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G Buttin

Publications and source records attributed to G Buttin.

At least 55 records · Page 3Linked to original sources
PubMed1984

Adenosine-resistant Chinese hamster fibroblast variants with hyperactive adenosine-deaminase: an analysis of the protection against exogenous adenosine afforded by increased activity of the deamination pathway.

The activity of purine salvage and interconversion enzymes was examined in two sublines of Chinese hamster cells--RA11 and RA41--isolated on the basis of their resistance to adenosine concentrations toxic to wild-type CCL39 cells. Adenosine deaminase (ADA) activity was found to be two times higher in RA11 and three times higher in RA41 than in CCL39. Inhibition of ADA activity by coformycin reduced the level of adenosine resistance but did not restore wild-type sensitivity, indicating that a second defect contributes to the adenosine-resistant phenotype of these variants; evidence was indeed obtained for the presence in both lines of additional alterations protecting them against the lethal depletion of phosphoribosylpyrophosphate (Ishii and Green, 1973) imposed by adenosine to wild-type cells. To gain better insight into the influence of ADA hyperactivity on adenosine resistance, a procedure was developed for the specific isolation of variants with increased levels of ADA activity. Cell lines with 3-5 times and then 100-500 times the wild-type ADA activity were stepwise recovered. These investigations confirmed that amplification of ADA can efficiently contribute in protecting cells against high concentrations of exogenous adenosine. The variants isolated by this procedure again manifested, in addition to amplification of ADA activity, another alteration decreasing their sensitivity to adenosine. A possible mechanism accounting for the frequent isolation of variants that coexpress ADA hyperactivity and a second defect contributing protection against adenosine toxicity are considered.

Adenosine↗
PubMed1984

Coexpression of two thermosensitive defects in a Chinese hamster cell line. Manifestation of a G1 block associated with a mitosis defect.

Asynchronous cultures of ts12, an anchorage-dependent derivative of the thermosensitive Chinese hamster cell line ts111, show a rapid drop in [3H]thymidine incorporation with accumulation of the cells in the G1 and in the G2 phases of the cycle, when shifted from 34.5 to 39.4 degrees C. Shift-up experiments carried out after either isoleucine deprivation or synchronization at 39.4 degrees C, locate the execution point of a ts function in late G1 (2.5-3 h before S). However, stimulation of proliferation of a high density-arrested population allows a fraction of the cells to enter S. In addition to the G1 ts defect, ts12 expresses a slight cytokinesis defect at 39.4 degrees C (8-15% binucleate cells). The results suggest that altered processes are taking place at a post-metaphasic stage during the first hours after the shift-up. When populations are synchronized by a thymidine block and released at 39.4 degrees C, multinucleate cells in addition to binucleate cells are observed. Part of these multinucleate cells result from abnormal karyokinesis without inhibition of cytokinesis. Evidence is presented suggesting that excess thymidine allows the re-expression of the multinucleation phenotype of ts111.

Animals↗
PubMed1984

Monoclonal antibody directed against the 43 000 dalton v1 polypeptide from Torpedo marmorata electric organ.

The acetylcholine receptor (AcChoR) plays an essential role at the vertebrate neuromuscular junction and in the fish electric organ: it mediates the opening of the post-synaptic ion channel by the neurotransmitter acetylcholine (AcCho). The supra-molecular organization of the AcChoR within the subsynaptic membrane is highly characteristic. In the course of the maturation of the cholinergic synapse during embryogenesis, the AcChoR molecules change from being diffusely distributed and metabolically labile to densely-packed and stable aggregates. A 43 000 dalton protein which is associated with the Torpedo electrocyte synapses and is present in membrane highly enriched in AcChoR (SDS-PAGE) has been postulated to be involved in the immobilization and stabilization of the AcChoR in the subsynaptic membrane. However, when analysed on two-dimensional isoelectric focusing-SDS-PAGE gels, the 43 000 dalton protein band was shown to be composed of three distinct spots referred to as v1, v2 and v3. The three polypeptides had different peptide maps. Furthermore, the subcellular localisation of the 43 000 dalton protein is not clearly established and contradictory conclusions, based on indirect evidence, have been drawn. Thus, a further investigation of the 43 000 dalton proteins appeared necessary. Monoclonal antibodies against the 43 000 dalton proteins were developed in CBA mice and characterized using the horseradish peroxidase immunoreaction as a screening assay. One of the monoclonal antibodies was directed exclusively against the v1 polypeptide. It reacted only with membrane fractions containing the v1 polypeptide and did not label the cytoplasmic fraction prepared from adult Torpedo electric organ.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Lack of adenosine deaminase activity in cultured murine cytotoxic T lymphocytes.

