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Biomedical subjects

P Boivin

Publications and source records attributed to P Boivin.

At least 19 recordsLinked to original sources
PubMed1992

Recombinant human granulocyte-macrophage colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation with unpurged and purged marrow in non-Hodgkin's lymphoma: a double-blind placebo-controlled trial.

The toxicity of autologous bone marrow transplantation (ABMT) is correlated to neutropenia. Although recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) seems to hold promise in accelerating neutrophil recovery, few analyses from randomized studies are presently available. Ninety-one patients with non-Hodgkin's lymphoma receiving high-dose ablative chemotherapy followed by ABMT with unpurged or purged marrow were included in a randomized, double-blind, placebo-controlled trial. Forty-four patients received 250 micrograms rhu GM-CSF (Escherichia coli)/m2 and 47 patients received placebo. Treatment was administered daily as continuous infusion from day of ABMT until the absolute neutrophil count (ANC) reached 0.5 x 10(9)/L for 7 days or until day 30, whichever was first. With rhu GM-CSF, 50% of the patients reached an ANC count greater than 0.5 x 10(9)/L at day 14 as opposed to day 21 with placebo (P less than .0001). Patients transplanted with marrow purged by mafosfamide also recovered earlier when treated with rhu GM-CSF (16 v 20.5 days, P = .013). The hospitalization duration was shorter in the rhu GM-CSF group (median, 23 v 28 days, P less than .05). No difference was observed in fever, number of infections, and antibiotic administration between the two groups. The major adverse event ascribed to rhu GM-CSF was a capillary leak syndrome in three patients graded as severe in two patients, moderate in one, and reversible in all three patients. In addition, one patient in the rhu GM-CSF group died suddenly with no explanation. In long term follow-up, the relapse rate was identical in both groups and there was no significant difference in the number of deaths at 1 year (12 with rhu GM-CSF v 9 with placebo), although deaths seemed to occur slightly earlier in the rhu GM-CSF group. We conclude that after ABMT with purged or unpurged marrow, rhu GM-CSF (E coli) significantly reduces neutropenia duration and hospitalization stay. A positive causative relation between the study drug and/or its mode of application with an increased toxicity as compared with GM-CSF from other sources and/or other modes of application cannot be deduced from the experiences in this study. Additional randomized trials would be necessary for an appropriate answer.

Adolescent↗
PubMed1992

Spectrin beta Tandil, a novel shortened beta-chain variant associated with hereditary elliptocytosis is due to a deletional frameshift mutation in the beta-spectrin gene.

An Argentinian family with hereditary elliptocytosis (HE) associated with a shortened beta-spectrin (Sp) chain was studied. As with most of the other shortened Sp beta-chains that have been described, this variant, called SpTandil, has impaired ability to participate in Sp dimer self-association, has lost its ability to become phosphorylated, and is associated with the presence of increased amounts of the alpha I 74-Kd fragment after partial tryptic digestion of Sp. The 3' ends of the beta-Sp gene of affected patients were analyzed. cDNA was prepared by reverse transcription of peripheral blood mRNA and amplified by the polymerase chain reaction (PCR) using primers corresponding to sequences 400 bp apart on the cDNA, spanning the last three exons (X, Y, Z) of the beta-Sp gene. Agarose gel electrophoresis of the cDNA amplification showed the presence of one band, the size of which was apparently the same as the band amplified from mRNA of a normal control. cDNA from one HE patient was subcloned and sequenced. Several clones showed the presence of a 7-bp deletion at codon 2041 in exon X. Genomic DNA of all the affected members of the family were amplified by PCR using primers flanking the deletion and corresponding to sequences 128 bp apart on exon X. Analysis of the PCR products using electrophoresis on polyacrylamide gel showed the presence of 121- and 128-bp bands in all HE subjects, and an additional doublet migrating more slowly than the two bands, which corresponded to the presence of heteroduplexes. The mutation results in a shift of the normal reading frame and leads to a new amino acid sequence at the C-terminus of the mutant beta-Sp chain. A new in-frame stop codon is encountered downstream, leading to premature chain termination. The identification of the molecular defect in Sp beta Tandil provides information regarding the region of the beta-Sp chain that is important for both Sp dimer self-association and an indication of potential sites of phosphorylation of the chain.

