Sequencing the erbA gene of avian erythroblastosis virus reveals a new type of oncogene.

Avian erythroblastosis virus (AEV) contains two distinct oncogenes, erbA and erbB . The erbB oncogene, which is homologous to a portion of the epidermal growth factor receptor, is related to the src family of oncogenes and efficiently transforms erythroblasts, whereas erbA potentiates the effects of erbB by blocking the differentiation of erythroblasts at an immature stage. This "potentiator" was sequenced; the amino acid sequence deduced from it was clearly different from the sequences of other known oncogene products and was related to carbonic anhydrases. These enzymes participate in the transport of carbon dioxide by erythrocytes, the precursors of which are main targets of avian erythroblastosis virus. A src-related oncogene such as erbB in synergy with an activated specific cell-derived gene such as erbA can profoundly affect early erythroid differentiation.

Differential reduction of the inter-chain disulfide bonds of rat immunoglobulin E: relation to biological activity.

Monoclonal rat IgE was reduced over a range of dithiothreitol (DTT) concns. The number of disulfide bonds reduced and their location in the IgE molecule were studied. One millimolar DTT was found to split the two inter-heavy-chain disulfide bonds of the C epsilon 2 domain while increasing DTT concn to 10 mM split the two inter-heavy-light-chain disulfide bridges. Therefore, the sensitivities to reduction of disulfide bonds in rat IgE were found to be the opposite of those in human IgE. In addition, the results indicated the absence, in rat IgE, of the intra-epsilon-chain labile disulfide bond of the C epsilon 1 domain, which is reduced by 2 mM DTT in human IgE. Circular dichroism studies showed significant modifications, mainly of tertiary structure, for rat IgE reduced with 10 mM DTT, but not for IgE reduced with 1 mM DTT. The ability to block passive sensitization with reaginic antibody was not modified when IgE was reduced with 1 mM DTT (which split the two inter-heavy-chain disulfide bonds), but was lost when inter-heavy-light-chain bridges were reduced with 10 mM DTT. In addition, a non-covalent epsilon-chain dimer was found to have the same blocking activity as native IgE (or IgE reduced with 1 mM DTT). Therefore, the results suggest that reduction of most or all the inter-chain disulfide bonds, in rat as in human IgE, induces changes in quaternary structure, more especially in the relationship between the Fab and Fc parts of the molecule, leading to steric blockade, by Fab, of the binding sites for mast cells present on Fc.

[Characterization of L-threonine deaminase activity of guinea pig liver induced by a high protein diet]. / Caractérisation de l'activité L-thréonine désaminasique du foie de cobaye induite par un régime hyperprotidique.

In a foregoing paper, we demonstrated that under equilibrated diet conditions, guinea pig liver L-threonine deaminase activity should be allocated to two distinct enzymes: a specific L-threonine deaminase without activity toward L-serine and a L-serine deaminase having a secondary activity toward L-threonine. In the present work, we observed that a high protidic diet caused an elevation of total threonine deaminase activity. Thus purification of guinea pig liver L-threonine deaminase was attempted, using ultracentrifugation, salt precipitation, heat treatment, ion exchange chromatography on DEAE Sephacel, Sephadex G 200 molecular sieve, 2 amino-2 methyl-1 propanol linked CH 4B Sepharose chromatography. The weak variations of the ratios of specific activities respectively toward L-threonine and L-serine observed at each stage of the purification procedure indicated that both activities are very likely supported by a single enzyme preexisting in the liver of guinea pigs fed an equilibrated diet. No isoenzyme was evidenced by polyacrylamide gel electrophoresis or DEAE Sephacel chromatography. Moreover, our purification procedure demonstrated that not only inducible L-threonine deaminase guinea pig liver activity was due to L-serine deaminase, but also that an initially existing specific L-threonine deaminase activity paradoxically disappeared with a protein rich diet.

Proteolysis of rat IgG subclasses by Staphylococcus aureus V8 proteinase.

Monoclonal IgG belonging to the four rat IgG subclasses (IgG1, IgG2a, IgG2b, IgG2c) and some IgG subclasses from normal rat serum were subjected to enzymatic degradation with Staphylococcus aureus V8 proteinase. The results show that only one subclass, IgG2b, is significantly cleaved by the enzyme, with the release of two main products identified as F(ab)2 and Fc-like fragments. This unique susceptibility of the IgG2b subclass represents therefore an easy means of identification and also offers a simple procedure for a preparation of F(ab)2 fragments from monoclonal IgG2b antibodies.

