann, Manfred E. (1970). "A Mechanism of the Irreversible Inactivation of Bovine Pancreatic Ribonuclease by Diethylpyrocarbonate. A General Reaction of Diethylpyrocarbonate with Proteins". European Journal of Biochemistry 13 (3): 519–25. doi:10.1111/j.1432-1033.1970.tb00955.x. PMID 5444158.

[sigma-4] "FAQ about DEPC". Sigma-Aldrich. Retrieved 12 August 2012.

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案内

diethyl pyrocarbonate