Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract.

RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.     

Initiation of histone synthesis by f-met-tRNAf in an ascites cell-free system

Formyl-[³⁵S]methionine is incorporated into histones synthesized by a mouse ascites cell-free system supplemented with histone mRNA and f-[³⁵S]met-tRNA_f from yeast. Most of the [³⁵S]methionine incorporated can be shown to be at the N-terminus by Edman degradation after deformylation. This indicates that methionine can be used as the initiator amino acid for histones in this cell-free system.     

Transmembrane ferricyanide reductase activity in Ehrlich ascites tumor cells

A transmembrane ferricyanide reductase activity was assayed in intact Ehrlich ascites tumor cells. Kinetic measurements gave a Km of 0.14 mM and a Vmax of 0.31 mumol/min per 10(6) cells. In short-term batch experiments, this activity was enhanced in the presence of 10 mM lactate, a source of cytosolic NADH. The transmembrane redox activity was accompanied by alkalinization of the cytosol. Both ferricyanide reduction and proton extrusion were diminished in the presence of 0.2 mM amiloride. Several cytotoxic drugs significantly inhibited the ferricyanide reductase activity at concentrations at which they show antineoplastic activity.     

Fc-receptor-bearing cells in spleen of mice injected with cell-free Ehrlich ascites fluid (EAF)

The kinetics of different cell populations (T and B) and subpopulations (one bearing easily releasable FcR and one bearing stable FcR) was followed in spleens of mice after one single i.p. injection of EAF. The number of FcR bearing cells doubled within 2-7 days after EAF injection. This increase was due to cells bearing temperature sensitive FcR and was accompanied by the doubling of theta positive cells. These results, supported by the demonstration of doubly labeled (theta+FcR+) cells, suggest that EAF injected into normal mice causes the appearance of T-cells expressing easily releasable FcR. These cells, according to Fridman et al. (1977) are suppressor cells. Maximal increase of theta positive cells and of cells with temperature sensitive FcR detected in vitro coincided with the maximum of the suppressive activity of EAF detected in vivo.     

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC₅₀ = 5.0 μM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8–12 h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 μg kg^?1 violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 μg kg^?1 for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.     

Parathyroid mRNA directs the synthesis of pre-proparathyroid hormone and proparathyroid hormone in the Krebs ascites cell-free system.

Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ～ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [³⁵S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, ($\text{i}$), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and ($\text{ii}$) the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH₂-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system.     

Multi-hour translation of mRNA in a cell-free system

In this study, we demonstrate that mRNA molecules can serve as an efficient template for cell-free translation through a combination of methods to protect them from nucleolytic digestion. Removal of major endonucleases activity from cell extract, the addition of a stemloop structure at the 3??-end of the mRNA and continuous reloading of ribosomes onto mRNA were found to be crucial for maintaining the functional integrity of mRNA during cell-free synthesis. When these three approaches were combined, mRNA-directed protein synthesis continued over 15 h, leading to the production of 2.6 mg/mL of encoded protein. The methods for direct translation of mRNA presented herein will provide a useful option for deciphering genetic information, including the fields of mRNA display and materialization of metagenomic information.