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A Michaud

Publications and source records attributed to A Michaud.

At least 37 records · Page 2Linked to original sources
PubMed1997

Substrate dependence of angiotensin I-converting enzyme inhibition: captopril displays a partial selectivity for inhibition of N-acetyl-seryl-aspartyl-lysyl-proline hydrolysis compared with that of angiotensin I.

Angiotensin I-converting enzyme (ACE) is composed of two highly similar domains (referred to here as the N and C domains) that play a central role in blood pressure regulation; ACE inhibitors are widely used in the treatment of hypertension. However, the negative regulator of hematopoiesis, N-acetyl-seryl-aspartyl-lysyl-prolyl (AcSDKP), is a specific substrate of the N domain-active site; thus, in addition to the cardiovascular function of ACE, the enzyme may be involved in hematopoietic stem cell regulation, raising the interest of designing N domain-specific ACE inhibitors. We analyzed the inhibition of angiotensin I and AcSDKP hydrolysis as well as that of three synthetic ACE substrates by wild-type ACE and the N and C domains by using a range of specific ACE inhibitors. We demonstrate that captopril, lisinopril, and fosinoprilat are potent inhibitors of AcSDKP hydrolysis by wild-type ACE, with K(i) values in the subnanomolar range. However, of the inhibitors tested, captopril is the only compound able to differentiate to some degree between AcSDKP and angiotensin I inhibition of hydrolysis by wild-type ACE: the K(i) value with AcSDKP as substrate was 16-fold lower than that with angiotensin I as substrate. This raises the possibility of using captopril to enhance plasma AcSDKP levels with the aim of normal hematopoeitic stem cell protection during chemotherapy and a limited effect on the cardiovascular function of ACE.

Angiotensin I↗
PubMed1996

Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity.

Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains). In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris. The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains. Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant. Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE delta CHO). This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin 1 and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE. However, AnCE delta CHO was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activity under the same conditions. Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis. A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.

Amino Acid Sequence↗
PubMed1995

Cell surface localization of proteolysis of human endothelial angiotensin I-converting enzyme. Effect of the amino-terminal domain in the solubilization process.

Angiotensin-converting enzyme (ACE) belongs to the type I class of ectoproteins and is solubilized by Chinese hamster ovary cells transfected with the full-length human ACE cDNA. ACE release in Chinese hamster ovary cells involves a proteolytic cleavage occurring in the carboxyl-terminal region, between Arg-1137 and Leu-1138. The subcellular localization of ACE proteolysis was established by pulse-chase experiments, cell surface immunolabeling, and biotinylation of radiolabeled mature proteins. The proteolysis of ACE takes place primarily at the plasma membrane. The solubilization of ACE is less than 2% within 1 h, is increased 2.4-fold by phorbol esters, but is not influenced by ionophores. An ACE mutant lacking the transmembrane domain and the cytosolic part (ACE delta COOH), is secreted at a faster rate without a carboxyl-terminal cleavage, and phorbol esters or ionophores have no effect on its rate of production in the medium. Therefore, the proteolysis of ACE is dependent on the presence of the membrane anchor and suggests that the secretase(s) involved is also membrane-associated. An ACE mutant lacking the amino-terminal domain (ACECF) is secreted 10-fold faster compared with wild-type ACE. The solubilization of ACECF occurs at the plasma membrane and is stimulated 2.7-fold by phorbol esters, and the cleavage site is localized between Arg-1227 and Val-1228. The amino-terminal domain of ACE slows down the proteolysis and seems to act as a "conformational inhibitor" of the proteolytic process, possibly via interactions with the "stalk" of ACE and the secretase(s) itself.

Amino Acid Sequence↗
PubMed1995

The hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin-converting enzyme.

Angiotensin I-converting enzyme (ACE) is a zinc-dipeptidyl carboxypeptidase, which contains two similar domains, each possessing a functional active site. Respective involvement of each active site in the degradation of the circulating peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a negative regulator of hematopoietic stem cell proliferation, was studied by using wild-type recombinant ACE and two full-length mutants containing a single functional site. Both the N- and C-active sites of ACE exhibit dipeptidyl activity toward AcSDKP, with Km values of 31 and 39 microM, respectively. However, the N-active site hydrolyzes the peptide 50 times faster compared with the C-active site, with kcat/Km values of 0.5 and 0.01 microM-1.s-1, respectively. The predominant role of the N-active site in AcSDKP hydrolysis was confirmed by the inhibition of hydrolysis using a monoclonal antibody specifically directed against the N-active site. The N-domain specificity for AcSDKP will aid the identification of specific inhibitors for this domain. This is the first report of a highly specific substrate for the N-active site of ACE, with kinetic constants in the range of physiological substrates, suggesting that ACE might be involved via its N-terminal active site in the in vivo regulation of the local concentration of this hemoregulatory peptide.

