PubMed Health⌕ Search

Biomedical subjects

C Poyart

Publications and source records attributed to C Poyart.

At least 73 records · Page 4Linked to original sources
PubMed1995

Chronic septic arthritis and osteomyelitis in a prosthetic knee joint due to Clostridium difficile.

A case of chronic septic arthritis and osteomyelitis in a prosthetic knee joint due to Clostridium difficile is reported. A knee prosthesis was installed in a 16-year-old boy for surgical treatment of an osteosarcoma of the femur. Later, the patient suffered a traumatic closed fracture of his patella, and a sterile fluid was aspirated. One month later, the joint displayed inflammation. Culture of the articular fluid yielded a nontoxigenic Clostridium difficile strain. Despite several attempts using conservative medical treatment with penicillins and ornidazole, Clostridium difficile strains with the same antibiotic susceptibility pattern were repeatedly isolated from the joint over an eight-month period. The foreign material was then ablated, and finally, the patient's leg was amputated one year after Clostridium difficile was first isolated. The possible sources of contamination in our case and other reported cases of extraintestinal infection due to Clostridium difficile are discussed.

Adolescent↗
PubMed1995

[Expression of recombinant human hemoglobin in plants].

Human utilization of recombinant proteins of therapeutical interest, as hemoglobin, implies that the transgenic host allows a low cost production of the active proteins with minimal risks of pathogen contamination. In this regard, the use of transgenic plants could be of great interest. In particular, the systems based on plants could be one of the most economical transgenic system, compared with the others, because biomass obtention in fields is not expensive.

Carbohydrate Sequence↗
PubMed1995

[Recombinant human hemoglobin with low oxygen affinity: additional effects of two mutations].

The search for human Hb variants exhibiting a low oxygen affinity without requiring 2,3-diphosphoglycerate, together with a low oxygation rate, is of an increased interest in the view of producing an artificial oxygen carrier. We have synthesized the recombinant Hb beta 41Phe-->Tyr (rHb beta F41Y) which exhibits a low oxygen affinity due to the stabilization of the deoxy state of tetrameric Hb [1]. Interestingly, the autooxydation rate for this mutant is similar to that for Hb A. We have associated the mutation beta F41Y with the naturally occurring beta 82Lys-->Asp substitution (Hb Providence) known to be responsible for a low oxygen affinity [2]. The second-site mutation further decreases the oxygen affinity of the rHb beta F41Y. The effects of the beta F41Y and K82D mutations are additive, resulting in a four fold decrease in oxygen affinity of the artificial mutant Hb beta F41Y-K82D, compared to Hb A. In spite of the marked decrease in oxygen affinity, the autooxydation rate is 2- to 3 fold larger than that of Hb A. These data show that it is possible to adjust the oxygen binding properties of human Hb by using protein engineering methods. Because of the low oxygen affinity coexisting with a moderately increased autooxydation rate, this variant is a good candidate for the development of a Hb-based oxygen carrier.

Allosteric Regulation↗
PubMed1995

[Transfer of functional properties from bovine hemoglobin to human hemoglobin].

Bovine hemoglobin (Hb) has been proposed as a potential blood substitute because of its low intrinsic oxygen affinity in the absence of chloride anions and 2,3-diphosphoglycerate. The use of bovine blood as a source of Hb does not eliminate the risks of viral infections. Biotechnology techniques allow to produce modified recombinant Hbs. We have engineered human Hb mutants with the aim of mimicking the functional properties of bovine Hb. The argument for this work resides in the crystallographic studies and in the comparison of human and bovine beta globin sequences. The mutant recombinant Hbs exhibit the heterotropic effects of bovine Hb do not exhibit the low intrinsic oxygen affinity of bovine Hb.

Animals↗
PubMed1995

Oxygen delivery and autoxidation of hemoglobin.

