Biomedical subjects

R Rosa

Publications and source records attributed to R Rosa.

At least 55 records · Page 3Linked to original sources

PubMed1994

[Surgical treatment of hepatic hydatidosis. Current trends].

The authors report their experience relative to 33 patients with hepatic hydatidosis surgically treated during the years 1987 through 1993. Various surgical techniques were employed: subtotal (21 cases) and total (5 cases) cystopericystectomy, partial pericystectomy (12 cases), hepatic resection (2 cases). The postoperative rate complication was 30% (4 cases of cholangitis, 4 biliary fistulas, 1 suppuration of residual cavity and 1 colonic fistula). The postoperative morbidity was higher in patients treated with partial pericystectomy. There was no postoperative mortality.

Adolescent↗

PubMed1993

[Hemolytic anemias due to enzymopathies].

The most inherited enzymopathies associated with hemolytic anaemias are due to enzyme deficiencies (as a consequence of either synthesis reduction or impairement of the enzyme function). Such deficiencies can disturb some metabolism of the red cells. The most of enzyme deficiencies are involved in glycolysis that represents the main source of energy for this cell. Generally the genetic transmission of these enzymopathies is recessive autosomal with the exception of two cases (glucose 6-phosphate dehydrogenase and phosphoglycerate kinase) which are X-chromosome-linked. It stands to reason that the only treatment of these enzymopathies is dependent on gene therapy, a fine future method. Nevertheless prenatal diagnosis can be considered in some cases that display particularly severe symptoms.

Anemia, Hemolytic, Congenital↗

PubMed1993

Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89.

Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗

PubMed1992

Compound heterozygosity in a complete erythrocyte bisphosphoglycerate mutase deficiency.

Erythrocyte bisphosphoglycerate mutase (BPGM) deficiency is a rare disease associated with a decrease in 2,3-diphosphoglycerate concentration. A complete BPGM deficiency was described in 1978 by Rosa et al (J Clin Invest 62:907, 1978) and was shown to be associated with 30% to 50% of an inactive enzyme detectable by specific antibodies and resulting from an 89 Arg-->Cys substitution. The propositus' three sisters exhibited the same phenotype, while his two children had an intermediate phenotype. Samples from the family were examined using polymerase chain reaction and allele-specific oligonucleotide hybridization and sequencing techniques. Amplification of erythrocyte total RNA from the propositus' sister around the 89 mutation indicated the presence of two forms of messenger RNAs, a major form with the 89 Arg-->Cys mutation and a minor form with a normal sequence. Sequence studies of the propositus' DNA samples indicated heterozygosity at locus 89 and another heterozygosity with the deletion of nucleotide C 205 or C 206. Therefore, the total BPGM deficiency results from a genetic compound with one allele coding for an inactive enzyme (mutation BPGM Créteil I) and the other bearing a frameshift mutation (mutation BPGM Créteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Créteil I mutation.

Arginine↗

PubMed1992

Anti-muscarinic activity of a family of C11N5 compounds isolated from Agelas sponges.

In a search for potential target sites for C11N5 compounds obtained from marine sponges of the genus Agelas we evaluated their interaction with muscarinic acetylcholine receptors from rat brain membranes. In competition experiments with 3H-QNB these compounds displayed the following rank order of potency: sceptrin greater than oroidin greater than or equal to dibromosceptrin greater than or equal to clathrodin. Sceptrin (50 microM) was shown to be a competitive inhibitor of 3H-QNB binding as revealed by Scatchard analysis. The results demonstrate the ability of these compounds to interact with multiple target molecules in the micromolar range.

Alkaloids↗

PubMed1992

Structural modeling of the human erythrocyte bisphosphoglycerate mutase.

Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

Amino Acid Sequence↗

PubMed1991

Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase.

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.

Bisphosphoglycerate Mutase↗

PubMed1990

Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase.

The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.

Amino Acid Sequence↗

PubMed1990

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

Amino Acid Sequence↗

PubMed1990

Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships.

2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.