Formation of biologically inactive polymers is responsible for the thermal inactivation of rat IgE.

The structural changes induced by heating rat IgE at 56 degrees C and relationship with loss of cytotropic activity were examinated in the present study. Circular dichroism spectrum of IgE heated at 56 degrees C showed irreversible changes in the peptide bond spectral regions: increase in beta-sheet structure, but no significant modifications in the aromatic side chain region. Thus, circular dichroism studies did not suggest important perturbations of the tertiary structure of the IgE molecule. Parallel studies with F(ab')2-epsilon fragment did not show significant alterations of either peptide bond or aromatic side chain spectral regions. Analysis of IgE heated at 56 degrees C by polyacrylamide gradient gel electrophoresis showed the presence of large amounts of polymeric material. Polymerization of IgE was found to increase with time of heating at 56 degrees C and to depend on protein concentration; polymerization was decreased at temperatures lower than 56 degrees C. A relationship between loss of cytotropic activity and the proportion of polymeric material in the heated IgE solutions was observed. Isolated polymeric molecules produced by heating showed considerable decrease in cytotropic activity whereas monomer isolated from heated IgE was found biologically active. The ability to form polymers is an intrinsic property of the carboxy-terminal domains C epsilon 3 and C epsilon 4, as the F(ab')2-epsilon fragment did not polymerize upon heating at 56 degrees C. A model of thermal inactivation of rat IgE is proposed in which aggregation of the carboxy-terminal domains of the epsilon-chain does not allow interaction of these domains with the monovalent IgE receptor of mast cells.

Phylogenetic studies of cardiac myosins from amphibia to mammals.

Comparison between pig atrial and ventricular myosins was performed on the light chains (using SDS-PAGE) and on the heavy chains (using Ca2+-ATPase measurements and NTCBA peptide mapping). Light chain composition of pig cardiac myosins was compared to three other species ones (frog, chicken and human). Up to birds, atrial and ventricular myosin light chain composition was identical whereas in mammals atrial and ventricular myosin light chain composition was different; likewise the heavy chains. Six cardiac myosin isoenzymes have been thus characterized. No correlation can be established between cardiac myosin light chain pattern and species evolution.

63Ni[II]-incorporation into lung and liver cytosol of Balb/C mouse. An in vitro and in vivo study.

The kinetic studies of 63Ni[II]-incorporation in whole tissue show that after 6 days lung has the highest affinity to nickel of all studied organs. The same observation was made when 63Ni[II]-incorporation was carried out by 7 daily injections. In both experiments, kidneys take the second place in the relative distribution of nickel. For the studies of Ni-binding proteins in lung and liver cytosols, three different types of 63Ni[II]-incorporation were performed: (i) in vitro incorporation, (ii) kinetic study of in vivo incorporation where the animals were sacrificed after 30 min, 1, 2 and 4 h after a single i.p. injection of 63NiCl2, (iii) in vitro incorporation by 7 daily i.p. injections of 63NiCl2 with sacrifice of the animals 24 h after the last injection. Several Ni-binding proteins could be observed without regard to the type of incorporation performed. In liver cytosol most of these proteins can be revealed in vitro as well as in vivo. The in vivo labelled proteins are not the same at the beginning of the incorporation (30 min) and after an elapsed period (1 to 2 h). In lung cytosol in vitro and in vivo incorporation gave different results: although an intense labelling occurs after in vitro incubation, only few proteins are labelled after in vivo incorporation, two of which are only revealed after continuous exposure to 63Ni[II]. Most of the labelled proteins of lung and liver cytosols can be recovered after different fractionation experiments. These investigations confirm that nickel is preferentially bound to lung and demonstrate that nickel-incorporation is different in lung and in liver cytosols. It is shown here that several cellular proteins are implicated in the nickel-transport. The evolution of this phenomenon suggests the existence of a nickel-metabolism in the cell.

The amino-acid sequence of histone H2A from cuttlefish Sepia officinalis.

The amino acid sequence of cuttlefish testis histone H2A (124 residues) was established from structural data obtained by automated sequencing of large peptides generated by the cleavage of the protein with V8 staphylococcal protease or by limited chymotryptic hydrolysis. Compared to the calf thymus homologous histone, cuttlefish H2A shows 14 substitutions (most of them conservative) and 5 deletions. Extensive evolutionary changes were mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few punctual changes are observed in the central region (residues 18-118), which interacts strongly with histone H2B to form the dimer H2A-H2B.