Amino Acid Sequence↗
PubMed1992

Inherited haemolytic anaemia created by insertional inactivation of the alpha-spectrin gene.

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. By in situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the alpha-spectrin gene locus.

Anemia, Hemolytic↗
PubMed1992

Purification of erythrocyte protein 4.1 by selective interaction with inositol hexaphosphate.

Protein 4.1 is a multifunctional structural protein occupying a strategic position in the erythrocyte membrane. It is present in the erythrocyte membrane skeleton and in many nonerythroid cells. This report describes a novel method for purifying this protein based on its selective interaction with inositol hexaphosphate dimagnesium tetrapotassium salt. This interaction was discovered in the course of chromatography of high-salt extract of inside-out membrane vesicles on Procion orange MX-2R-Sepharose. The new procedure is simple and selective and produces protein 4.1 with better yield than that obtained with a previously published procedure. The purified protein 4.1 has the same immunoreactivity and the same alpha-chymotryptic digest profile as protein 4.1 purified by published methods and is fully functional in enhancing the interaction between F-actin and spectrin dimers.

Chemical Precipitation↗
PubMed1992

Elliptocytosis-associated spectrin Rouen (beta 220/218) has a truncated but still phosphorylatable beta chain.

Spectrin Rouen (beta 220/218) is a novel variant, carrying a shortened beta chain with an apparent molecular weight of 218 kDa. It was detected in a French family. All affected members suffered from haemolytic hereditary elliptocytosis. As other shortened beta chain variants described before, the beta Rouen chain is truncated at its carboxyl terminus. Spectrin Rouen is associated with a defect in spectrin dimer self-association and with an abnormally high amount of the alpha I 74 kDa peptide following partial tryptic digestion. Dimer reconstitution experiments from normal and abnormal purified Sp subunits indicated that the increased alpha I 74 kDa fragment is induced by the altered beta chain. However, spectrin Rouen is different from other mutants with a truncated beta chain in several respects: its amount is low (less than 10%) and the spectrin dimer self-associated defect is mild. Critically, the beta Rouen chain has retained the ability of undergoing phosphorylation, even though it is modified in its C-terminal region. These results, compared to those obtained with beta 220/214 spectrin Le Puy and beta 220/216 spectrin Nice, allowed better localization of the beta chain sites that can be phosphorylated by a membrane-bound casein kinase.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

[Clinical and biochemical study of 9 patients with hereditary elliptocytosis].

Using clinical, functional and biochemical criteria, we studied the red blood cell membrane of nine caucasic patients from four families with hereditary elliptocytosis (HE). From a clinical point of view, seven cases were classified as compensated mild HE in whom anemia and splenomegaly are absent and reticulocytosis is slightly elevated or normal. Two cases were uncompensated mild HE with anemia, reticulocytosis, splenomegaly and erythrocytic fragmentation. (Table 1). Three individuals from one family displayed a significant reduction of protein 4.1 by polyacrylamide gel electrophoresis; one of them was uncompensated HE. The patterns of limited tryptic digestion and dimer/tetramer proportion of spectrin were normal. The study of red cell deformability by ectacytometry revealed that the cell deformability under isotonic conditions was decreased in all HE patients and the curve obtained had trapezoidal shape (Fig. 3). We found that the deformability index correlated well with the degree of anemia, but no correlation was observed between clinical findings, morphological phenotype and specific molecular etiology. According to our knowledge, this is the first report on molecular and functional studies in HE in Argentina.

Adult↗
PubMed1991

A splice site mutation of the beta-spectrin gene causing exon skipping in hereditary elliptocytosis associated with a truncated beta-spectrin chain.

