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J Thillet

Publications and source records attributed to J Thillet.

At least 55 records · Page 3Linked to original sources
PubMed1985

Oxidation and cross-linking of human hemoglobins by activated oxygen species.

The effect of activated oxygen species on human hemoglobins was studied. All radicals induced polymerization in Hb A both intermolecular and by cross-linking of subunits (intramolecular). However, a system producing mainly superoxide ion gave the most important changes. An oxidation step is necessary to produce polymerization since in the case of cyanmet Hb A (where there is no possible oxidation) no polymerization occurs. The effect of O2- on blocked SH beta 93 Hbs or on the abnormal Hbs tested was practically identical to that on Hb A although their autoxidation rates were modified. Consequently the action of radicals is different from autoxidation processes and the modified residues in the abnormal hemoglobins are not involved in the action of superoxide ion on Hb. The kinetics of oxidation of Hb by H2O2 followed two steps: the first is the oxidation of oxy Hb to ferri Hb and the second is hemichrome formation. This last step is independent of the presence of H2O2 since it is not inhibited by catalase. The kinetics of oxidation to ferri Hb were of second order and the rate constant was found to be 16 M-1 sec-1.

Cross-Linking Reagents↗
PubMed1984

Expression of the mouse dihydrofolate reductase cDNA in B. subtilis: a system to select mutant cDNAs coding for methotrexate resistant enzymes.

With the aim to obtain a cDNA coding for a mammalian methotrexate resistant dihydrofolate reductase (Dhfr) a plasmid ( pQS1 ) harboring the mouse wild type Dhfr cDNA was constructed and used to transform a methotrexate sensitive bacteria: B. subtilis. A plasmid, pQS4 , expressing large amount of Dhfr in both E. coli and B. subtilis was isolated through a two steps selection with two substrate analogues, trimethoprim followed by methotrexate. This new plasmid has a 54 bp duplication including the beta-lactamase promoter and a deletion of 564 bp removing the 5' end of the beta-lactamase coding region. These changes create a new -35 region TTGAAA and a potentially stronger binding site for both E. coli and B. subtilis 16S ribosomal RNA. pQS4 transformed B. subtilis were then grown in the presence of high level of methotrexate and resistant mutants isolated. One of them, pQS6 , which codes for an enzyme about 50 times more resistant to methotrexate than the wild type Dhfr was sequenced. It shows that a point mutation replaces the glutamine residue at position 35 by a proline.

Animals↗
PubMed1984

Transformation of teratocarcinoma stem cells and fibroblasts with various vectors containing the Eco.gpt gene as a selection marker.

Eco.gpt, which codes for xanthine guanine phosphoribosyltransferase (XGPRT), when placed under the control of SV40 early genes regulating sequences (pSV2gpt) selects transformed teratocarcinoma cells with a low efficiency. The SV40 promoter may not function efficiently in teratocarcinoma stem cells, as suggested by the fact that such cells do not support SV40 T antigen expression. We have tested whether one could change the efficiency of gpt as a dominant selective marker in transformation by several operations. (1) Deletion of 121 base pairs (bp) upstream the bacterial coding sequence gpt (pQS14) did not make any difference. (2) Replacement of the SV40 regulating sequences by the HSV tk regulating sequences (pQS15) resulted in ten times fewer transformants with PCC4 teratocarcinoma cells as well as with L cells. No XGPRT activity was detectable in cultures 48 h after transfection. (3) Reintroduction of the PvuII-HindIII SV40 fragment (which contains an enhancing sequence together with the origin of replication and the early promoter of the virus) into the pQS15 vector, either in 5' or 3' from tk-gpt composite gene (pQS20 and pQS22) allows selection of ten to twenty times more transfected PCC4 or L cells colonies and restores transient XGPRT activity upon transfection. Whatever the vector used, the transformation frequency of PCC4 teratocarcinoma cells remains ten times lower than that of L cells. It appears that the presence of the SV40 PvuII-HindIII fragment in the vector increases cell transformation even with PCC4 cells and that the low frequency obtained with pSV2pgt is likely not due to the use of the SV40 early promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Effects of anions on the ligand-linked subunit assembly of human hemoglobin: the mutual effects of Cl- and EDTA.

