Self-inactivation

To address biosafety concerns, self-inactivating lentiviral vectors and stable packaging cell lines have been developed. 

LO activity is found in a variety of mammalian cells, most notably leukocytes, with the enzymes from neutrophils and the rat basophilic leukemia (RBL-1) cell line being the most studied several have been purified and cloned [5]. 5-LO is a cytosolic enzyme which requires calcium, ATP and additional uncharacterized cell components for optimal activity. Characterization and kinetic studies with 5-LO enzymes are often difficult, because of self-inactivation and complex cofactor requirements. 

FIGURE 12-14 Self-inactivation of Gs.The steps are further described in the text. The protein s intrinsic GTPase activity, in many cases stimulated by RGS proteins (regulators of G protein signaling), determines how quickly bound GTP is hydrolyzed to GDP and thus how long the G protein remains active. 

A. Fassati, A. Bardoni, M. Sironi, D. J. Wells, N. Bresolin, G. Scarlato, M. Hata-naka, S. Yamaoka, and G. Dickson, Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression, Hum. Gene Ther. 9 2459 (1998). 

Valderrama B, Vazquez-Duhalt R (2005) Electron-balance during the oxidative self-inactivation of cytochrome c. J Mol Catal B Enzym 35 41—44... 

In contrast, metal and stractural analyses of the second group of FALS mutants show that these proteins are fully capable of binding metals and retain enzymatic activity, if they are properly folded. Thus, they have been termed wild-type-like. It is postulated that these mutants have higher prooxidant activity (possibly via bicarbonate-mediated inactivation, see Scheme 2). Self-inactivation could lead to metal loss and similar aggregation effects described for the metal-binding-region mutants. 

Iwakuma T, Cui Y, Chang LJ (1999) Self-inactivating lentiviral vectors with U3 andU5 modifications. Virology 261 120-132. 

Yu, S. R, von Ruden, T., Kantoff, P. W., Garber, C., Seiberg, M., Ruther, U., Anderson, W. F., Wagner, E. F. and Gilboa, E. (1986). Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. 

Abu-Soud, H., Wang, J, Rousseau, DL, Fukuto, JM, Ignarro, LJ. and Stuehr, DJ. 1995. Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. J Biol Chem 270 22997-23006. 

Major concerns in the use of retroviral vectors are the possibility of vector mobilization and recombination with defective (endogenous) retroviruses in the target cell. This led to the development of self-inactivating vectors (SIN) (38). In these vectors, the viral promotor and enhancer regions in the 3 U3 are deleted, thus preventing LTR-driven transcription in the transduced cells. Furthermore, transgene expression in these vectors is exclusively driven by an internal promotor, which improves the use of regulatory and tissue-specific promoters. 

Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, et al. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72 9873-80... 

Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. 1998. Development of a self-inactivating lentivirus vector. J. Virol. 72 8150-57... 

Schnell T, Foley P, Wirth M, Munch J, Liberia K. 2000. Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum. Gene Ther. 11 439 17... 

Although for many years lipoxygenase activity in fish was discounted (14-17), plant lipoxygenases were easily demonstrated, and became well-accepted. Research into the biosynthesis of the six-, eight-, and nine-carbon volatile aroma compounds of mushrooms (27, 56) and cucumber/melon-fruits (37, 57-58) showed the concerted activities of both site-specific lipoxygenases and hydroperoxide lyases. Because animal lipoxygenases exhibit self-inactivation properties (59-60), early experimentation failed to detect an active lipoxygenase from fish, and this led to conclusions that autoxidation was responsible for the... 

Although little is known about many of the important enzymic parameters of the flavor generating systems that are endogenous in fish, the self-inactivation or suicidal nature of animal... 

The relative ratios of alcohols and carbonyls for the six-, eight- and nine-carbon volatiles in fish (23-24) and oysters (26) parallel those encountered in cucumber fruits (37) and mushrooms (27, 56) if the two systems are combined. Therefore, the use of plant-based enzyme systems for the controlled generation of fresh seafood flavors and aromas has been under consideration in our laboratory as a means to overcome some of the self-inactivating problems associated with fish lipoxygenases. 

Accounting for half of the oxidizing equivalents based on Ru(tpy) (bpy)O implies that about 50% of the total Ru(IV)0 reacts via a self-inactivation pathway ( x)> which has been demonstrated for polypyridyl oxidants 129). This self-inactivation pathway results from the bimolecular reaction of two oxoruthenium complexes to produce Ru(II) and oxidized polypyridyl ligands, as discussed earlier and shown in Scheme 5. Thus, observations imply that in terms of Scheme 10, kx > k(i > sugar Ru(IV)0. This ordering makes the prediction that a less powerful oxidant will be reduced via the kx and Hq pathways, but not via as long as the rates of the various processes are... 

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