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Biomedical subjects

V Picard

Publications and source records attributed to V Picard.

29 records · Page 2Linked to original sources
PubMed1995

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Animals↗
PubMed1995

Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the beta-antithrombin III isoform with enhanced heparin affinity.

Two antithrombin III (ATIII) isoforms occur naturally in human plasma. The alpha-ATIII isoform has four N-linked oligosaccharides attached to asparagines 96, 135, 155, and 192. The beta-ATIII isoform lacks carbohydrate on asparagine-135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does alpha-ATIII. Two isoforms are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant beta' isoform similarly binds heparin with higher affinity than the recombinant alpha' isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. On the basis of database and in vitro glycosylation studies suggesting that N-X-S CSs are utilized less efficiently than N-X-T CSs, we hypothesized that the beta-ATIII isoform might result from inefficient core glycosylation of the N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of alpha' and beta' molecules, the N-X-T variant produced alpha' exclusively, while the N-X-A variant produced beta' exclusively. Thus, serine in the third position of the N135 CS is responsible for its "partial" glycosylation and leads to production of beta-ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Human thrombin variable region 1, including E39, is involved in interactions with alpha 1-antitrypsin M358R and protein C.

We used an antithrombin autoantibody (IgG D), the epitope of which encompasses ABE1 and amino acids located within variable region 1, to study thrombin interactions with R358 alpha 1-AT and protein C. IgG D inhibited the thrombin interaction with R358 alpha 1-AT, while hirugen had no effect, indicating that the interaction of R358 alpha 1-AT with thrombin may involve the VR1 subsite. We also obtained evidence that VR1 may be involved in the activation of protein C by thrombin in the absence of thrombomodulin. Moreover, IgG D attenuated the inhibitory effect of calcium ions during protein C activation by thrombin, probably by masking E39 within the VR1 site.

Amino Acid Sequence↗
PubMed1995

Immunosuppressive activities of 6(5H)-phenanthridinone, a new poly(ADP-ribose)polymerase inhibitor.

6(5H)-phenanthridinone, a recently identified poly(ADP-ribose)polymerase (PARP) inhibitor, is able, at micromolar concentrations, to inhibit concanavalin A-induced lymphocyte proliferation and to potentiate the effect of gamma radiation upon murine spleen cells. When added at the onset of a mixed lymphocyte culture, this compound strongly depresses the induction of primary allogeneic (anti-H2k) cytotoxic T-lymphocytes (CTLs). Lymphokine-activated killer (LAK) induction was also found to be impaired by the PARP inhibitor. Taken together, these results clearly indicate that PARP plays a key-role in immune reactions involving cytotoxicity and that 6(5H)-phenanthridinone could be considered as a potent immunomodulator.

Acridines↗
PubMed1994

An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation.

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion-binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration-dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma-thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion-binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.

Anions↗
PubMed1994

A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase.

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

DNA-Directed DNA Polymerase↗
PubMed1994

Activation-induced exposure of the thrombin anion-binding exosite. Interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite-specific antibody.

The activation of serine protease zymogens involves conformational changes that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.

Amino Acid Sequence↗
PubMed1991

Comparative evaluation of five agglutination techniques and a new miniaturized system for rapid identification of methicillin-resistant strains of Staphylococcus aureus.

The speciation of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant diagnostic problem when rapid identification methods such as slide agglutination tests, are used, because of the high proportion of false-negative reactions. 150 perfectly identified MRSA strains were tested on 5 commonly used agglutination reagents ("Bacto staph latex test", "Monostaph", "Pastorex staph", "Staphaurex", and "Staphyslide test") in comparison with a new micromethod ("RAPIDEC staph") which detects a type of staphylocoagulase within 2 hours by a fluorescence test. The "RAPIDEC staph" reagent enabled identification of all the MRSA while the agglutination tests gave poorer results: "Monostaph" correctly identified 64.6% of strains, "Staphyslide", 59.3%, "Bacto staph latex test", 44.6%, "Pastorex staph", 38.6% and "Staphaurex", 28.6%. These results show that agglutination slide tests are not reliable enough for the identification of MRSA which are more and more encountered in hospital wards. The authors recommend not to use slide agglutination methods. They suggest the tube test for coagulase which is the reference technique, although it is time-consuming and not well standardized. The results of this evaluation encourage the use of the "RAPIDEC staph" reagent since it is an easy-to-use, reliable technique for the rapid identification of Staphylococcus aureus.

Agglutination Tests↗
PubMed1983

Effect of acute and chronic 17 beta-estradiol treatment on serotonin and 5-hydroxyindole acetic acid content of discrete brain nuclei of ovariectomized rat.

Increased serotonin content of the dorsal raphe nucleus and of the substantia nigra were seen following acute (12 h or 24 h) administration of 17 beta-estradiol to ovariectomized rats. No changes were seen in numerous other nuclei of forebrain or hindbrain, and no changes occurred during chronic (1 month) estradiol administration. Changes in the concentration of the major serotonin metabolite, 5-hydroxyindole acetic acid, paralleled those of serotonin. These results show that, under precisely defined conditions and in specific locations only, estradiol can modulate serotonergic neuronal systems.

Animals↗
PubMed1983

Perturbation of rat brain serotonergic systems results in an inverse relation between substance P and serotonin concentrations measured in discrete nuclei.

Since substance P (SP) has been demonstrated to coexist with serotonin (5-HT) in the same population of neurons in the descending raphe system, we have studied the possibility of interactions between these neurotransmitters in other brain areas. Brain nuclei were punched from frozen 300-micron slices of rat brain and extracted with 0.1 M HCIO4 or 2 M acetic acid prior to assay, respectively, of 5-HT content by HPLC with electrochemical detection or SP content by specific radioimmunoassay. Ten days after injection of rats with the 5-HT neurotoxin P-chloroamphetamine (PCA, 10 mg/kg, B.W., i.p.) or 3 days after 5-HT synthesis blockade with p-chlorophenylalanine (PCPA, 300 mg/kg, B.W., i.p.), the 5-HT content of all brain nuclei studied was reduced by means of, respectively, 50% and 81%. In PCA-treated animals, the SP content of the periaqueductal grey matter was significantly increased; PCPA treatment caused, in addition, large increases in the SP content of five other brain nuclei. Blockade of 5-HT receptors by methysergide (15 mg/kg for 5 days) did not significantly change 5-HT levels or turnover, but resulted in 50-200% increases in the SP content of 10 of the 28 brain nuclei studied. Significant decreases in the SP content of numerous areas were seen following treatments (pargyline 30 mg/kg, alone or in combination with 5-hydroxytryptophan, 60 mg/kg) that simultaneously increased 5-HT levels. These results illustrate the modulation of distinct SP-containing systems of the rat brain by perturbation of central serotoninergic pathways and indicate a reciprocal relationship between the SP and 5-HT concentrations of numerous brain nuclei, in particular n. striae terminalis, n. raphe dorsalis, n. accumbens, n. septi, substantia grisea centralis, and n. raphes medianus.

Animals↗