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Biomedical subjects

H Trachsel

Publications and source records attributed to H Trachsel.

At least 73 records · Page 4Linked to original sources
PubMed1982

Association of an Mr 50,000 cap binding protein with the cytoskeleton in BHK cells.

A monoclonal antibody (anti-CBP antibody) is shown to be directed against cap binding protein(s) (CBP) by virtue of its ability to inhibit the translation of capped reovirus mRNA in a cell-free system derived from L-cells and inhibit the specific (cap analogue-inhibited) cross-linking of proteins to the oxidized 5' terminal cap structure of reovirus mRNA. Anti-CBP antibody reacts with an Mr 50,000 polypeptide in rabbit reticulocyte polysomes and this polypeptide appears to be associated with the 5' cap structure of mRNA. In BHK-21 cells immunofluorescence microscopy reveals that the antibody reacts with a fibrous network extending through the cytoplasm in a radial arrangement. The network behaves like intermediate filaments in colchicine-treated cells suggesting a direct or indirect linkage of CBP with intermediate filaments. The association of CBP with a cytoskeletal element is further confirmed by isolation of proteins from Triton X-100-extracted cells and identification of CBP in the cytoskeletal fraction with anti-CBP antibody. The major polypeptide reacting with anti-CBP antibody is an Mr 50,000 component. Tryptic peptide mapping shows that this polypeptide is related to an Mr 24,000 polypeptide identified as cap binding protein in earlier experiments [Sonenberg, Morgan, Merrick & Shatkin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4843-4847].

Animals↗
PubMed1981

In vitro translation in reovirus- and poliovirus-infected cell extracts. Effects of anti-cap binding protein monoclonal antibody.

Monoclonal antibodies directed against rabbit reticulocyte cap binding proteins were used as probes to study cap-dependent translation initiation in mock-infected and virus (poliovirus or reovirus)-infected cells. The antibodies inhibited capped mRNA translation, but did not have any effect on the translation of naturally uncapped mRNAs in uninfected cell-free extracts prepared from either L-cells or HeLa cells. Cell-free extracts prepared from either reovirus-infected L-cells or poliovirus-infected HeLa cells did not support capped mRNA translation. However, translation of naturally uncapped mRNAs in the latter extracts was comparable to that observed in extracts from uninfected cells. Thus, translation of mRNAs in extracts from either reovirus- or poliovirus-infected cells proceeds most probably via a cap-independent mechanism, since it is not inhibited by antibodies directed against cap binding proteins.

Animals↗
PubMed1981

Probing the function of the eucaryotic 5' cap structure by using a monoclonal antibody directed against cap-binding proteins.

A monoclonal antibody directed against cap-binding proteins was used to elucidate the possible mechanism by which cap-binding proteins function in initiation of eucaryotic translation. The monoclonal antibody preparation employed in this study exhibited a marked differential effect in inhibiting the translation of folded, capped eucaryotic-mRNAs to a far greater extent than naturally uncapped mRNAs or native capped mRNAs that do not possess extensive 5' end secondary structure. These findings were consistent with the effects of the antibody on initiation complex formation with three different types of reovirus mRNA: native reovirus mRNA; inosine-substituted reovirus mRNA, which has a relaxed secondary structure; and bromouridine-substituted reovirus mRNA, in which base pairing is enhanced relative to regular reovirus mRNA. The extent that translation initiation complex formation was inhibited by the monoclonal antibody directly correlated to the degree of secondary structure present in the mRNA. Binding of bromouridine-substituted reovirus mRNA to ribosomes was inhibited to the greatest extent, while binding of inosine-substituted reovirus mRNA was not inhibited at all in the reticulocyte lysate system or was slightly inhibited in a wheat-germ system. These results support the hypothesis that cap-binding proteins are involved in unwinding of the 5' terminal, secondary structure of many eucaryotic mRNAs, thus facilitating the attachment of ribosomes to mRNA.

Animals↗
PubMed1980

Skin test and lymphocyte stimulation in delayed hypersensitivity against Staphylococcus aureus antigens. Development of hypersensitivity.