We studied a French kindred with hereditary elliptocytosis associated with a spectrin variant (spectrin LePuy) containing a beta-spectrin chain that is truncated at its C terminus (Dhermy, D., Lecomte, M., Garbarz, M., Bournier, O., Galand, C., Gautero, H., Feo, C., Alloisio, N., Delaunay, J., and Boivin, P. (1982) J. Clin. Invest. 70, 707-715). The structure of the 3' end of the beta-spectrin gene, the region encoding the C terminus of beta-spectrin, was determined. Nucleotide sequencing of amplified genomic DNA revealed a mutation at position +4 (A----G) of the 5' donor consensus splice site of the intron following the third-to-last exon (exon X) in one beta-spectrin allele of a heterozygous patient. Agarose gel electrophoresis of polymerase chain reaction-amplified cDNA revealed an extra band of lower molecular weight, suggesting that the shortened beta-spectrin chain of spectrin LePuy arises from aberrant mRNA splicing. Nucleotide sequencing of the shorter cDNA amplification product revealed that the sequences encoding exon X were absent. Southern blotting of cDNA amplification products confirmed this result. The skipping of exon X causes a shift in the normal reading frame resulting in the encoding of a new amino acid sequence at the C terminus of the mutant beta-spectrin chain. A new in-frame stop codon is encountered following a single residue of this novel sequence.

Amino Acid Sequence↗
PubMed1991

The synthesis and accumulation of membrane protein 4.1 in Friend erythroleukemia cells.

The effect of extensive differentiation on the synthesis and accumulation of protein 4.1 were studied on Friend erythroleukemia cells grown in suspension and on fibronectin coated dishes. Whole membranes of Friend erythroleukemia cells (FELC) contained a protein 4.1a and 4.1b doublet of Mr 76 and 74 kDa and two minor bands of Mr 105 and 43 kDa that cross-reacted with anti-human protein 4.1 IgG. These proteins were present even in uninduced cells. The synthesis of protein 4.1 was maximal after 4 days of induction in both suspension culture and in fibronectin-coated dishes whereas the protein 4.1 continued to accumulate until the seventh day. More protein 4.1 accumulated in cells grown on fibronectin-coated dishes, at each stage of differentiation, than in cells grown in suspension. The protein 4.1a/4.1b ratio changed during differentiation. The amounts of protein 4.1b increased progressively after induction until the protein 4.1a/4.1b ratio was similar to that of mouse mature erythrocyte. The protein 4.1a/4.1b ratio appears to be an internal marker of erythroid differentiation.

Animals↗
PubMed1991

Immobilization of perfluoroalkylated enzymes in a biologically active state onto Perflex support.

Perflex has been introduced by E. I. du Pont de Nemours and Co., Inc., as a new fluorocarbon-based technology for protein immobilization. Due to the hydrophobic character of the support, however, significant loss of enzymatic activity may occur upon immobilization of certain enzymes, which appears to be due to a large conformational change of the protein ("inversion"). Pretreatment of the Perflex support with a neutral fluorosurfactant lessened the surface hydrophobicity, thus decreasing the hydrophobic interaction between the support and the protein. Modification of enzymes with a high number of fluorocarbon residues, which forms a hydrophobic "envelope" around the protein, also appears to prevent enzyme inactivation upon immobilization on Perflex support. Moreover, preactivation of the support with either perfluorooctylpropylisocyanate or reactive poly(fluoroalkyl) sugar reagents greatly improves the enzyme particle activity by increasing the amount of immobilized enzyme. Fluorosurfactant treatment of the support activated with perfluorooctylpropylisocyanate improves the retention of activity for sensitive enzymes such as alpha-chymotrypsin and increases the wetability and ease of handling of the Perflex particles.

Adsorption↗
PubMed1990

[Iron overload in congenital hemolytic anemia caused by pyruvate kinase deficiency. A major late complication].