The level of adenosine deaminase (ADA) activity was investigated in various populations of IL 2-dependent, cultured cytotoxic T lymphocytes (CTL), from bulk cultures as well as from CTL lines (CTL-A and CTL-B types). The study of C57BL/6 derived, cytotoxic bulk cultures yielded the following mean values of ADA activity: 12,500 U/mg in the cortical, immature region of the thymus, 1500 U/mg in the immunocompetent, cortisone-resistant medullary thymocytes, and 2000 U/mg in the T cell population from the spleen. These results are in agreement with previous studies on separated T lymphocyte populations of known origin and further indicate that a fall in ADA activity accompanies T cell maturation. ADA activity was measured in C57BL/6-derived CTL-A lines obtained from the thymic and splenic bulk cultures. All lines were characterized by a very low level of ADA activity, compared with the T cell bulk cultures freshly initiated from the thymic medulla or from the spleen, and to a variety of T tumor lines established in long term culture. Some showed undetectable ADA activity (less than or equal to 20 units/mg), whereas others maintained significant activity (50 to 500 U/mg). No correlation was found between the residual ADA activity level and the killing activity, at the time of the enzyme assay. Identical properties were observed for CTL-B cloned lines of various genetic backgrounds. These results suggest that the level of ADA activity of the CTL in the mouse is lower than the average value of mature T cells of the thymic medulla, and might constitute a differentiation marker specific to the CTL population. A possibility remains that low ADA activity levels in these CTL lines may be the consequence of an extinction of the ADA gene during in vitro growth, as it is observed for the cytotoxic activity itself. In either case, a low ADA activity level is a remarkable property of IL 2-dependent CTL clones, when compared to various established T tumor lines, which exhibit high and stable ADA levels during long term in vitro growth (5000 to 15,000 U/mg).

Adenosine Deaminase↗
PubMed1983

ABPC 48 cross-reactive idiotopes in BALB/c mice. Natural and levan-induced expression.

Using monoclonal antiidiotypic antibodies, we developed a sensitive binding assay that detects molecules with one or with two idiotopes of the ABPC48 idiotype. ABPC48 cross-reactive idiotypes were thus shown to be present in substantial amounts in sera of nonimmunized mice. Levan binding sites are found on these idiotypes. During the life time of the mice, the natural anti-levan titer increases while ABPC48 idiotypic expression remains constant, suggesting different controls for these two activities. On the other hand, ABPC48 cross-reactive idiotypes participate--as minor components--in the response that follows a deliberate immunization with bacterial levan. This induction process is likely to reflect the selection of idiotopes expressed by the B cell clones preactivated in sera of nonimmunized mice rather than the activation of silent clones. We suggest that a similar situation might explain the reported emergence of ABPC48 idiotypes in animals primed with antiidiotypic antibodies and subsequently stimulated with levan.

Animals↗
PubMed1983

Increased inhibition of HGPRT by IMP and GMP and higher levels of PRPP in an 8-azaguanine - hat resistant mutant of Chinese hamster cells.

In this paper an 8-azaguanine resistant mutant of Chinese hamster cells able to grow in HAT medium is analyzed. Kinetic analysis of hypoxanthine-guanine-phosphoribosyl-transferase show that the enzyme of the mutant has an altered affinity for both phosphoribosyl-pyrophosphate and the inhibitors IMP and GMP. Furthermore we show that mutant cells exposed to inhibitors of de novo purine synthesis have increased levels of phosphoribosyl-pyrophosphate probably due to decreased utilization by the de novo pathway. These results explain the ability of the mutant to grow both in 8-azaguanine and HAT medium.