The development of delayed hypersensitivity against staphylococcal antigens in guinea pigs was observed from day 3 to day 50 after sensitization with staphylococcal homogenate in Freund's incomplete (FIA) and Freund's complete adjuvant (FCA). Skin test reactivity, stimulation of lymph node lymphocytes, and peripheral blood lymphocytes and the titre of precipitating antibodies were followed during this period. Maximal skin test reactivity as well as maximal lymphocyte responsiveness occurred at day 21 after sensitization in FCA-sensitized guinea pigs. In FIA-sensitized animals highest skin reactivity was observed at day 14 and maximal lymphocyte stimulation 35 days after sensitization. Precipitating antibodies reached a plateau at day 20 in plasma of animals sensitized with FCA and at day 35 in FIA-sensitized animals.

Animals↗
PubMed1980

Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells.

It was previously shown that the poliovirus-induced inhibition of translation of capped mRNAs can be reversed by a protein found in preparations of the eukaryotic initiation factor eIF-4B [Rose, J. K., Trachsel, H., Leong, K. & Baltimore, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2732--2736]. This "restoring factor" has now been purified from a high-salt wash of rabbit reticulocyte ribosomes by taking advantage of its tight association with factor eIF-3 at low salt concentrations. It did not copurify with the major Mr 80,000 polypeptide of eIF-4B preparations but did copurify with a Mr 24,000 polypeptide previously shown to bind to the cap structures of mRNAs [Sonenberg, N., Rupprecht, K. M., Hecht, S. M. & Shatkin, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 4345--4349]. Both the electrophoretic mobility and the tryptic peptide pattern of the restoring factor were indistinguishable from those of the cap-binding protein, and the restoring factor could be crosslinked to the 5'-terminal cap on mRNA. Thus, is appears that poliovirus inhibits cellular protein synthesis by inactivation of some crucial property of the cap-binding protein.

Carrier Proteins↗
PubMed1979

Initiation of mammalian protein synthesis. The multiple functions of the initiation factor eIF-3.

The protein synthesis initiation factor eIF-3 (a multicomponent protein complex) was labelled with 32P by phosphorylation with a protein kinase present in a partially purified 'hemin-controlled repressor' preparation. The interaction of the labelled factor with the 40 S ribosomal subunit during the course of initiation was followed. It binds to the 40 S subunit in the absence of other initiation factors and inhibits the Mg2+-dependent reassociation of the 40 S with the 60 S ribosomal subunit. It stimulates the binding of the ternary complex (eIF-2, GTP, Met-tRNAf) to the 40 S subunit, and earlier work (Trachsel, H., Schreier, M.H., Erni, B. and Staehelin, T. (1977) J. Mol. Biol. 116, 745-767) also showed it to be essential for the subsequent binding of mRNA. The factor is released from the 40 S initiation complex during the 60 S subunit joining reaction.

Animals↗
PubMed1979

Characterization of rabbit reticulocyte factor(s) that stimulates the translation of mRNAs lacking 5'-terminal 7-methylguanosine.

Purified reticulocyte initiation factors were assayed for their ability to stimulate the translation of uncapped vesicular stomatitis virus (VSV) mRNA relative to capped VSV mRNA in reticulocyte lysates. Both eIF-3 and eIF-4B preparations contained such an activity. However, at least some of the activity was due to the presence of an as yet unidentified factor that contaminated the eIF-3 and eIF-4B preparations. A polypeptide of apparent molecular weight 24,000 that can be specifically cross-linked to the oxidized 5'-terminal cap structures of reovirus and other viral mRNAs co-purifies with this new factor, and may be a component of it. This stimulatory activity present in our eIF-4B preparations was found to be greater when lower concentrations of KCl were present in the reaction mixture, possibly explaining why uncapped VSV mRNA is translated relatively more efficiently at low K+ concentrations.

Animals↗
PubMed1979

Purification of seven protein synthesis initiation factors from Krebs II ascites cells.

Seven protein synthesis initiation factors were isolated from Krebs II ascites cells using the procedures developed for the purification of the corresponding factors from rabbit reticulocytes. The ascites factors display identical characteristics in ion exchange chromatography and sucrose density gradient sedimentation. Based on their profiles in SDS polyacrylamide gels, the ascites factors have polypeptide profiles and molecular weights identical to those of the reticulocyte factors. Most significantly, each ascites factor is competent in replacing its corresponding reticulocyte factor in a reconstituted in vitro protein synthesizing system which is dependent on all seven factors.

Animals↗
PubMed1979

Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.