Twelve patients (5 women and 7 men, aged from 19 to 54 years) presenting with congenital, non-spherocytic haemolytic anaemia due to erythrocyte pyruvate kinase (PK) deficiency were investigated for systemic iron overload 18 to 27 years after the diagnosis was made. One patient had, beside PK deficiency, idiopathic haemochromatosis demonstrated by the HLA A3 and B14 markers. Another, 21-year old male patient had received more than 100 blood transfusions. In both patients, blood ferritin levels were as high as 5,584 and 9,665 g/litre respectively. Among the remaining 10 patients, 9 had biochemical signs of iron overload, such as high serum iron levels, reduced total siderophilin saturation capacity and blood ferritin levels of about 1,500 g/litre. Hepatic histology could be obtained from 5 patients and showed significant iron overload with cirrhosis in one case and clear-cut portal fibrosis in 3 cases. In all but the patient with multiple transfusions the iron overload was unrelated to transfusions, being present in their absence, usually during the 3rd and 4th decades of their life. The finding of iron overload requires preventive measures such as limitation of transfusions and elimination of iron by deferoxamine therapy.

Adult↗
PubMed1990

[Systematic blood transfusions in adult homozygote sickle-cell anemia. Study of 11 cases followed for 5 to 10 years].

Eleven patients (5 men and 6 women) with sickle-cell anaemia were treated with systematic repeated blood transfusions to avoid the sickling crises and complications of the disease and were followed up for 5 to 10 years. On average, the patients received 22 erythrocyte concentrates per year, to a mean individual total of 180 sediments. Each blood transfusion was accompanied by an intravenous infusion of deferoxamine 2 grams. Systematic blood perfusions unquestionably had beneficial effects on the frequency of sickling crises and complications, as well as on the patients' quality of life and psycho-social status. On the other hand, the iron overload could not be avoided, and it sometimes resulted in haematochromatosis (2 patients). At the moment, only continuous deferoxamine infusions could prevent this from happening and enable blood transfusions to be more widely used, provided its indications, restricted to the most severe forms of the disease, are carefully weighed. In this series of patients, the risk of viral contamination seemed to be relatively low, and in particular no HIV seroconversion was detected.

Adolescent↗
PubMed1990

In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (calpain I).

1. In whole ghosts, ankyrin, protein 4.1, protein band 3 and spectrin are lysed by purified calpain I in the presence of calcium. 2. Limited calpain lysis of purified ankyrin results in several peptides, including a 85 kD peptide bearing the ankyrin interaction site for the protein band 3 internal fragment (43 kD), and a 55 kD peptide carrying the ankyrin-spectrin interaction site. 3. These peptides are differently phosphorylated: the 85 kD by cytosol casein kinase, and the 55 kD by membrane casein kinase. 4. Protein 4.1 lysis mainly produces a 30 kD peptide resistant to proteolysis. 5. The spectrin beta-chain is more sensitive to calpain cleavage than the alpha chain; both chains seem to be cleaved in a similar sequential manner. 6. Limited proteolysis of spectrin dimer does not impede tetramerization in vitro.

Ankyrins↗
PubMed1990

Severe recessive poikilocytic anaemia with a new spectrin alpha chain variant.

A severe neonatal haemolytic anaemia was observed in a propositus from the West Indies. Frequent blood transfusions were required until complete splenectomy was carried out. Blood smears showed predominant poikilocytosis with spherocytes and microcytes as observed in hereditary pyropoikilocytosis. Erythrocytes were completely fragmented after incubation at 45 degrees C. The two asymptomatic parents had normal haematological profiles. The erythrocyte membrane electrophoretic patterns of the splenectomized propositus and her parents were normal. The propositus had a moderate defect in the spectrin (Sp) dimer self-association. Limited tryptic digestion of the propositus' Sp under standard conditions (0 degrees C, 20 h, enzyme-substrate ratio of 1/100) revealed an increased sensitivity to tryptic digestion. The major features detected by one- and two-dimensional electrophoresis gels of Sp tryptic digests were the absence of high molecular weight peptides from the Sp alpha II (48 kDa and 35 kDa peptides) and Sp alpha III (52 kDa peptide) domains with increased amounts of the lower molecular weight peptides from the Sp alpha II and Sp alpha III (29 kDa peptide and 47 kDa peptide) domains respectively. Kinetic studies of Sp tryptic digestion (10 min to 36 h) confirmed the increased tryptic susceptibility of Sp. Immunodetection with specific anti-alpha-chain domain antibodies showed that the highest molecular weight peptides from the alpha II and alpha III domains are released early in digestion, but disappear quickly, with an increase in their corresponding smaller peptides. The molecular weights of the peptides corresponding to the 48 kDa and 35 kDa peptides from the alpha II domain are slightly modified. Peptides from the alpha I and alpha IV domains showed no significant abnormalities. The Sps of both parents were normal. These results indicate that the patient has a novel Sp alpha chain defect, which is probably located in the region of the alpha II domain which adjoins the alpha III domain.