We have studied the mutual effects of chloride ion and EDTA on the dimer-tetramer assembly of human deoxyhemoglobin and oxyhemoglobin. It is found that these two anions have similar but interdependent effects. In low C1- (.01 M) increasing concentrations of EDTA are found to decrease both forward and reverse rate constants for deoxyhemoglobin, whereas no effect is observed at 0.1 M C1-. These results suggest that binding of anions at the alpha 1 beta 2 intersubunit contact may stabilize both the dimeric and tetrameric forms of the deoxy molecule, thus inhibiting both the dissociation and reassociation reactions. The overall effects of EDTA and low C1- on the dimer-tetramer equilibrium constants are found to be distinctly different in deoxy and oxyhemoglobins with a major effect on the oxy form. These findings establish validity of the results from previous thermodynamic studies carried out in approximately physiological C1- concentrations along with the small amounts of EDTA which are used to minimize artifacts of oxidation. As observed for deoxyhemoglobin, it is found that in 0.1 M C1- ion there is no further effect of EDTA on the oxyhemoglobin dimer-tetramer equilibrium.

Adult↗
PubMed1982

Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.

The sites of energy transduction within the human hemoglobin molecule for the regulation of oxygen affinity have been determined by an extensive study of the molecule's energetic response to structural alteration at individual amino acid residues. For 22 mutant and chemically modified hemoglobins we have determined the total free energy used by the tetrameric molecule for alteration of oxygen affinity at the four binding steps. The results imply that the regulation of oxygen binding affinity is due to energy changes which are mostly localized at the alpha 1 beta 2 interface. They also indicate a high degree of "internal cooperativity" within this contact region--i.e., the structural perturbations at individual residue sites are energetically coupled. Cooperativity in ligand binding is thus a reflection of cooperativity at a deeper level--that of the protein-protein interactions within the alpha 1 beta 2 interfacial domain.

Allosteric Regulation↗
PubMed1982

Binding of 21 thiol reagents to human hemoglobin in solution and in intact cells.

The reactivity of the cysteine-beta 93 residue of human hemoglobin was investigated in order to define the optimal structure of potential antisickling agents. The properties of 21 thiol reagents were compared with regard to (a) their binding rate to hemoglobin in solution and within intact cells; (b) the modification of the oxygen dissociation curve of intact cells and (c) the effect on methemoglobin formation in solution or within intact cells. The results show the very different behaviors of these reagents.

Adult↗
PubMed1981

Functional studies of two new abnormal hemoglobins with their mutation located at intersubunit contacts: Hb Hotel Dieu beta99 (G1) Asp replaced by Gly and Hb Pitie Salpetriere beta34 (B16) Val replaced by Phe.

The functional properties of two new abnormal hemoglobins with high oxygen affinity were studied. Hb Hôtel Dieu beta99 (G1) Asp replaced by Gly is situated in the alpha1beta2 contact. Hb Pitié Salpétrière beta34 (B16) Val replaced by Phe is situated in the alpha1beta1 contact. Both hemoglobins exhibited similar functional properties with a 10-fold increased oxygen affinity, a decreased cooperativity, a decreased Bohr effect and a normal or slightly decreased 2,3-diphosphoglycerate effect. The structure-function relationship is discussed in the light of these results.

2,3-Diphosphoglycerate↗
PubMed1981

The structure of hemoglobin Creteil (beta 89 Ser replaced by Asn) is similar to that of abnormal human hemoglobins having sequence changes at Tyr 145 beta.

