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Biomedical subjects

H Amesz

Publications and source records attributed to H Amesz.

9 recordsLinked to original sources
PubMed1987

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2.

The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzyme-linked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A--Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondrial phospholipase A2) antibodies.

Animals↗
PubMed1985

Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane.

Six monoclonal antibodies directed against the trimeric form of outer-membrane pore protein PhoE of Escherichia coli K-12 were isolated and characterized. All six antibodies bind to PhoE protein in intact cells and as isolated trimers, but not to dodecyl-sulphate-denatured monomers. Cross-reaction with the related pore proteins OmpF protein and OmpC protein was not observed. A hybrid pore protein in which the amino-terminal 74 amino acids of PhoE protein have been replaced by the corresponding part of OmpF protein is able to bind the six monoclonal antibodies. Five of the antibodies bind to the PhoE proteins of thirteen different Enterobacteriaceae when expressed in E. coli K-12, whereas the other antibody recognizes PhoE protein from nine of these strains. Four monoclonal antibodies are able to block adsorption of the PhoE-protein-specific phage TC45 to its receptor on whole cells. None of the antibodies has any effect on the uptake rate of the antibiotic cefsulodin through PhoE protein pores. Five antibodies are able to direct the complement-mediated killing of PhoE-protein-carrying cells. It is concluded that the six monoclonal antibodies recognize at least three distinct cell-surface-exposed epitopes whose specificity is determined by the carboxy-terminal 256 amino acids of PhoE protein.

Antibodies, Monoclonal↗
PubMed1979

A comparison of the initiation factors of eukaryotic protein synthesis from ribosomes and from the postribosomal supernatant.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernantant. A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.

Animals↗
PubMed1979

Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.

Animals↗
PubMed1979

Postribosomal complexes containing eukaryotic initiation factor eIF-2.

Eukaryotic initiation factors are found in the post-ribosomal subunits. We have analyzed the factor activities from the supernatant by means of zonal centrifugation followed by Sepharose-heparin affinity chromatography. They exist both as free factors, sedimenting in a broad range from 4 to 7S, and complexed with other protein(s) with a sedimentation value of 16-20S. This complexed fraction contains besides eIF-2 another activity which exhibits a profound stimulation on amino acid incorporation in crude lysates and appears to counteract the heme-regulated inhibitor.

Animals↗
PubMed1979

The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.

Animals↗
PubMed1977

Initiation of protein synthesis in eukaryotes. Binding to Sepharose-heparin and partial purification of initiation factors from Krebs II ascites cells.

By means of affinity chromatography of lysates from Krebs II ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained. The fraction is eluted from the column at 350 mM KCl using a linear gradient and displays a number of activities, i.e. binding of Met-tRNAfMet to form a ternary complex with GTP; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, MettRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns. Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors.

Animals↗
PubMedDate not supplied

Studies on a possible relationship between alterations in the cytoskeleton and induction of heat shock protein synthesis in mammalian cells.

Heat shock-induced alterations in protein synthesis and the cytoskeleton of two mammalian cell types have been investigated. A hyperthermic treatment of 30 min at 43 degrees C causes an accumulation of heat shock proteins (HSPs). The apparent molecular weights of HSPs of Reuber H35 hepatoma cells and of N2A neuroblastoma cells are 28 000, 65 000, 68 000, 70 000, 84 000, 100 000 D and 68 000, 70 000, 84 000 and 100 000 D respectively. Hyperthermia induces the disruption of microfilaments in hepatoma cells. Microtubules and intermediate filaments (vimentin and cytokeratin) remain intact. In neuroblastoma cells microfilaments remain intact whereas microtubules become disorganized after heat shock. As a result vimentin is found as a perinuclear aggregate. These cells were still able to synthesize heat shock proteins after pretreatment with cytoskeleton disrupting drugs such as dihydroxycytochalasin B and colchicine. Therefore it is concluded that the alterations in the cytoskeleton observed after the heat treatment are unlikely to be the cause of heat shock protein synthesis. Our results suggest that these heat shock-induced alterations in the cytoskeleton can be considered as a part of the heat shock response.

Animals↗