UV differential study of the histones H2A-H2B-H3-H4 octamer.

The individual calf thymus histones H2A, H2B, H3 and H4, the dimer H2A-H2B, the tetramer (H3-H4)2 and the octamer (H2A-H2B-H3-H4)2 were studied by differential UV absorption i.e. observing absorption shifts of tyrosyl residues due to thermal perturbations. The histone octamer was studied in 2 M NaCl pH 7.5 a condition under which it is stable, as demonstrated by Eickbush and Moudrianakis [18]. In addition these authors suggested that the interactions which maintain the four histones as an octamer involve the weak association of one (H3-H4)2 tetramer with two H2A-H2B dimers and might be due essentially to histidine-lysine or histidine-tyrosine hydrogen bonds. We performed the study of the octamer by UV differential absorption using the tyrosyl residues as a natural probe to follow their interaction with different residues in their neighbourhood. The main result obtained shows that the tetramer (H3-H4)2 has all its tyrosyl residues exposed to the solvent whereas the octamer has no tyrosine exposed, suggesting that with this polymer no DNA-tyrosine interactions could take place.

Tryptic cleavage of rat IgG: a comparative study between subclasses.

Monoclonal rat IgG belonging to the 4 rat IgG subclasses, and some IgG subclasses isolated from normal rat serum were subjected to enzymatic degradation with trypsin. Differences in the products of tryptic digestion were observed according to the IgG subclass. IgG2b and IgG2c were degraded mainly into Fab and Fc fragments. IgG1 and IgG2a appeared resistant to such cleavage. However, tryptic digestion of the latter two produced two fragments separated only in dissociating media. Results of the studies suggest that one of the fragments (mol. wt. 13,000) probably consists of most of the variable domain of the gamma heavy chain, while the second (mol. wt. 120,000) consists of the IgG deleted of the VH regions.

Flow cytofluorometric analysis of the effects of new nitrosourea derivatives on proliferation of EMT 6 tumor cells "in vitro".

Examined by flow cytofluorometric analysis, the DNA distribution of EMT 6 tumor cells was highly perturbed after one hour of in vitro incubation with: RPCNU, RFCNU, chlorozotocin (CZT) or 185 (CNCC), four new nitrosourea derivatives. After the treatment with chlorozotocin (20 micrograms/ml) and CNCC (50 micrograms/ml), most of cells were in G2 + M phase and this accumulation lasted more than 48 hours without any restoration before 72 hours. RPCNU (20 micrograms/ml) and RFCNU (50 and 65 micrograms/ml) induced and accumulation of cells in G2 + M phase during 24 hours. The normal state was regained after 48 hours. These reduced rate of progression of the cells through S phase and the G2 block observed after exposure to the new compounds, should, in part, explain their antitumoral activity.

Study of the 10-nm-filament fraction isolated during the standard microtubule preparation.

The cold non-depolymerizable fractions obtained during the standard procedure for the isolation of microtubules from ox brain stem-cerebral hemispheres and spinal cord have been studied. The cerebral-hemisphere preparation was composed of 10-nm filaments but also contained large amounts of membranes. The polypeptide content included tubulin, microtubule-associated proteins and minor proteins corresponding to the neurofilament triplet of proteins of mol.wt. 210 000, 160 000 and 70 000 respectively. The brain-stem preparation contained more 10-nm filaments than membranes. The polypeptide content consisted of the neurofilament triplet (35%), tubulin (30%) and minor proteins. In contrast, the spinal-cord preparation was mainly composed of 10-nm filaments, free of membranes and containing essentially the neurofilament protein triplet (64%). These filaments appeared very similar to the peripheral-nervous-system neurofilaments described by several authors. Since the best neurofilament from the central nervous system often contained less than 15% of the neurofilament protein triplet, our spinal-cord preparation is an improvement on the usual neurofilament preparation. This simple and rapid method gave large amounts of 10-nm filaments (100 mg per 100 g of spinal cord) characterized by the absence of membranous material, a low content of tubulin and the 50 000-mol.wt.-protein component, and a high content of neurofilament peptides. Thus, the presence of tubulin in 10-nm filament preparations seems to be related to the contaminant membranous material and not to be linked to the interaction in vitro of tubulin or microtubules with neurofilaments, as has been suggested previously.