Rabbit reticulocyte

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 hours, becoming young red blood cells and concomitandy losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by addition of Ca +. The system is then pro-... 

Jackson, R. J., and Hunt, T. (1983). Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 96, 50-74. 

Inhibitory constants for inhibition of natural globin mRNA translation in a rabbit reticulocyte lysate system. For the tri-, tetra-, and pentaphosphate series, each value for Kx was normalized by dividing with the value for Kj for the cap analog standard for the series m7Gp3G, m7Gp4G, and m7GpsG, respectively. 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Dasso, M. C., and Jackson, R. J. (1989). On the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system. Nucleic Acids Res. 17, 3129—3144. 

Translation extracts Rabbit reticulocyte lysates (RRL) (Promega), wheat germ (WG) extract (Promega), bacterial S30 extract (Promega). Extracts from Krebs-2 cells were prepared as described (Svitkin and Sonenberg, 2004). 

Preparation of Krebs-2 translation extracts Krebs-2 extracts are an ideal system to screen for compounds that inhibit translation because they faithfully recapitulate the cap dependency and the cap-poly(A) synergism associated with eukaryotic mRNA translation (Svitkin and Sonenberg, 2004), unlike standard rabbit reticulocyte lysates (RRL) (Borman et al., 2000). Furthermore, the translation of many types of IRESes is supported in Krebs-2 extracts. The use of commercially available translation competent extracts prepared from RRL, wheat germ, and E. coli is extremely useful in assessing selectivity of inhibitors identified in primary screens. 

Borman, A. M., Michel, Y. M., and Kean, K. M. (2000). Biochemical characterization of cap-poly(A) synergy in rabbit reticulocyte lysates The eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5r-end. Nucleic Acids Res. 28, 4068-4075. 

An important characteristic of mammalian 15-LOX is its capacity to oxidize the esters of unsaturated acid in biological membranes and plasma lipoproteins without their hydrolysis to free acids. Jung et al. [19] found that human leukocyte 15-LOX oxidized phosphatidylcholine at carbon-15 of the AA moiety. Soybean and rabbit reticulocyte 15-LOXs were also active while human leukocyte 5-LOX, rat basophilic leukemia cell 5-LOX, and rabbit platelet 12-LOX were inactive. It was suggested that the oxygenation of phospholipid is a unique property of 15-LOX. However, Murray and Brash [20] showed that rabbit reticulocyte... 

LOX-catalyzed oxidation of LDL has been studied in subsequent studies [26,27]. Belkner et al. [27] showed that LOX-catalyzed LDL oxidation was not restricted to the oxidation of lipids but also resulted in the cooxidative modification of apoproteins. It is known that LOX-catalyzed LDL oxidation is regio- and enantio-specific as opposed to free radical-mediated lipid peroxidation. In accord with this proposal Yamashita et al. [28] showed that LDL oxidation by 15-LOX from rabbit reticulocytes formed hydroperoxides of phosphatidylcholine and cholesteryl esters regio-, stereo-, and enantio-specifically. Sigari et al. [29] demonstrated that fibroblasts with overexpressed 15-LOX produced bioactive minimally modified LDL, which is probably responsible for LDL atherogenic effect in vivo. Ezaki et al. [30] found that the incubation of LDL with 15-LOX-overexpressed fibroblasts resulted in a sharp increase in the cholesteryl ester hydroperoxide level and a lesser increase in free fatty acid hydroperoxides. 

A series of indolizines 281 and azaindolizines 282 were screened as possible inhibitors of 15-lipoxygenase (15-LO) from soybeans and rabbit reticulocytes. Most compounds studied were significantly more active than quercitin (IC50 51 pi) <2003BML5409>. The indolizine and azaindolizine sulfonates were particularly studied and showed high activity <2005BMC5409>. 