Anemia, Hemolytic, Congenital↗
PubMed1990

Abnormal tryptic peptide from the spectrin alpha-chain resulting from alpha- or beta-chain mutations: two genetically distinct forms of the Sp alpha I/74 variant.

Limited tryptic digestion of native spectrin (Sp) has revealed several variants in hereditary pyropoikilocytosis (HPP) and in a subset of patients with hereditary elliptocytosis (HE). In most cases, tryptic peptide corresponding to the alpha I (N-terminal) 80 kD domain is wholly or partially replaced by smaller fragments. These variants are provisionally designated according to the molecular weight of the most prominent new peptide. Partial amino acid sequences of the abnormal peptides and DNA analysis of the alpha-spectrin gene have shown that most variants result from substitution or insertion of an amino acid in the alpha I-domain. However, similar investigations did not detect any such abnormality in the spectrin alpha I-domain of an HE black kindred with one of the spectrin variants called Sp alpha I/74. In this kindred, restriction fragment length polymorphism studies and transmission of the genetic polymorphism relative to the alpha II-domain excluded the involvement of the alpha-chain in the pathological process. To ascertain whether the abnormal alpha I 74 kD peptide might be caused by a beta-chain mutation, we reconstituted hybrid dimers combining normal and HE Sp-chains. The tryptic peptide patterns of spectrin hybrid dimers containing HE alpha-chain and control beta-chain showed a normal 80 kD tryptic product. In contrast, the hybrid dimer containing normal alpha-chain and HE beta-chain gave rise to increased 74 kD peptide at the expense of the 80 kD, demonstrating that the mutation in this family resides in the beta-chain. The same method was used to show that in two other unrelated white kindreds, the elevated 74 kD peptide arose from a Sp alpha-chain defect. Thus an alteration in tryptic susceptibility within the N-terminal domain of the spectrin alpha-chain can be directed by a mutation in the beta-chain. The hybridization technique affords a definitive means of distinguishing between alpha- and beta-chain mutants.

Amino Acid Sequence↗
PubMed1990

Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association.

alpha I/74 hereditary elliptocytosis (HE) is a subgroup of HE in which patients exhibit an impaired self-association of spectrin dimers and an abnormal proteolytic cleavage of the alpha I domain of spectrin. We studied a family in which the proband presented with a severe neonatal hemolytic anemia with poikilocytosis. Biochemical analysis of erythrocytes from the proband and his family members allowed us to ascertain a diagnosis of homozygosity for alpha I/74 HE in the proband and heterozygosity in his parents and several of their offspring. Results of polymorphism linkage analysis suggested that the defect in this family was located in beta rather than alpha spectrin. We analyzed the 3' end of the beta-spectrin gene of the proband and detected a mutation that changes a codon for alanine to one for proline. Allele-specific oligomer hybridization on slot blots of DNA from other family members confirmed the presence of the mutation only in members heterozygous for the disorder. This is the first example of a point mutation in the beta-spectrin chain that is associated with defective spectrin dimer self-association and an abnormal proteolytic cleavage of the alpha chain. Based on this finding, we propose a model for the mechanism of interaction between the alpha- and beta-spectrin chains.

Amino Acid Sequence↗