Search PubMed⌕ Search

Biomedical subjects

A Petit

Publications and source records attributed to A Petit.

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

Multiple substitutions at position 104 of beta-lactamase TEM-1: assessing the role of this residue in substrate specificity.

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

Amino Acid Sequence↗
PubMed1995

Inhibition of angiotensin II-stimulated inositol phosphate production by D2-dopamine receptor is calcium-dependent in human trophoblastic cells.

We previously reported that dopamine (DA) acted via D2-dopamine receptors in human trophoblastic cells to inhibit All-stimulated inositol phosphate (InsP) accumulation. However, the mechanism by which DA inhibited All-stimulated InsP accumulation is still unknown except that this inhibitory effect was sensitive to Pertussis toxin (PTX). In this study, we characterize this DA-mediated inhibition of All-stimulated InsP production in human placenta. Freshly isolated human term placental cells were prelabeled with myo-[2-3H]inositol and incubated with various stimuli in the presence of 10 mM LiCl. All (10(-6) M) stimulated 1.5 times the human trophoblastic cell InsP production whereas DA (10(-4) M) inhibited this All-stimulated InsP production by 54 +/- 7%. This inhibitory effect was mimicked by bromocriptine (53 +/- 3% of inhibition), a D2-dopamine agonist. We recently reported that bromocriptine inhibited human placental adenosine 3',5'-cyclic monophosphate (cAMP) production. Increasing the intracellular concentration of cAMP levels by adding forskolin did not modify the effect of DA and bromocriptine on InsP accumulation. On the other hand, the effect of DA and bromocriptine on All-stimulated InsP production were greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the D2-dopaminergic mediated inhibition was prevented by treatment of cells with the Ca2+ ionophore ionomycin (10(-5) M) and was mimicked either by removal of Ca2+ from incubation medium (53 +/- 6%) or by blockage of voltage-gated Ca2+ channels with nifedipine (51 +/- 7%). Our data indicate that the inhibitory effect of D2-dopamine agonists on All-stimulated InsP production is an indirect event probably due to the DA-inhibition of calcium influx.

Angiotensin II↗
PubMed1995

Systemic mastocytosis associated with chronic myelomonocytic leukemia: clinical features and response to interferon alfa therapy.

Systemic mastocytosis is a rare disease that shows marked heterogeneity in clinical manifestations and prognosis. It may be associated with hematologic disorders. We describe a patient with systemic mastocytosis associated with chronic myelomonocytic leukemia accompanied by ascites, pleural effusion, and development of skin lesions along a surgical scar. The disease responded well to interferon alfa therapy. This is the second report of successful treatment of mastocytosis with interferon alfa and the first associated with a hematologic malignancy.

Ascites↗
PubMed1995

A new Agrobacterium strain isolated from aerial tumors on Ficus benjamina L.

Crown gall tumors, collected from branches of 1-year-old weeping fig (Ficus benjamina L.) trees, yielded both tumorigenic and nonpathogenic agrobacteria. On the basis of classical diagnostic tests, the nonpathogenic strains were identified as Agrobacterium tumefaciens, whereas the tumorigenic strains could not be assigned to any of the known terrestrial Agrobacterium spp. The tumorigenic strains also differed from other members of the genus by producing more acid from mannitol. According to cluster analysis of carbon substrate oxidation (GN Microplate; Biolog, Inc.) and fatty acid content, the tumorigenic fig strains were distinct from strains of A. tumefaciens, Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi. Furthermore, they had unusual opine metabolism, inducing tumors that synthesized nopaline and three recently discovered opines: chrysopine (d-lactone of N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-5-oxo-L-proline. The nonpathogenic A. tumefaciens strains present in the same tumors were unable to degrade any of the opines tested. The phylogenetic position of the tumorigenic fig strain AF3.10 was inferred from comparing its rrs (i.e., 16S rRNA gene) sequence with those from the type strains of Agrobacterium and Rhizobium species. The analysis showed that strain AF3.10 clustered with A. tumefaciens and A. rubi but not with A. vitis and was far removed from A. rhizogenes. However, the sequence was significantly different from those of A. tumefaciens and A. rubi to suggest that the tumorigenic fig strain may be a new Agrobacterium species that is as different from A. tumefaciens and A. rubi as these two species are from one another.

Arginine↗
PubMed1995

Stimulation of intracellular calcium concentration by adenosine triphosphate and uridine 5'-triphosphate in human term placental cells: evidence for purinergic receptors.