In normal deoxyhemoglobin A, the beta chain COOH-terminal peptide adopts a well ordered structure which is needed for the full expression of allosteric action. Our crystallographic studies of deoxyhemoglobin Creteil (beta 89 Ser replaced by Asn), a variant hemoglobin characterized by high oxygen affinity and a very low level of allosteric function, show that replacement of Ser 89 beta by asparagine causes severe disordering of the beta chain COOH-terminal tetrapeptide. This results, as shown by our spectroscopic studies, in the destabilization of the quaternary structure of deoxyhemoglobin Creteil. We find, furthermore, that the changes in tertiary structure observed in deoxyhemoglobin Creteil are common to other variant hemoglobins having similar functional abnormalities but very different changes in primary structure. In particular, direct comparison of the difference electron density map of deoxyhemoglobin Creteil with that of deoxyhemoglobin Nancy (beta 145 Tyr replaced by Asp) suggests that these two abnormal hemoglobins may have the same mechanism of dysfunction despite the very different nature of their respective sequence changes.

Amino Acid Sequence↗
PubMed1981

Hereditary pyruvate kinase abnormalities associated with erythrocytosis.

A familial erythrocytosis is described. The propositus is a 52-year-old man who was found, on routine testing, to have an erythrocytosis with a Hb level of 19.8 g/dl. Physical examination and laboratory findings revealed an associated hemolysis. The P50 of the stripped hemolysis was normal. The 2,3-DPG level was reduced to 15% of normal but that of ATP was increased more than twofold. Red cell diphosphoglycerate mutase activity was normal as were the levels of all red cell glycolytic and hexose monophosphate shunt enzymes with the exception of pyruvate kinase (PK). The level of the latter was elevated to 24.5 U/g (normal: 6.2 +/- 1.1). The electrophoretic pattern of hemolysate PK was abnormal, showing three additional bands, one of these migrated as the M2 isozyme. The findings were similar in the propositus's daughter and her daughter and in one of the two brothers of the propositus. The second brother and the son of the first brother was exhibited erythrocytosis but their red cell 2,3-DPG levels were normal. In addition, the level of their RBC PK was reduced to between 50% and 60% of normal and the abnormal electrophoretic bands were absent. The erythrocytosis appears to be inherited as an autosomal-dominant trait. The relationship between the PK abnormalities appears complex.

Adenosine Triphosphate↗
PubMed1981

Effect of some physical features and of amino acid substitutions on the mechanical precipitation of hemoglobin.

In 1973 Asakura demonstrated that, following mechanical shaking, oxy-Hb S was much less stable than oxy-Hb A (Nature 244: 437, 1973). We have studied the mechanical stability of Hb Crétil (beta 89 Ser leads to Asn), Hb Hope (beta 136 Gly leads to Asp), Hb Strasbourg (beta 23 Val leads to Asp), and the hybrid Hb S/Stanleyville-II (beta 6 Glu leads to Val: alpha 78 Asn leads to Lys) by the method of Roth et al. (Blood 45:377, 1975). Hb Créteil, Hb Hope, and Hb S/St-II were sensitive to mechanical shaking, while Hb Strasbourg was more stable than Hb A, a hitherto undescribed finding. The precise mechanisms responsible for this precipitation are not known. From comparisons with published results, but excluding thermosensitive Hbs, we conclude that: - standard methods for isolation of hemoglobins modify its mechanical stability, - alpha mutation increases the mechanical stability of Hb S in the hybrid Hb S/St-II, - some mutations produce a more stable hemoglobin than Hb A.

Amino Acids↗
PubMed1980

[Clinical and biological studies of an hybrid S/Stanleyville II hemoglobin (alpha 2 78 Asn replaced by Lys beta 2 6 Glu replaced by Val) (author's transl)].

A report of a doubly heterozygous case for both haemoglobins: Hb Stanleyville II (alpha 78 replaced by Lys) and Hb S (beta 6 Glu replaced by Val) with Hb hybrid S/St II. A 29-year-old woman from Zaïre was found to have four haemoglobins: Hb A, Hb Stanleyville II, Hb S and Hb hybrid S/St II. The clinical and hematological effects of this combination were similar to those of heterozygous sickle cell anaemia. Substitution of Lysin for Asparagin at residue alpha 78 reduced the tendency to polymerization and increased mechanical stability. These findings demonstrated involvement of this site in intermolecular interactions and explained the moderate severity of the sickle cell anaemia syndrome.

Adult↗