Protein Synthesis in a Cell-Free Extract from Staphylococcus aureus

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.     

Reduction of Hexavalent Chromium by Cell-Free Extract of Bacillus sphaericus AND 303 Isolated from Serpentine Soil

Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S₁₂, S₃₂, and S₁₅₀). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S₁₅₀) concentration equivalent to 4.56 mg protein/mL, 300 μM Cr(VI) and pH 6.0 after 30 min incubation at 30°C. The enzyme was heat labile; 80% of its activity was lost when exposed at 70°C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a V_max of 1.432 μmol Cr(VI)/mg protein/min and K_m of 158.12 μM Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity over control was recorded with 1.0 μM NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 μM Co(II), which retained 93% activity over control.     

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h?1), Cr(VI) concentration (2 to 8 mg L?1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h?1 did not affect the rate of Cr(VI) reduction, but above 6 mL h?1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h?1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h?1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L?1 at flow rates of 3 to 6 mL h?1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.     

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h-1), Cr(VI) concentration (2 to 8 mg L-1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h-1 did not affect the rate of Cr(VI) reduction, but above 6 mL h-1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h-1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h-1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L-1 at flow rates of 3 to 6 mL h-1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.     

Cloning and Characterization of Flagellin Genes and Identification of Flagellin Glycosylation from Thermophilic Bacillus Species

Flagellin glycosylation was identified in Bacillus sp. PS3 and Geobacillus stearothermophilus. In vivo complementation showed that these flagellin genes did not restore the motility of a Bacillus subtilis flagellin mutant, whereas the genes encoding non-glycosylated flagellin from Geobacillus kaustophilus and Bacillus sp. Kps3 restored motility. Moreover, four types of flagellins expressed in B. subtilis were not glycosylated. We speculate that glycosylation is required for flagellar filament assembly of these bacilli.     

Synthesis of Bacteriophage M13-Specific Proteins in a DNA-Dependent Cell-Free System II. In Vitro Synthesis of Biologically Active Gene 5 Protein

It is shown that gene 5 protein of bacteriophage M13 is one of the major proteins synthesized in vitro under the direction of M13 replicative-form DNA. By means of DNA-cellulose chromatography, this protein has been purified to homogeneity and its biological characteristics have been compared with those of its native counterpart. Like native gene 5 protein, the purified, in vitro-synthesized protein binds tightly and selectively to single-stranded, but not to double-stranded, DNAs. These results suggest that truly functional gene 5 protein is made in the cell-free system.     

In Vitro Cytotoxic Activity of Total Flavonoid from Equisetum Arvense Extract

Background:Normally happening substances like flavonoids are regarded as active candidates for the treatment and prevention of cancer The purpose of this study was to see how Iraqi E. arvense total flavonoid affected cell lines biologically and human lung fibroblast normal cell line (WISH).Methods:Plant powder was extracted by reflex apparatus, then thin-layer chromatography (TLC) was used to determine total flavonoids. Cytotoxicity assay (MTT) was used to determine the cytotoxic activity of the prepared plant against human breast cancer (MCF-7), cells human cervix cancer (HELA), human colon cancer (Caco-2) and human lung fibroblast normal cell line (WISH).Results:The flavonoids Rutin, Quercetin, Kaempferol, and luteolin were detected using the Thin Layer Chromatography (TLC) technique. In contrast to the negative control, the extract inhibited cell growth to a highest of 82.158% for MCF-7 and 61.360% for Caco-2 at the concentration (100 µg/ml), and (54.880%) for Hela cell line at the concentration (100 µg/ml). In addition, the concentration (6.25 µg/ml) of total flavonoid extract produced a decrease in the growth of the normal WISH cell line to reach (1.094%).Conclusion:Equisetum arvense contain high amounts of flavonoids, the qualification of some flavonoids compounds was detected using TLC. The total flavonoids showed significant cytotoxic activity against various types of cancer cell lines and normal cell line in vitro, the antitumor activity was highly efficient in a dose and cell type dependent manner.Key Words: Equisetum arvense L, Caco2, Hela, Total flavonoid, MCF-7, WISH     

Effects of Different RNAs and Components of the Cell-Free System on In Vitro Synthesis of Sindbis Viral Proteins

Cell-free extracts from Krebs ascites cells and rabbit reticulocytes synthesized a variety of viral-specific proteins when programmed with several different kinds of Sindbis viral RNAs. The RNAs included purified virion RNA (42S) and two species (26S and "33S") of purified intracellular viral messenger RNAs from viral-infected BHK cells. Proteins formed in vitro were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rate-zonal centrifugation in urea-sucrose gradients, two-dimensional tryptic peptide fingerprints, and immunoprecipitation with rabbit anti-Sindbis virus serum. The only major identifiable protein formed in vitro was viral capsid, but the relative amount of capsid produced was determined by the mRNA, the source of cell-free extract, and the components of the cell-free system. Virion RNA directed synthesis of larger-molecular-weight proteins than did intracellular viral RNAs, and some of this protein was distinct from that formed by the smaller viral RNAs. Indirect evidence is presented for in vitro synthesis of viral envelope proteins.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

Inhibition of Sphingolipid Synthesis by Cycloserine In Vitro and In Vivo

Abstract: d - and l -cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes, l-Cycloserine was shown to be 100 times more inhibitory than the d-isomer for the brain microsomal enzyme in vitro. In vivo , l -cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of l -cycloserine on a regimen of intraper-itoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.     