Animals↗
PubMed1979

A new purification scheme for elongation factor 1 from rabbit reticulocytes and investigation of the homology of the subunits with those of initiation factor 2.

The aim of this work was to compare the subunits of the elongation factor EF-1 and the initiation factor eIF-2 from rabbit reticulocytes. We devised a simple procedure for the purification of EF-1: stepwise chromatography on heparin-Sepharose, separation of the heavy form by sucrose gradient centrifugation, and a final step of stepwise chromatography on RNA-Sepharose. The heparin-Sepharose column also clearly separated EF-1 and EF-2 within one chromatographic step. The EF-1 was 350-fold puried and the yield was 10%. This preparation showed after electrophoresis on polyacylamide gels in the presence of sodium dodecyl sulfate three bands corresponding to those described by others as the subunits, with Mr of 54000, 49000 and 29200. An additional band of Mr 34000 was present but no others. The 49000-Mr and 34000-Mr bands corresponded exactly in molecular weight to two of three subunits of eIF-2. A more detailed comparison was therefore made of all subunits of EF-1 and eIF-2. This was done by examination of chymotryptic fingerprints on polyacrylamide gel electrophoresis. No evidence for homology between EF-1 and eIF-2 was found. However, the two larger subunits of eIF-2 had a majority of chymotryptic fragments in common, thus indicating some homology between these polypeptides.

Animals↗
PubMed1979

[Delayed hypersensitivity against staphylococcal antigens in guinea pigs (author's transl)].

Delayed type hypersensitivity against staphylococcal antigens could be induced in guinea pigs by injecting the animals with a staphylococcal homogenate in Freund's adjuvant in all four foot pads and the nuchal skin. Maximal skin reactivity, tested intracutaneously, was observed 21 days after sensitization. Highest stimulation of lymph node cells and peripheral blood lymphocytes was also obtained 21 days after sensitization. When comparing cell walls, cell materials (nonsoluble material obtained after the separation of the cell wall fraction) and soluble fraction (obtained after sedimentation of all nonsoluble material, the strongest skin test reactions occurred after testing intracutaneously with the cell wall fraction.

Animals↗
PubMed1978

Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation.

The eukaryotic initiation factor eIF-2 forms a ternary complex with Met-tRNAf and GTP. This complex binds to the 40S ribosomal subunit in the absence of mRNA and mRNA binding factors. Highly purified eIF-2 from rabbit reticulocytes was labeled with 125I by using the Bolton-Hunter reagent or with [gamma-32P]ATP by using the heme-regulated translational inhibitor protein kinase. The labeled eIF-2 was bound, together with equimolar amounts of Met-tRNAf and GTP, to the 40S subunit. In the presence of mRNA, mRNA binding factors, and 60S ribosomal subunits (complete initiation assay), eIF-2 was released from the 40S initiation complex in the subunit joining reaction. GTP also was released in this step and probably was hydrolyzed in the reaction that is dependent upon eIF-5 and the 60S subunit. The function of phosphorylated eIF-2 in initiation of protein synthesis is discussed.

Animals↗
PubMed1978

Inhibition of translation by poliovirus: inactivation of a specific initiation factor.

Translation of vesicular stomatitis virus (VSV) mRNA, like host mRNA translation, is inhibited in cells infected with poliovirus. To study the mechanism of poliovirus-induced inhibition of protein synthesis, we prepared extracts from poliovirus-infected and uninfected HeLa cells. Poliovirus mRNA was translated in lysates from both infected and uninfected cells, while VSV mRNA was translated only in the lysate from uninfected cells. Addition of purified translation initiation factors to the extract from infected cells showed that one factor, eIF-4B, could restore VSV mRNA translation in the infected lysate, but did not increase poliovirus mRNA translation. Further experiments involving translation of VSV mRNA in mixed extracts from poliovirus-infected and uninfected cells showed (i) that there was not an excess of an inhibitor of VSV mRNA translation in the infected lysate, but (ii) that an acitivity that caused a slow inactivation of eIF-4B was present in the infected lysate. Inactivation of eIF-4B appears to be the mechanism by which poliovirus infection causes a selective inhibition of translation.

Cell-Free System↗
PubMed1978

Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.

To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.

Adenosine Triphosphate↗
PubMed1977

Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis.

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.

Adenosine Triphosphate↗