Animals↗
PubMed1983

Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ.

Subsynaptic membrane fragments prepared from Torpedo marmorata electric organ contain, in addition to the acetylcholine receptor polypeptides, a major protein band of apparent molecular mass 43,000 daltons. On two-dimensional gels, this band yields three spots referred to as v1, v2, and v3. Monoclonal antibodies against the 43,000-dalton proteins were developed in CBA mice. One of them reacted exclusively with the v1 polypeptide but not with v2 and v3. Staining by the "immunogold" reaction followed by observation by electron microscopy showed that this antibody exclusively labeled the innervated membrane of T. marmorata electroplaque on its cytoplasmic face. Electroblots of one-dimensional gels of membrane preparations from 80-mm embryo electric organ were prepared. After reaction with the anti-v1 monoclonal antibody, a strongly stained 43,000-dalton band was revealed.

Animals↗
PubMed1983

Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.

Animals↗
PubMed1983

[Production of anti-L-histidine decarboxylase monoclonal antibodies].

Hybridomas secreting monoclonal antibodies directed against Rat gastric L-histidine decarboxylase (H.D.) have been obtained from a partially purified preparation and screened by immunoprecipitation of the enzyme activity. They cross react with Rat brain H.D., indicating at least that these two enzymes share common antigenic determinants.

Animals↗
PubMed1982

Probing yeast RNA polymerase A subunits with monospecific antibodies.

Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.

Animals↗
PubMed1982

Specificity and idiotypic analysis of monoclonal antibodies directed against the MOPC460 idiotype.

Eight syngeneic anti-idiotypic hybridomas (IDMs) have been obtained against the BALB/c myeloma protein MOPC460 which displays anti-TNP activity. The study of their anti-idiotype specificity allowed us to distinguish them into two groups which define the presence of at least two idiotypic determinants or idiotopes in the MOPC460 idiotype. The biochemical analysis of the monoclonal antibodies is consistent with this dichotomy. This analysis, in fact, showed a striking correlation between anti-idiotypic specificity and biochemical characteristics of the monoclonal antibodies. Consequently, the idiotypic specificities of three of these hybridomas were studied. In accordance with what is expected, our results clearly indicate a strong idiotypic similarity for hybridomas belonging to the same group and a lack of idiotypic cross-reactivity.

Animals↗
PubMed1982

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

AMP Deaminase↗
PubMed1981

Switched allotype expression in an immunoglobulin-nonsecreting rabbit lymphoid cell line fused with rabbit gangliocytes.

The Simian virus 40-transformed rabbit spleen cell TRSC-1 synthesizes intracellular whole IgG molecules of the alb4 allotype. Two hypoxanthine-guanine phosphoryl transferase-deficient mutants were derived from this line. One of these, TRSC-1-8, was used in somatic cell fusion experiments together with gangliocytes from a rabbit immunized against beta-galactosidase. Out of nineteen hybrid clones surviving in selective medium, only one, L17, was shown to produce free gamma chains which express the a2 allotype of the donor rabbit rather than the al marker of the parents TRSC-1-8 line. The inability to restore IgG secretion in hybrids suggests that dominant regulatory controls are exerted by the TRSC-1 genome on Ig reduction. This supports the notion that the TRSC-1 line originated from a splenocyte that had not reached the final plasmocyte differentiation stage at the time of viral transformation.

Animals↗
PubMed1981

Idiotype-anti-idiotype interactions and the control of the anti-beta (2 leads to 6) polyfructosan response in the mouse: specificity and idiotypy of anti-ABPC48 anti-idiotypic monoclonal antibodies.

