Protein synthesis in brine shrimp embryos. Regulation of the formation of the ternary complex (Met-tRNAf X eIF-2 X GTP) by two purified protein factors and phosphorylation of Artemia eIF-2.
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We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co-eIF-2(A) and Co-eIF-2(B). These factors are essential for ternary complex formation and binding of [35S]-Met-tRNAf to 40-S ribosomal subunits with 15-30 microgram eIF-2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co-eIF-2(A) is composed of a single polypeptide of Mr 65 000, whereas Co-eIF-2(B) contains polypeptides of Mr 105000 and 112000. Co-eIF-2(A) is sensitive to 4.5 microM aurintricarboxylic acid but Co-eIF-2(B) requires approximately 15 microM aurintricarboxylic acid to give 50% inhibition of ternary complex formation. The stimulatory activity of both factors is abolished by pretreatment of the proteins with N-ethylmaleimide. Artemia eIF-2 rapidly bonds [3H]GDP or [3H]GTP and at 15 degrees C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co-eIF-2(A) and Co-eIF-2(B) have no effect on the exchange or the amount of nucleotide bound. The small subunit (Mr 43 000) of Artemia eIF-2 is phosphorylated in the presence of the rabbit reticulocyte heme-repressible kinase. Tryptic digestion of [32P]phosphorylated eIF-2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF-2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF-2 is active in ternary complex formation, in AUG-dependent binding of [35S]Met-tRNAf to 40-S ribosomal subunits and in cell-free protein synthesis. Both Co-eIF-2(A) and Co-eIF-2(B) stimulate ternary complex formation with phosphorylated eIF-2. A kinase that phosphorylates the small subunit of eIF-2 is present in the post-ribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos. (+info)
Photochemical cross-linking of initiation factor-3 to Escherichia coli 30 S ribosomal subunits.
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Initiation factor-3 has been photochemically cross-linked to Escherichia coli 30 S ribosomal subunits by means of near-ultraviolet (> 285 nm) irradiation. The cross-linking was judged to be specific since noncovalent binding was required for subsequent cross-linking and was prevented by the presence of 0.5 M NH4Cl or 0.5 mM aurintricarboxylic acid. Covalent attachment reached its maximum level of 8.5 to 11% of the noncovalently bound IF-3 after 60 min of irradation. Cross-linking was unaffected by the presence of the photosensitizer, acetone (5 or 10%), but was reduced to 30 to 40% of its maximum level by addition of 5 mM dithiothreitol, a free radical scavenger. Analysis of the 14C-labeled IF-3 x 30 S subunit covalent complex revealed that the IF-3 was distributed between the protein and RNA of the subunits in a 3:1 ratio. The target proteins have been identified as S7, S11, S12, S18, and S21 by immunochemical techniques. (+info)
Evaluation of standard and modified M-FC, MacConkey, and Teepol media for membrane filtration counting of fecal coliforms in water.
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MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined. (+info)
Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate.
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DRB triphosphate inhibits activity of isolated RNA polymerase B, and, to a lesser extent, that of polymerase A. The same holds true for transcription in isolated nuclei. It does not act as an initiation inhibitor. In all cases, high concentrations of DRB triphosphate are required. Cells do not phosphorylate DRB to a measurable extent. hn RNA resistant to DRB is initiated with both ATP and GTP in the presence of the drug. These experiments render the hypothesis unlikely that DRB triphosphate in the cell specifically interferes with the initiation reaction of polymerase B. (+info)
Reconstitution into liposomes of membrane proteins involved in ribosome binding on rough endoplasmic reticulum. Ribosome-binding capacity.
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A membrane protein fraction having a high affinity for polyribosomes was isolated from microsomal membranes of rat liver and was incorporated into liposomes made from microsomal lipids to evaluate the polyribosome-binding capacity of the reconstituted liposomes, with the following results. (1) The polyribosome binding to the reconstituted liposomes depended on the amounts of polyribosomes added to the binding mixture. (2) Liposomes made from lipids alone did not bind any polyribosomes. (3) The polyribosome-binding capacity of the reconstituted liposomes was very sensitive to proteolytic enzyme and strongly inhibited by addition of 0.1 mM-aurintricarboxylic acid or by increasing KCl concentration. These results suggest that the binding mechanism of polyribosomes to the reconstituted liposomes is much like that for rough microsomal membrane stripped of endogenous polyribosomes. (+info)
Protein synthesis in rabbit reticulocytes. Demonstration of the requirements for eIF-2 and Co-eIF-2A for peptide chain initiation using immune sera.
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Antisera to eIF-2 and Co-eIF-2A have been prepared by immunizing two chickens separately with homogeneous preparations of either eIF-2 or Co-eIF-2A. Addition of anti-eIF-2 or anti-Co-eIF-2A to reticulocyte lysates strongly inhibits protein synthesis and, in each case, protein synthesis inhibition is reversed by the addition of the corresponding homogeneous factor. Protein synthesis inhibition by anti-Co-eIF-2A is not reversed by eIF-2 at any concentration tested indicating an absolute requirement for Co-eIF-2A in protein synthesis. Also, in partial peptide chain initiation reactions, the addition of anti-Co-eIF-2A inhibits Co-eIF-2A stimulation of ternary complex formation by eIF-2 and Co-eIF-2A protection of ternary complexes in the presence of aurintricarboxylic acid. (+info)
Cell nucleus and DNA fragmentation are not required for apoptosis.
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Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death. (+info)
Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes.
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In rat hepatocytes, DNA damage by t-butyl hydroperoxide (tBOOH) was measured by using the fluorimetric analysis of alkaline DNA unwinding. The electrophoretic profile of genomic DNA suggests single rather than double DNA strand breaks formation. Oxidative DNA modifications, measured as increased 8-hydroxy-deoxyguanosine content, were not detected. Lysis of hepatocytes and DNA strand breaks induced by tBOOH did not correlate, indicating that both processes are not interconnected. Since o-phenanthroline prevents against tBOOH-mediated effects on both DNA and membrane integrity, we discussed about a putative role of iron. (+info)