Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.
The metabolic disposition of etodolac (etodolic acid) was studied after oral and intravenous administration of the 14C-labeled or unlabeled drug to rats and dogs, and after oral administration of the drug to man. In all species, peak serum drug levels were attained within 2 hr after dosing. In rats and dogs, virtually all of the oral dose was absorbed; etodolac represented 95% of the serum radioactivity in rats and 75% in dogs. Serum levels in all species were generally dose-related. The elimination portion of the serum drug concentration/time curves was characterized by several peaks, which in rats were shown to be due to enterohepatic circulation. Tissue distribution studies in rats showed that radioactivity localized primarily in blood vessels, connective tissue, and highly vascularized organs (liver, heart, lung, and kidney) and that the rate of elimination of radioactivity from tissues was similar to that found in the serum. The apparent elimination half-life of etodolac averaged 17 hr in rats, 10 hr in dogs, and 7 hr in man. Etodolac was extensively bound to serum proteins. Liver microsomal cytochrome P-450 levels were unaltered in rats given etodolac daily for 1 week. The primary route of excretion in rats and dogs was via the bile into the feces. Preliminary biotransformation studies in dogs showed the presence of the glucuronide conjugate of etodolac in bile, but not in the urine. Glucuronide conjugates were not seen in the rat. Four hydroxylated metabolites in rat bile were tentatively identified. It was concluded that, in rats and dogs, etodolac is well absorbed, is subject to extensive enterohepatic circulation, undergoes partial biotransformation, and is excreted primarily into the feces.U
Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.
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Two synthetic pathways are described leading to a novel series of pyrrolo[4,3,2-de]isoquinolines. Some of these derivatives show high activity in preventing reserpine-induced ptosis and in lowering the blood pressure of spontaneously hypertensive rats, In a continuation of investigations of novel peri-fused tricyclic heterocyclic systems,l two synthetic routes to the pyrrolo[4,3,2-de]isoquinoline system (e.g., 3-1 1, see Scheme I) have been developed and various reactions of this system have been studied. This report describes the use of these routes for the preparation of a variety of pyrrolo[4,3,2-de]isoquinolines bearing substituents at one or more of positions 1, 3, 4, and 5, as well as a derivative containing a ring fused at the 4 and 5 positions. Some of these derivatives have been evaluated pharmacologically and the results are discussed. Scheme I R2 1. R = H 8 2, R = Me 3-11Chemistry. The first synthetic route to the pyrrolo[4,3,2-delisoquinoline system (Scheme I) involved a n internal Mannich reaction of 4-aminomethylindole ( 1 ) 2 or 4-methylaminomethylindole (2)s with aliphatic or aromatic aldehydes to afford a series of 3-substituted and 3,4-disubstituted 1,3,4,5-tetrahydropyrrolo[4,3,2-de]isoquinolines 3-11. Their structures and physical data are collected in Table I.?The second synthetic pathway (Scheme 11) consisted of' the conversion of methyl indole-4-carboxylate5 (12) uia a Vilsmeier reaction to the 3-formyl derivative 13, oximation to the aldoxime 14, and reduction of 14 and reduction of 14 catalytically with palladium in the presence of hydrochloric acid to afford the key intermediate, methyl 3-aminomethylindole-4-carboxylate hydrochloridewhich could be isolated and characterized by nmr spectroscopy but for which an analysis was not obtained because of the ease with which it converts to 16. Thus, treatment of 15 with sodium methoxide in ethanol at room temperature gave the 3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5( 1H) -one (16) in 84% yield. Alternatively, when the aldoxime 14 was reduced in acetic acid using platinum as the catalyst and the reaction mixture made alkaline during the work-up procedure, 16 was obtained directly but in only 22% yield.$ :After this work had been completed, the preparation, by a similar route. of 3 and I 1 was reported in the patent literature.* SchemeI1 MeOOC R M a O C CH,NH,. HCI I I I 1 12, R = H 13, R = CHO 14, R=CHNOH 15 H H 16 17Reduction of lactam 16 with diborane afforded the unsubstituted form of the ring system, 17, which made accessible for pharmacological screening a series of monoand disubstituted derivatives bearing alkyl and dialkylaminoalkyl groups on the nitrogen atoms. These transformations of 17 are shown in Scheme I11 and involved the use of conventional series of reactions. Thus, the 1-alkyl compounds 21 and 22 were obtained from 17 uia the benzyloxycarbonyl derivatives 18-20. The 1,4-dialkyl derivative 24 was prepared from 21 uia acylation and reduction. Alternatively, the 1,4-dimethyl analog 27 could be obtained from 17 by 1-alkylation of th...
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