2,3-Diphosphoglycerate↗

PubMed1989

Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism.

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.

Amino Acid Sequence↗

PubMed1989

Isolation, characterization, and structure of a mutant 89 Arg----Cys bisphosphoglycerate mutase. Implication of the active site in the mutation.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is a trifunctional enzyme which displays synthase, mutase, and phosphatase activities. The purification, characterization, and structural study of an abnormal form of the enzyme, isolated from a patient which we reported earlier (Rosa, R., Prehu, M. O., Beuzard, Y., and Rosa, J. (1978) J. Clin. Invest. 62, 907-915), is described. The abnormal enzyme, present at 50% of the level of the normal enzyme as estimated by immunological methods, showed elevated electrophoretic mobility and hybridized with erythrocyte phosphoglycerate mutase (EC 5.4.2.1.) in the same manner as the normal control. The mutant enzyme was unstable at 55 degrees C and could be protected against thermal instability by 0.5 mM glycerate 2,3-bisphoshate but not by either glycerate 3-phosphate or glycolate 2-phosphate. Two of the three functions of the mutant enzyme were distinct from those of the normal protein. The specific activity of the synthase was 0.57% of normal and that of the mutase 4.1%. By contrast, the specific phosphatase activity was not affected by the mutation. However, the phosphatase activity of the mutated protein was markedly less stimulated by glycolate-2-phosphate than that of the control. High performance liquid chromatography analysis of tryptic peptides derived from the mutant enzyme showed an abnormal profile with the absence of two peaks normally containing the T12 and T13 peptides and without the appearance of a supplementary peak. Amino acid sequence and mass spectrometric analysis demonstrated the substitution of Arg----Cys residue in position 89 producing an uncleaved T12-T13 present in the same peak as the T6. Considered together, our data suggest that Arg-89 is located at or near the active site of bisphosphoglycerate mutase and that this residue is probably involved in the binding of monophosphoglycerates.

Amino Acid Sequence↗

PubMed1989

Blood glucose partition and levels of glycolytic enzymes in erythrocytes and somatic tissues of penguins.

1. A comparative study was carried out on blood glucose partition and glucose metabolism of penguin erythrocytes and somatic tissues. Pygoscelidae penguins (Pygoscelis antarctica and P. papua) were used in these experiments. 2. Blood glucose partition was established by assaying whole blood and plasma glucose in several individuals of the gentoo and chinstrap penguins. 3. It was found that almost all the whole blood sugar is compartmentalized at the plasma site, the red blood cells being ineffective in regard to glucose metabolism. 4. Levels of hexokinase, phosphoglucose isomerase, phosphofructokinase, fructose bisphosphate aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, phosphopyruvate hydratase (enolase), pyruvate kinase, alpha-glycerolphosphate dehydrogenase and fructose bisphosphate phosphatase were estimated in the erythrocytes of both gentoo and chinstrap penguins, the same determinations being carried out also on the somatic tissues (leg muscle, breast muscle, heart muscle, liver and brain) of the gentoo.

Animals↗

PubMed1989

Comparative study of human M2-type pyruvate kinases isolated from human leukocytes and erythrocytes of a patient with red cell pyruvate kinase hyperactivity.

M2-type pyruvate kinases (M2-PK) have been isolated from human leukocytes and from the erythrocytes of a patient with erythrocyte PK hyperactivity. The kinetic characteristics of the patient erythrocyte M2-PK were similar to those of leukocyte M2-PK except for the Hill coefficient of phosphoenol pyruvate kinetics that showed little difference in the values. The patient erythrocyte M2-PK displayed complete immunological identity with leukocyte M2-PK in immunodiffusion, immunoblotting and immunoneutralization. The sensitivity to proteolysis by trypsin and the electrophoretic migration in different conditions were similar for the M2-PK of both origins. These results suggest an identity between this M2-PK abnormally present in erythrocytes and the M2-PK from leukocytes.

Chromatography, Ion Exchange↗

PubMed1989

Incidence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Tunisian populations.

Screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency was performed on male students originating from several towns in Tunisia. Three hundred and twenty-five assays were made, allowing calculation of the mean value and standard deviation of G6PD (6.32 +/- 0.72 U/g Hb), the incidence of the deficiency (1.84%) and its geographic distribution in Tunisia. G6PD electrophoresis in 54 subjects showed marked predominance of the B+ type (96.2%) compared with the A+ type (1.96%). Three deficient subjects displayed an electrophotetic mobility identical to that of the A+ type of G6PD.

Adult↗

PubMed1988

Sequence of the human erythrocyte phosphoglycerate mutase by microsequencer and mass spectrometry.

We have previously reported the isolation in pure form of the human erythrocyte phosphoglycerate mutase isozyme B. We now report the sequence of the whole protein and the identification of its N-terminal blocking group. The protein tryptic peptides of phosphoglycerate mutase isozyme B were isolated by high performance liquid chromatography and their sequence determined by microsequencing. The sequence and the nature of the blocking group of the N-terminal tryptic peptide was shown to be N-acetyl-Ala-Ala-Tyr-Lys by mass spectrometry. Overlaps of the tryptic peptides were obtained by studying the V8 Staphylococcus aureus protease peptides of the aminoethylated phosphoglycerate mutase isozyme B either by microsequencing or by mass spectrometry. The procedure used allowed us to obtain the sequence on a very small amount of material and in a short period of time. Our data agree well with those derived from the cDNA nucleotide sequence described by Sakoda et al. (Sakoda, S., Shanske, S., DiMauro, S., and Schon, E. A. (1988) J. Biol. Chem. 263, 16899-16905). In addition, our data directly indicate that the initiation codon does not introduce a methionine as N-terminal amino acid and allowed the identification of the acetyl N-terminal group.

Amino Acid Sequence↗

PubMed1988

Isolation and characterization of the human 2,3-bisphosphoglycerate mutase gene.

The human 2,3-bisphosphoglycerate mutase gene was isolated from genomic libraries and analyzed by Southern blots and DNA sequencing. The transcription initiation site was localized by primer extension as well as by S1 protection of the mRNA. The gene extends over 22 kilobase pairs; it is composed of two introns (8.8 and 11.5 kilobase pairs long) and three exons (84, 662, and 965 base pairs long). The second exon correlates with a functional subdomain of the protein, as shown by comparison with the yeast phosphoglycerate mutase structure. The sequence TAGAAAA was found 30 bases upstream from the transcription initiation site and could be analogous to the TATA box. A sequence homologous to the CCAAT box was found twice, at positions -75 and -178. There is no GC-rich sequence or GC box in the 5'-flanking region of the gene. Northern blot analysis indicates that the 2,3-bisphosphoglycerate mutase mRNA is detected mainly in erythroid tissues and cell lines, although it is also present in low amounts in a nonerythroid tissue. A comparison of the 5'-upstream sequences with other promoters active only in erythroid cells did not reveal any common signal that could be responsible for the "erythroid promoter."

Amino Acid Sequence↗

PubMed1988

Comparative effects of two sulfhydryl reagents on the activities of a multifunctional red cell enzyme: bisphosphoglycerate mutase.

The effects of two sulfhydryl reagents on the three activities of bisphosphoglycerate mutase have been compared. Under N-ethylmaleimide treatment all the activities were inhibited except for 60% of the non-stimulated phosphatase. With iodoacetamide the mutase and the stimulated-phosphatase activities were completely inhibited whereas the non-stimulated phosphatase and 60% of the synthase activities were unaffected. 2,3-bisphosphoglycerate protected all the activities of the enzyme against inactivation by the two sulfhydryl reagents whereas 3-phosphoglycerate protected them only against iodoacetamide. 2-phosphoglycolate had an identical effect to that of 3-phosphoglycerate except for its effect on the non-stimulated phosphatase activity, which was slightly enhanced under N-ethylmaleimide treatment.

2,3-Diphosphoglycerate↗