Recent data suggest an important role for calcium (Ca2+) in human placental endocrinology. Thus, the regulation of Ca2+ influx seems to be implicated in the modulation of human placental lactogen and hCG release. A possible mechanism of influx regulation is through receptor-operated channels. One of the most characterized receptor gating Ca2+ channels, the ATP receptor, stimulates the intracellular calcium concentration ([Ca2+]i) in various tissues. The aim of this study was to determine whether ATP receptors gating Ca2+ channels are also present in placental cells. We thus determined the effect of ATP on [Ca2+]i in human term trophoblastic cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. ATP stimulated a 4.3 +/- 0.4 (+/- SE)-fold increase in [Ca2+]i, with a half-maximal effective concentration (EC50) of 1.5 mumol/L. The pharmacological activation profile suggests the presence of purinergic P2u receptors (nucleotide receptors), because uridine 5'-triphosphate (UTP) also stimulated [Ca2+]i (4.0-fold increase, with an EC50 of 10 mumol/L). The ATP-stimulated [Ca2+]i was partly sensitive to pertussis toxin; we observed a 58% inhibition of ATP-induced [Ca2+]i with the toxin without effect on basal [Ca2+]i. The ATP- and UTP-stimulated [Ca2+]i declined with time in the presence of ATP (or UTP). The rate of deactivation was rapid (t1/2, < 60 s with 10(-5) mol/L ATP) and concentration dependent. The deactivation occurring during one application of ATP or UTP resulted in a diminution of subsequent responses. The recovery was incomplete even with long waiting times (up to 30 min). ATP and UTP also stimulated inositol phosphate production with EC50 values of 11 and 15 mumol/L, respectively, but not human placental lactogen or hCG release in experiments in which known secretagogues were effective. The results suggest the presence in human term placental cells of P2u receptors pharmacologically similar to those observed in other tissues, especially in the pituitary and amnion. The physiological significance of this stimulation of [Ca2+]i by ATP and UTP in the human placenta remains to be investigated.

Adenosine Triphosphate↗
PubMed1995

[Stenoses of the supra-aortic trunks associated with aortic supravalvular stenosis in a 3-year-old girl. Long-term surgical result].

Supravalvular aortic stenosis may be associated with arterial lesions. This condition is rare and its natural history is not well known. The authors report the case of a child operated in 1981 for supravalvular aortic stenosis and stenoses of the supraaortic vessels. The operation consisted of enlarging the ascending aorta with a patch and resection-reimplantation of both carotids without interposition of prosthetic material. Twelve years later, the haemodynamic result was the same as that of the postoperative control. This case tends to prove the low evolutive potential of this condition and the long term benefits of this surgical technique.

Aorta↗
PubMed1995

The use of digoxigenin-labelled probes to detect DNA sequences specific for plant pathogenic bacteria.

Southern blot hybridization is a valuable method in the assessment of the pathogenicity of bacterial strains or isolates. It is also a powerful tool for the demonstration of the presence of foreign DNA sequences in the genome of genetically-engineered plant cells. In this respect, cold, digoxigenin-labelled DNA probes can be used in place of classical radioactive probes, whether hybridizations are performed on bacterial genomic or plasmidic DNA, or on plant genomic DNA. The versatility of this cold labelling makes it suitable for the detection of unique bacterial genomic or plasmid sequences, even though these are located on large plasmids. The sensitivity of this cold probe technique also permits the detection of subpicogram quantities of DNA in plant genomic preparations. Their long term storage stability allows them to be frequently re-used over long periods of time, making this technique quite cost efficient.

Bacteria↗
PubMed1994

D2-dopamine agonists inhibit adenosine 3':5'-cyclic monophosphate (cAMP) production in human term trophoblastic cells.

We previously reported that dopamine (DA) acted via D2-dopamine receptors on human trophoblastic cells to inhibit basal and hormone-stimulated secretion of human placental lactogen (hPL). We also described that these DA effects were coupled with inhibition of calcium influx. The present study examines the interaction of placental D2-dopamine receptor with adenylate cyclase (AC). Incubations of isolated human term trophoblastic cells with R(-)-propylapomorphine (NPA), (+/-)-PPHT, and bromocriptine (3 different D2 agonists) led to time- and dose-dependent inhibitions of cAMP production as determined by measuring the conversion of [2-3H]-ATP into [2-3H]-cAMP. The maximal inhibition was reached after 15 min of incubation and was 33 +/- 1 (SE) %, 29 +/- 3% and 31 +/- 1% for bromocriptine (10(-5) M), NPA (10(-7) M) and (+/-)-PPHT (10(-8) M) respectively. However, the time- and dose-dependent curves were biphasic with NPA and (+/-)-PPHT and the inhibition of cAMP production was abolished at higher agonist concentrations or after time incubations longer than 15 min. These inhibitions were receptor specific since they were reversed by spiperone and haloperidol, two specific--dopamine antagonist, and by butaclamol (mix D2/D1-dopamine antagonists) but not by alpha- and beta-adrenergic, D1- and D4-dopaminergic, and 5-HT2-serotonergic antagonists. The results reported here suggest that human placental D2 receptors interact with AC to inhibit its activity. Also, bromocriptine seems a better agonist for the characterization of dopaminergic effects on human placenta.