Seventeen hybridomas, secreting monoclonal anti-idiotypic antibodies (IDA) directed against the BALB/c ABPC48 idiotype, were isolated from one immunized BALB/c mouse. Several IDA also bind another Balb/c idiotype: UPC10. ABPC48 and UPC10 are both myeloma proteins with a beta (2 leads to 6)-polyfructosan (levan) specificity. The binding of every IDA to the ABPC48 idiotype can be completely inhibited by levan molecules, but at different concentrations. Mutual inhibition assays between the IDA made it possible to define six groups of IDA which bind at least three different idiotopes of ABPC48. Sixteen IDA have been studied by means of mouse anti-anti-idiotypic antibodies (Ab3) directed against two of them, IDA3 and IDA10. Anti-IDA3 Ab3 recognize idiotopes particular to IDA3 which are not found on other monoclonal anti-idiotypic antibodies (Ab2). Anti-IDA10 AB3 cross-reacts with several monoclonal Ab2, including Ab2 with different spectrotypes belonging or not to the same isotype and Ab2 with different specificities for the ABPC48 idiotype. Some IDA10 idiotopes are present in the polyclonal anti-ABPC48 antibody response of BALB/c, A/J and CBA mice showing that they are recurrent and that their expression is not linked to a particular Igh-C haplotype. In contrast, IDA3 idiotopes are not detected in the same anti-ABPC48 antisera.

Animals↗
PubMed1981

The potentiation of adenine toxicity to Chinese hamster cells by coformycin: suppression in mutants with altered regulation of purine biosynthesis or increased adenylate-deaminase activity.

When added to medium containing coformycin (2 microM or above), adenine is lethal to Chinese hamster fibroblasts at the concentration inhibiting de novo purine biosynthesis (Debatisse and Buttin, '77b). Rescue by hypoxanthine suggested that cells die of IMP starvation when the analog can turn off deamination of both adenosine and adenylate. As predicted from this hypothesis, two classes of variants resistant to the mixture of coformycin + adenine have been isolated: Class 1 variants have altered control of de novo IMP biosynthesis; they fall into two subclasses on the basis of their resistance to adenosine. Class 2 variants have a 6-10-fold increased level of AMP-deaminase (E.C.: 3.5.4.6); their growth in the selective medium is temperature-dependent, a property accounted for by the observation that cell growth in the presence of coformycin imposes a gradual thermodependent decay of specific AMP-deaminase activity in both wild-type and variant lines. This control by coformycin of AMP-deaminase activity is unaltered in mutants deficient in the four activities of adenosine-kinase. APRT, HGPRT and deoxycytidine-kinase. Most of the resistant variants are unstable and exhibit either increased or reduced resistance, depending on prolonged growth in selective or normal medium.

AMP Deaminase↗
PubMed1981

Characterization of ribonucleotide reductase induction in BHK-21/C13 Syrian hamster cell line upon infection by herpes simplex virus (HSV).

Ribonucleotide reductase is an essential enzyme in mammalian DNA replication. In quiescent BHK-21/C13 cells exhibiting a low level of ribonucleotide reductase activity, infection with herpes simplex virus (HSV) resulted in the early induction of an altered ribonucleotide reductase. The extent of the induction was dependent upon the m.o.i. and could be diminished or prevented by u.v. treatment of the viral stock, or by inhibitors of mRNA synthesis or protein synthesis. The induction followed the same course of synthesis as viral thymidine kinase and DNA polymerase, and could thus be classified with them as a beta polypeptide. These results suggested that the new activity was produced as a consequence of the virus genome expression. Comparisons of the properties of ribonucleotide reductase extracted from exponentially growing BHK-21/C13 cells showed that the HSV-induced enzyme differed from the cellular isozyme by its insensitivity to inhibition by dTTP, dATP or araATP and its resistance to high salt concentrations. On the other hand, the virus-induced enzyme and the cellular isozyme exhibited a similar sensitivity to hydroxyurea. Therefore, the reported inhibition of HSV DNA replication by hydroxyurea could be the result of inhibition of both HSV-induced and cellular reductase activities.

Ammonium Sulfate↗