Hemoglobin can be considered to exist in active and inactive states. When the iron atom is in the ferrous form, the protein is active and can bind oxygen reversibly; high and low affinity substates account for the cooperativity of ligand binding. However, like bulk metal, the iron can rust. The oxidation to the ferric form (metHb) leads to an inactive protein. The oxidation rate will therefore limit the useful lifetime of oxygen transporters; this is especially critical for cell free Hb solutions. This problem is compounded by the fact that Hb is a tetramer. A single oxidized heme effects the other three subunits within the same tetramer. The statistics are different from those for a monomeric system. For example, a random distribution of 20% ferric iron would imply 58% of the tetramers with at least one oxidized subunit. The oxidized subunits remain liganded (with water or OH) and will change the oxygenation curve for the neighbouring subunits. Since the tetramer will no longer make a full transition to the deoxy state, the oxygenation curves are shifted towards higher affinities. Thus the oxygen delivery will decrease for two reasons: the direct loss of active hemes, and the secondary influence on the remaining ferrous subunits. Control of the oxidation rate is also complicated by the intercorrelation of parameters. The rate is globally correlated with the oxygen affinity; it is also increased for hemoglobin dimers relative to tetramers. One must therefore accept a compromise of these parameters, in order to obtain a useful transporter.

Biological Transport↗
PubMed1995

Hb Arta [beta 45 (CD4) Phe-->Cys]: a new unstable haemoglobin with reduced oxygen affinity in trans with beta-thalassaemia.

The interaction of rare Hb variants with beta(0)-thalassaemia results in a quasihomozygous state where the erythrocytes contain the variant as the only major adult Hb component. Such a situation is a unique model that enables functional studies even in the case of a neutral variant that could not be isolated from Hb A. We report here an unusual patient carrying Hb Arta, a novel Hb variant [beta 45 (CD4) Phe-->Cys], in trans with beta(0)-thalassaemia gene (beta(0) 39). The aminoacid substitution at the critical CD corner of this Hb molecular renders the molecule unstable. In addition, haem is displaced in a position that favours the deoxy (T) conformation of the variant, but less than in Hb Cheverly [beta 45 (CD4) Phe-->Ser], and results in a p50 of 43 mmHg (pH 7.4, 37 degrees C) in the red cells with preservation of cooperativity. Solution studies of the almost pure Hb Arta show a 50% decrease in oxygen affinity and normal cooperativity; the Bohr effect and the interaction with organic phosphates are similar to those of Hb A. Hb Arta retains both normal homo- and heterotropic effects allowing a well-preserved oxygen transport in vivo despite a mild anaemia.

Adult↗
PubMed1995

Conjugative transposition of Tn916-related elements from Enterococcus faecalis to Escherichia coli and Pseudomonas fluorescens.

We studied the ability of transposons Tn916, Tn1545, and Tn916-Km, a Tn916 derivative expressing kanamycin resistance, to be conjugatively transferred from Enterococcus faecalis to various gram-negative bacteria. Our results demonstrate that these types of elements can carry out conjugative transposition from the chromosome of E. faecalis to those of Escherichia coli and Pseudomonas fluorescens and that the accomplishment of this event depends on the donor potential of the E. faecalis transposon delivery strain. Since the tet(M) gene does not confer a selectable level of tetracycline resistance to gram-negative bacteria such as E. coli, the presence of another marker(s) readily expressed in these recipients is required for the detection of this type of transfer. Conjugal transfer of Tn916-Km from E. faecalis to E. coli is not restricted by the EcoK restriction system, nor does it depend on the presence of functional homologous recombination system and integration host factor proteins in the recipient bacteria.

Base Sequence↗
PubMed1995

Interaction of 2 amino acid substitutions within the same beta chain of human hemoglobin: the examples of Hb Corbeil and Hb Villeparisis.