Amino Acid Sequence↗
PubMed1995

Recent advances in knowledge of the structure and function of the angiotensin I converting enzyme.

AIM: To review the structure and function of the angiotensin I converting enzyme (ACE), focusing on recent results from studies using a wide range of molecular biological techniques. ACE STRUCTURE AND FUNCTION: ACE is an ectoenzyme expressed as two isoenzymes in mammals, a larger somatic form found in endothelial, epithelial and neuronal tissues and a smaller form in germinal tissues. Both forms have similar enzymatic activities but differ in size and immunological properties. The somatic form of ACE is composed of two highly homologous domains (amino and carboxyl domains) while the germinal form contains only one domain. Somatic ACE has two functional catalytic sites, both dependent on a zinc cofactor. Each ACE domain has also been shown to interact differently with competitive inhibitors. MECHANISM OF ACE ANCHORAGE AND SOLUBILIZATION: The mechanism for anchoring ACE to the cell membrane has also been reported, and the solubilization step outlined. The relationship between the membrane-bound and soluble forms has been investigated, and the physiological relevance of this mechanism discussed. GENETIC STRUCTURE: The structure of the ACE gene has been determined and the distribution in cells and different tissues has been reported in various studies. CONCLUSION: All results have indicated that there are important functional and structural differences between the two domains, but at present ACE cannot be considered a true bifunctional enzyme, even though an exclusive substrate has been identified for the amino domain.

Animals↗
PubMed1993

Proteolytic release of human angiotensin-converting enzyme. Localization of the cleavage site.

Angiotensin-converting enzyme (EC 3.4.15.1, ACE) is a transmembrane protein with a short carboxyl-terminal cytoplasmic domain, a 17-amino acid hydrophobic anchor domain, and a large N-terminal extracellular region containing two catalytically homologous domains. An active soluble form of ACE circulates in human plasma and is produced in culture medium of Chinese hamster ovary (CHO) cells transfected with the full-length human ACE cDNA. The mechanism of ACE release in CHO cells involves a post-translational proteolytic cleavage occurring in the carboxyl-terminal region. The carboxyl terminus of the secreted recombinant ACE, AGQR, was established by carboxyl-terminal microsequencing and corresponds to a cleavage site between Arg-1137 and Leu-1138. Two independent studies confirmed this proposed cleavage site: amino acid analysis of a carboxyl-terminal peptide derived from soluble ACE and immunocharacterization of membrane-bound and soluble ACE with antibodies raised against three peptides located along the carboxyl-terminal ACE sequence. In order to assess the importance of Arg-1137, this amino acid was mutated to a glutamine residue. This mutation did not prevent the secretion of ACE, suggesting that the solubilizing enzyme can accommodate this change or can use an alternative cleavage site. Finally, the production of soluble ACE in CHO cells appears to be proportional to the level of cellular ACE, implying that the solubilizing enzyme is not a limiting factor. In addition, the carboxyl-terminal sequence of the human plasma ACE was identified as AGQR, thus supporting the fact that a similar mechanism could operate in human vascular cells.

Amino Acid Sequence↗
PubMed1993

[Medical value of a respiratory mixture of oxygen and helium (Heliox)].

Lighter and more viscous than air, the oxygen-helium mixtures (heliox), well known to respiration physiologists, are used for functional exploration in the diagnosis of airways obstructions; they are also administered to deep-sea divers. However, it is usually little known that heliox can be used as therapeutic agent in the treatment of acute respiratory distress or severe disorders of ventilation caused by obstruction (e.g. inflammation). Heliox reduces the ventilatory work and increases alveolar ventilation, thereby improving haematosis and carbon dioxide elimination. The English-speaking authors, in particular, have a large experience of oxygen-helium mixtures in the treatment of such pathologies as severe asthma, small airways obstruction, cystic fibrosis, bronchoalveolitis, post-extubation pathologies, pharyngo-laryngeal oedema and drowning. The oxygen-helium mixture, therefore, participates efficiently, simply and inaggressively in the treatment of airway obstruction and acute respiratory distress.