Characteristics of a New Strain of Bacillus popilliae Sporogenic In Vitro

Conditions are described that led to the isolation of NRRL B-2309M, a strain of Bacillus popilliae which sporulates regularly in laboratory culture. Colonies grown on a medium formulated with yeast extract and the ingredients of Mueller-Hinton with phosphate, trehalose, and agar, produced 20% spores in 10 to 12 days. The quantity and kind of yeast extract determine the extent of sporulation, although there are other requirements for optimal growth and sporulation. Spore inocula free of viable vegetative cells are necessary to maintain sporogenicity since asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain, NRRL B-2309N, is also asporogenic in larvae, but lethal, owing to vigorous vegetative growth. Strain B-2309M is infective when vegetative cells or spores are injected into Japanese beetle larvae but fewer spores are formed in vivo than when infections are caused by NRRL B-2309. The characteristics of four related strains of B. popilliae are tabulated.     

Oxidation and Reduction of D-Xylose by Cell-Free Extract of Pichia quercuum

The fermentation mechanism of the simultaneous production of D-xylonic acid and xylitol from D-xylose by Pichia quercuum was studied by using a cell-free enzyme preparation. Nicotinamide adenine dinucleotide phosphate (NADP)-dependent D-xylose dehydrogenase activity and NADP-dependent D-xylose reductase activity were detected, and the oxido-reduction reaction of D-xylose was able to couple through regeneration of NADP and NADPH to produce D-xylonic acid and xylitol.     

Breast Milk from Tanzanian Women Has Divergent Effects on Cell-Free and Cell-Associated HIV-1 Infection In Vitro

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-Lewis(X) from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding.     

Degradation of Urea Herbicides by Cell-Free Extracts of Bacillus sphaericus

N′-methoxy phenylurea herbicides are degraded by induced cells of Bacillus sphaericus ATCC 12123 by liberating carbon dioxide from the ureido portion of the molecule and leaving the corresponding aniline moieties. Cell-free extracts of B. sphaericus inactivate these herbicides in the same way as reported for whole cells. A 6.6-fold purification of the crude extract was achieved by a combination of salt fractionation with ammonium sulfate and column chromatography on diethyl-aminoethyl cellulose.     

Protein Synthesis by a Cell-Free Preparation from the Bird Malaria,Plasmodium lophurae*

SYNOPSIS. Cytoplasmic polyribosomes were isolated from the avian malaria parasite Plasmodium lophurae by lysis with 0.15% Triton X-100 followed by high speed centrifugation through a discontinuous sucrose gradient. Polyribosomes were protected from nuclease degradation using 100 μg/ml heparin or 50 μg/ml dextran sulfate. Cell-free incorporation of radioisotope-labeled amino acids required a pH 5 fraction (duck reticulocyte), Mg²⁺, and an energy-generating system. The protein synthesizing system was stimulated by the addition of polyuridylic acid. Optimum conditions for protein synthesis by the plasmodial system are described. The effects of drugs on the cell-free protein synthesizing system using duck reticulocyte and plasmodial ribosomes are reported.     

Biphasic Modulation by ACTH-Like Peptides of Protein Synthesis in a Cell-Free System from Rat Brain

Brain protein synthesis in a cell-free system was stimulated by 10(-8) M-ACTH1-24. This stimulatory effect was completely inhibited by aurintricarboxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N-terminal peptide sequence 4-10 exerted a biphasic modulation of cell-free protein synthesis, i.e., a stimulation at low concentrations (10(-8) and 10(-10) M) and an inhibition at a high concentration (10(-4) M). The D-isomer, ACTH4-10-7-D-phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4-10-7-L-phe at 10(-8) M and 10(-4) M.     

In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.     

Inhibition of Cell-Free Protein Synthesis by Hydrostatic Pressure

Pressure inhibition of cell-free polypeptide synthesis is manifested in the same manner as that observed in the intact cell: (i) starting at approximately 200 atm, there is a progressive inhibition with increasing pressures; (ii) there is complete inhibition at 680 atm; (iii) incorporation into polypeptide is instantaneously reversible after pressure release and proceeds at a rate parallel to an atmospheric control; and (iv) the volume change of activation (DeltaV*) is 100 cm(3)/mole. Peptide bond formation per se can occur at a pressure level which is totally inhibitory to polypeptide synthesis. The one investigated step in translation that is inhibited in an identical manner is the binding of aminoacyl-transfer ribonucleic acid (AA-tRNA) to the ribosome-messenger RNA (mRNA) complex. The volume change of activation (DeltaV*) calculated for the binding reaction is also 100 cm(3)/mole. Thus, the inability of AA-tRNA to bind to ribosomes and mRNA under pressure, possibly in conjunction with translocation, appears to be responsible for the observed inhibition of the translational mechanism.