Most frequently, extracts of either prokaryotic or eukaryotic origin as such from Escherichia coli, wheat germ or rabbit reticulocytes are employed for cost reasons and availability. While those based on E. coli are unable of post-translational protein modification, eukaryotic extracts do allow synthesis of glycosylated or phosphorylated proteins to some extent when additional components, such as microsomes for glycosylation are added. Care needs to be taken with cell-free systems recombinated from the individual components when a native protein is to be produced that does not fold spontaneously... 

R. Hough, G. Pratt, M. Reichsteiner, Ubiquitin-Lysosome Conjugates. Identification and Characterization of an ATP-Dependent Protease from Rabbit Reticulocyte Lysates , J. Biol. Chem. 1986, 261, 2400-2408. 

Hoeeman, L., Phatt, G., and Rechsteiner, M. Multiple forms of the 20 S multicatalytic and the 26S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate. J. Biol. Chem. 1992, 267, 22362-22368. 

Additional lipoxygenases are known which oxygenate different positions on the arachidonic acid chain. 12-LO, resulting in the formation of 12-HETE (7), is best known in platelets, while the 15-LO from soybean has been studied in detail for many years [8]. 15-HETE (8) is also produced by mammalian cells the enzymes from neutrophils and particularly rabbit reticulocytes are the best characterized. 

Dittmar KD, Hutchison KA, Owens-Grillo JK, Pratt WB. (1996) Reconstitution of the steroid receptor.hsp90 heterocomplex assembly system of rabbit reticulocyte lysate. J Biol Chem. 271, 12833-12839. 

The suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

Waxman, L., Fagan, J.M., and Goldberg, A.L. (1987). Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates. J. Biol. Chem. 262,2451-2457. 

Hershko, Rose and colleagues identified the ubiquitin system more than 20 years ago in a cell-free system from rabbit reticulocytes. They demonstrated... 

Dube P, Bacher G, Stark H, Mueller F, Zemlin F, van Heel M, Brimacombe R (1998) Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution. J Mol Biol 279 403-421... 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Figure 3. SDS-urea electrophoresis of proteinase Inhibitors I and II. Key Lane I, proteinase Inhibitor II Lane 2, proteinase Inhibitor I (both lanes stained with Coomassie blue) and Lane 3, [ S]preproteinase Inhibitors I and II synthesized in an in vitro rabbit reticulocyte system immunoprecipitated for Inhibitors I and II.

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

In a further experiment we assayed for the presence of a cap structure on the mRNAs for both Inhibitors I and II by competitive inhibition by 7-methyl-guanosine 5 -monophosphate (m G p) of the in vitro translation of these messengers. Concentrations of 40 pM m G p inhibited by 50% the in vitro translation of total tomato leaf poly(A)" " mRNA (Fig. 7A). This level is 40-fold lower than that required to similarly inhibit rabbit globin mRNA translated in a rabbit reticulocyte lysate (17) and 4-fold lower than that required to inhibit the same mRNA in a wheat germ system (18). It was of interest that the translation of Inhibitor I is inhibited to 50% by 20 pM m G p while 50% inhibition of Inhibitor II requires less than 10 pM (Fig. 7B). The basis of this difference is not understood but... 

Additional information <1-7, 11, 14, 15, 17-19, 21, 30> (<7> cell-free synthesis in mRNA-dependent rabbit reticulocyte lysate system [40] <2,4,5> high activities in tissues where turnover of energy from adenine nucleotides is great, e. g. muscle [3] <1-6,11,14,15> tissue distribution [3,46] <2,5> rabbit and human carry a minimum of 2 sets of isozymes within an individual one set in muscle, erythrocytes, brain and another in liver, kidney and spleen [3]) [3, 40, 46]... 

The regulation of translation through the phosphorylation of eIF-2 is best understood as it operates in the rabbit reticulocyte. Two protein kinases specific for the a subunit of eIF-2 have been purified from reticulocytes. One of these kinases, termed the heme-regulated inhibitor repressor (HRI), serves to coordinate the rate of hemoglobin synthesis (more than 90% of the total protein synthesized in the reticulocyte is hemoglobin) with the availability of hemin (the... 

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