Adult↗
PubMed1992

Study of the protein S system in HIV-infected patients: acquired protein S deficiency or unsuitable assays?

The plasma protein S (PS) system was studied in 120 HIV seropositive patients (CDC classification: group II: n = 35; group III: n = 6; groups IVA, IVC2 or IVE, n = 38; groups IVB, IVC1 or IVD, n = 41). Total PS antigen and C4b-binding protein (C4b-BP) values were not significantly different from control values. Free protein S levels assessed by an immunological method in the supernatant of PEG-precipitated plasma samples (PEG-fPS) were below the normal limit in 85 patients, lower values being found in patients with advanced HIV disease. No correlation was found between PEG-fPS levels and C4b-BP or total PS levels. At least 25 patients had a low functional PS value. Low functional levels of PS were found in each clinical group although there was no difference in the distribution of functional PS values among groups. Crossed immuno-electrophoresis showed an abnormal distribution of PS in some patients, but failed to confirm the marked decrease of free PS in patients with very low PEG-fPS. An impairment of the protein S system is observed in HIV-infected patients. However, the discrepancies found in some patients among the results of the various PS-related assays should lead to a cautious interpretation concerning the incidence of PS deficiency in HIV-infected patients.

Adult↗
PubMed1991

Expression and characterization of recombinant human angiotensin I-converting enzyme. Evidence for a C-terminal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes.

Chinese hamster ovary (CHO) cells have been transfected with either a full-length cDNA encoding human angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE) or a mutated cDNA, in which the last C-terminal 47 amino acids, including the putative transmembrane domain, are not translated. Cell lines expressing high levels of the wild-type ACE or the mutant were established. The cells transfected with the wild-type cDNA (CHO-ACE) express a membrane-bound ectoenzyme with an intracellular C terminus, as shown by indirect immunofluorescence using an antiserum (28A7) raised against a synthetic peptide corresponding to the deduced C terminus of ACE. This enzyme is structurally, immunologically, and enzymatically identical to human kidney ACE. In addition, CHO-ACE cells also produce a secreted form of the enzyme. Neither this secreted form nor the enzyme purified from human plasma is recognized by the antiserum 28A7, indicating that they undergo a truncation in the C-terminal region. On the other hand, the transfected cells expressing the C-terminally truncated mutant (CHO-ACE delta COOH) do not retain ACE in the plasma membrane, but secrete it into the medium. These results indicate that ACE is anchored to the plasma membrane by the predicted C-terminal transmembrane domain, and the secreted form is derived from the membrane-bound form by a post-translational proteolytic cleavage of the C-terminal region.

Amino Acid Sequence↗
PubMed1989

Effect of sex hormones on plasma T-kininogen in the rat.

The influence of sex hormones on rat plasma T-kininogen concentration was examined. The level of T-kininogen in the post-pubertal female rat is about 3-times that of the male animal. Female rats castrated as adults or 15 days after birth, had low T-kininogen concentrations, near those of male rats. In contrast, castration of mature or immature male animals induced no change in T-kininogen. Treatment of castrated female or male rats with 17 alpha-ethinylestradiol significantly increased the T-kininogen level, whereas administration of testosterone or progesterone had no effect. The influence of estrogen was specific for T-kininogen, since plasma HMW kininogen concentration was the same in male and female rats and was not affected by castration or sex hormone treatment. T-kininogen concentration was not significantly changed in pregnant rat between the 12th and the 20th day of pregnancy, but increased after parturition. It was high in the newborn rat at birth and then decreased similarly over the next 3 weeks in males and females. It continued to decrease in the males, reaching the level of the adult rat, but it increased in the female from 3-4 weeks of age and reached the adult level at about 6-8 weeks. These data indicate that natural estrogens have a physiological influence on the plasma level of T-kininogen in female rats whereas testosterone had no effect on either male or castrated female rats. HMW kininogen is not physiologically dependent on sex hormones.

Acute-Phase Proteins↗
PubMed1988

Effect of dexamethasone on kininogen production by a rat hepatoma cell line.