Only 16 hemoglobin (Hb) variants carrying 2 point mutations within the same polypeptide chain have been identified up to now. Two of those are reported in this paper. In Hb Villeparisis, the beta 77 (EF1) His-->Tyr mutation is identical to that of Hb Fukuyama while the other mutation, beta 80 (EF4) Asn-->Ser, has not yet been described. These 2 abnormalities are located almost within interacting distance. They lead to decreased oxygen affinity and, according to molecular simulations, to a remodeling of the surface of the molecule. Hb Corbeil associates 2 substitutions, that of Hb E [beta 26 (B8) Glu-->Lys], and of Hb Sherwood Forest [beta 104 (G6) Arg-->Thr], which are far from one another. The resulting properties consist only in the additive effects of both abnormalities. Hb Corbeil is, thus, a thalassemic Hb, like Hb E, and a molecule having altered oxygen binding properties, characterized by decreased heterotropic effects, similar to those of Hb Sherwood Forest. The simultaneous presence of more than 1 structural abnormality leads to a large spectrum of possible consequences on the function of the molecule, from local effects to a perturbation of intermolecular interactions. The genetic mechanism resulting in Hb with more than 1 point mutation is discussed: in some cases it is very likely that the second mutation arose on a chromosome already carrying a point mutation, in contrast a single mutational event involving a micro gene conversion has been proposed in other cases.

Allosteric Site↗
PubMed1994

Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) of Enterococcus faecalis in the apparent absence of pheromone induction.

Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAM beta 1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2 x 10(-7) and 5 x 10(-7) per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae. Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.

Cell Aggregation↗
PubMed1994

Heme-based sensors, exemplified by the kinase FixL, are a new class of heme protein with distinctive ligand binding and autoxidation.

FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia. We have overexpressed three FixL variants in Escherichia coli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein. One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric. We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates. FixL P50's for oxygen are 17-76 mmHg. FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo. Autoxidation rates are about 50 times faster than that of sperm whale myoglobin. The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen. Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters. We expect that some of the novel properties of FixL will be characteristic of the class.

Bacterial Proteins↗
PubMed1994

Formation of two hydrogen bonds from the globin to the heme-linked oxygen molecule in Ascaris hemoglobin.

We have tried to find out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P50 approximately 0.004 mmHg; 1 mmHg = 133 Pa). Following Kloek et al., we have synthesized the N-terminal globin domain of Ascaris hemoglobin in Escherichia coli [Kloek, A. P., Yang, J., Mathews, F. S. & Goldberg, D. (1993) J. Biol. Chem. 268, 17669-17671]. Like Kloek et al., we found its oxygen affinity to be as high as that of native Ascaris hemoglobin. We thought that this high affinity might be due to the heme-bound oxygen molecule being stabilized by two hydrogen bonds from the globin instead of the usual one. Ascaris hemoglobin has a distal glutamine instead of the more usual histidine as one of the potential hydrogen bond donors. In addition, it contains a tyrosine at position 10 of B helix (B10) in place of the leucine generally found there in vertebrate myoglobins and hemoglobins. Following the discovery of Carver et al. that sperm whale myoglobin with the replacement of leucine B10 by phenylalanine has a raised oxygen affinity, we have replaced tyrosine B10 in the N-terminal domain of Ascaris hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Phillips, G. N., Jr., & Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450]. Either of these replacements lowered the oxygen affinity about 100-fold, to the same level of that of human alpha-globin chains. These results are consistent with a hydrogen bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy of 2.7 kcal/mol.

Allosteric Regulation↗
PubMed1994

Direct evidence for the role of haem doming as the primary event in the cooperative transition of haemoglobin.

The study of cooperative ligand binding among the four subunits of haemoglobin has played a central role in the understanding of allosteric transitions in a large number of enzymes. Haem iron out-of-plane motion has been suggested to be the trigger for the cooperative transition of haemoglobin. To function as a trigger in a dynamic sense, haem-iron doming must be the first conformational change to occur following ligand dissociation. Here we present the first direct demonstration that haem-iron doming occurs on the same time scale as the breaking of the iron-ligand bond, thus establishing haem-iron doming as the primary event which lead to the R-->T transition in haemoglobin.

Allosteric Regulation↗