Cells, Cultured↗
PubMed1994

Coexistence of Meyerson's with Sutton's naevus after sunburn.

We described the simultaneous occurrence of perinaevic eczema (Meyerson's naevi) and Sutton's halo naevus in one patient. Characteristic clinical and histological features of perinaevic eczema were found around four benign melanocytic naevi on the limbs. In addition, the patient had a typical Sutton's naevus on the back. The association of Meyerson's naevi and Sutton's naevus has been reported only once in the past literature. Both diseases appeared a few weeks after a severe sunburn. The role of sun exposure in the development of eczematiform and/or vitiligoid reactions around melanocytic naevi is discussed.

Adult↗
PubMed1994

Labelling of D2-dopaminergic and 5-HT2-serotonergic binding sites in human trophoblastic cells using [3H]-spiperone.

We previously reported that dopamine (DA) inhibited the release of human placental lactogen (hPL) from human placental cells. We also demonstrated the presence of D2-dopamine receptors in membrane preparations of human term placenta. The aim of the present study was to characterize these D2 receptors on freshly isolated human trophoblastic cells. The binding of [3H]-spiperone to these cells showed a curvilinear Scatchard plot suggesting the presence of two classes of binding sites (Kd1 = 1.26nM; Kd2 = 44.3nM). Competition experiments showed the following inhibitory binding potencies: serotonin-2 (5-HT2) > or = D2 >>> alpha-adrenergic, beta-adrenergic, D1-dopamine, thus suggesting the presence of 5-HT2 binding sites. We have examined this possibility by blocking [3H]-spiperone binding to 5-HT2 receptors in the presence of 50nM ketanserin, a selective antagonist of 5-HT2 sites. Under this condition, the linear Scatchard plot obtained suggested a single population of homogeneous binding sites for [3H]-spiperone with a Kd of 0.55nM. To further characterize placental D2 receptors we conducted binding experiments with [3H]-raclopride, an more selective D2 antagonist. The linear Scatchard plot obtained with this ligand suggested one class of binding sites for [3H]-raclopride (Kd = 6nM) with the following inhibitory potencies: D2 >>> beta-adrenergic >> 5-HT2, D1, alpha-adrenergic. These results suggest an important paracrine function for DA in human placenta and show for the first time that [3H]-spiperone binds putative 5-HT2 receptors in human placenta.

Humans↗
PubMed1994

[Valvuloplasty of a tricuspid bioprosthesis by the Inoue's technique].

The authors report the case of a patient who had undergone tricuspid valve replacement with a bioprosthesis in 1985 after infectious endocarditis complicated by paradoxical embolism. The appearance of signs of right heart failure eight years later led to the diagnosis of stenotic degeneration of the bioprosthesis. Inoue balloon valvuloplasty was performed with no complications leading to significant improvement in the patient's condition.

Adult↗
PubMed1994

Comparison of omeprazole and famotidine on esophageal pH in patients with moderate to severe esophagitis: a cross-over study.

OBJECTIVES: This open cross-over study compared the effects on esophageal pH of omeprazole (O) (20 mg once a day) and famotidine (F) (40 mg b.i.d.) in 19 patients with proven acid gastroesophageal reflux (GER) complicated by erosive or ulcerated esophagitis. METHODS: Each drug was taken for 7 days. A wash-out interval of at least 3 days separated the two treatment periods in each subject. Twenty-four-hour pH measurements were performed in similar standardized conditions at 7 +/- 2 days, at the end of each period of treatment. RESULTS: Compared with the pretreatment results, both O and F reduced the following pH parameters: percent of time with esophageal pH < 4, total number of GER episodes, number of nocturnal GER episodes, and duration of the longest GER episodes. O was more effective than F except for the percent of time esophageal pH < 4 and the number of nocturnal GER episodes, not different between the two treatments. With O, GER was reduced to physiological value in 19 of the 19 patients and abolished in 13. With F, GER was normalized in 13 and abolished in four. The effects of both drugs and grading of esophagitis were not correlated. Both treatments were well-tolerated. CONCLUSIONS: In patients with acid GER complicated by ulcerative esophagitis, O, 20 mg daily, and F, 40 mg b.i.d. significantly reduced acid exposure. O was superior to F, but the latter drug improved significantly two important prognostic variables: the total percent of time pH < 4 and the nocturnal GER.

Adult↗
PubMed1993

Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes.

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.

Base Sequence↗
PubMed1993

The modulation of placental lactogen release by opioids: a role for extracellular calcium.

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 micrograms/ml streptomycin. 45Ca2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca2+ influx by ethylketocyclazocine (EKC) with an EC50 of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50,488 (kappa agonist) had the same stimulatory effect as EKC (221 +/- 25% of control). D-Ala2,NMe-Phe4,Gly-ol5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca2+ influx (128 +/- 5% of control, p > 0.05) whereas D-Ser2,Leu,Thr6)-enkephalin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