T Kininogen and High Molecular Weight Kininogen were characterized in the cell culture medium of Fao cells, a highly differentiated cell line derived from the Reuber H35 rat hepatoma. Immunoreactive T Kininogen and High Molecular Weight Kininogen identified by direct and specific RIAs were indistinguishable from standard kininogens. Immunoreactive T Kininogen was further identified by HPLC analysis of T kinin released after trypsin hydrolysis of the cell culture medium. The basal release rate of T kininogen was ten-fold higher than that of High Molecular Weight Kininogen. T Kininogen was not stored within the cells contrary to High Molecular Weight Kininogen. The production of the two kininogens in the cell medium was stimulated by dexamethasone up to five times in a dose-dependent manner. The specific antiglucocorticoid compound RU 38486 did not alter the basal rate of kininogen release by Fao cells, but abolished the stimulation by dexamethasone, indicating that dexamethasone exerts a true glucocorticoid type effect.

Animals↗
PubMed1988

Effect of thyroidectomy on rat T-kininogen.

The thyroid state affects the inflammatory reaction; in general, thyroid hormones increase and hypothyroidism decreases the inflammatory response. T-kininogen is an acute phase protein that inhibits cysteine proteases. The influence of thyroidectomy and 3,5,3'-triiodothyronine (T3)-treatment of thyroidectomized animals was examined on rat plasma T-kininogen concentration. Thyroidectomy increased the T-kininogen level three- to fourfold. A single administration of 10 micrograms T3/100 g body wt in 7-wk postthyroidectomy rats decreased it by approximately 50%. Daily treatment with 0.5 micrograms T3/100 g body wt prevented the increases in T-kininogen. The modifications in plasma T-kininogen concentration were confirmed by high-performance liquid chromatography analysis of the plasma kinins released by trypsin. They demonstrated that T-kinin was greatly and bradykinin slightly increased after thyroidectomy and that T-kinin concentration in T3-treated thyroidectomized animals was almost restored to the control level. The ability of thyroidectomized animals to produce a higher plasma T-kininogen compared with euthyroid animals was further documented when T-kininogen production was stimulated by laparotomy. This procedure resulted in a fourfold increase in T-kininogen in thyroidectomized animals compared with controls. The increase of plasma T-kininogen level after thyroidectomy results from a more active synthesis of the protein, since liver slices from thyroidectomized rats synthesized T-kininogen at a rate about two times higher than normal rats. These data may account at least in part for the diminution of the inflammatory response observed after thyroidectomy.

Animals↗
PubMed1987

A new affinity matrix for mineralocorticoid receptors.

The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.

Aldosterone↗
PubMed1986

Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution.

Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent.

Adrenal Cortex Hormones↗
PubMed1986

Follow-up of renal morphology and growth of 141 children operated for vesicoureteral reflux: a retrospective computerized study.

This study relates the postoperative evolution after ureterovesical reimplantation for vesicoureteral reflux (VUR) in 141 children who were ten years old or younger at the time of surgery. Renal growth and morphology were evaluated 2 and 5 years after surgery. We estimated renal growth by measuring the ratio of the bipolar parenchymal thickness to the total length of the kidney. We noticed that whatever the degree of reflux might have been, most of the kidneys partially or totally compensated for their growth failure. This growth resumption required many years to be completed. Surgical correction of VUR had favorable consequences on the radiologic aspect of pyelonephritic scars only on some of the kidneys: in these cases, the child's age appeared to be the only factor that had a statistical importance affecting the postoperative evolution of pyelonephritic scarring: the younger the children were at the time of surgery, the better the results obtained.

Age Factors↗
PubMed1986

In vitro comparison of ampicillin-chloramphenicol and ampicillin-cefotaxime against 284 Haemophilus isolates.

Since November 1982 at the Sainte-Justine Hospital in Montreal, ampicillin and cefotaxime were used in association as initial treatment (greater than or equal to 48 h) for childhood bacterial meningitis. In this report is described the in vitro interaction of the new regimen in comparison with that of the previous ampicillin-chloramphenicol combination against 284 Haemophilus isolates. Among the 156 ampicillin-susceptible, beta-lactamase-negative isolates, synergy was detected in 13 with ampicillin-cefotaxime, and antagonism was detected in only 1; in contrast, synergy was found in only 2 strains with ampicillin-chloramphenicol, and antagonism was found in 15. These differences were statistically significant (P less than 0.01). Such significant differences were not observed among the 128 ampicillin-resistant, beta-lactamase-positive Haemophilus isolates. The synergy of ampicillin-cefotaxime did not contribute to a decrease of the MIC of cefotaxime for 90% of isolates tested, whereas the antagonism of ampicillin-chloramphenicol did not contribute to increase the MIC of ampicillin for 90% of isolates tested.

Ampicillin↗