PubMed Health⌕ Search

Biomedical subjects

P Legendre

Publications and source records attributed to P Legendre.

At least 19 recordsLinked to original sources
PubMed2006

Effect of von Willebrand disease type 2B and type 2M mutations on the susceptibility of von Willebrand factor to ADAMTS-13.

BACKGROUND: von Willebrand disease (VWD) type 2 is associated with mutations in von Willebrand factor (VWF) that affect its secretion, multimeric pattern, affinity for platelet receptors and clearance of the protein. While increased proteolysis by a disintegrin-like and metalloprotease with thrombospondin type 1 motifs-13 (ADAMTS-13) has been clearly established for VWF type 2A, only little is known about VWF types 2B and 2M in this regard. OBJECTIVES: Sensitivity of wild-type (WT) and mutated recombinant (r) VWF to proteolysis by ADAMTS-13 was investigated to better understand the role of this process in the pathophysiology of VWD. METHODS: We used human rADAMTS-13-WT to digest 11 full-length recombinant forms of VWF carrying molecular abnormalities identified in patients with VWD type 2A (E1638K and P1648S), type 2B (InsM1303, R1306W, R1308P and V1314F) and type 2M (G1324A, E1359K, K1362T, R1374H and I1425F). RESULTS: Using low ionic strength conditions, all mutations induced increased proteolysis of rVWF by rADAMTS-13 as compared with rVWF-WT. The susceptibility of mutants decreased in the following order: type 2A > type 2B > type 2M > rVWF-WT. At physiological salt concentration (150 mm NaCl) the sensitivity of all rVWF to rADAMTS-13 was significantly decreased. However, type 2A and type 2B mutants still exhibited a significantly higher susceptibility to rADAMTS-13 than rVWF-WT, whereas type 2M mutants normalized. CONCLUSIONS: Type 2M mutants and rVWF-WT exhibit a similar sensitivity to rADAMTS-13-mediated proteolysis, in agreement with the normal multimeric pattern in vivo. In VWD type 2B, the spontaneous binding to platelets and excessive degradation by ADAMTS-13 of VWF high-molecular-weight multimers may account for their clearance from plasma.

ADAM Proteins↗
PubMed2006

Transforming growth factor alpha promotes sequential conversion of mature astrocytes into neural progenitors and stem cells.

An instability of the mature cell phenotype is thought to participate to the formation of gliomas, primary brain tumors deriving from astrocytes and/or neural stem cells. Transforming growth factor alpha (TGFalpha) is an erbB1 ligand overexpressed in the earliest stages of gliomas, and exerts trophic effects on gliomal cells and astrocytes. Here, we questioned whether prolonged TGFalpha exposure affects the stability of the normal mature astrocyte phenotype. We first developed astrocyte cultures devoid of residual neural stem cells or progenitors. We demonstrate that days of TGFalpha treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors. TGFalpha-generated radial glial cells support embryonic neurons migration, and give birth to cells of the neuronal lineage, expressing neuronal markers and the electrophysiological properties of neuroblasts. Lengthening TGFalpha treatment to months results in the delayed appearance of cells with neural stem cells properties: they form floating cellular spheres that are self-renewing, can be clonally derived from a single cell and differentiated into cells of the neuronal lineage. This study uncovers a novel population of mature astrocytes capable, in response to a single epigenetic factor, to regress progressively into a neural stem-like cell stage via an intermediate progenitor stage.

Animals↗
PubMed2006

Frequency-dependent modulation of glycine receptor activation recorded from the zebrafish larvae hindbrain.

In vertebrates, most glycinergic inhibitory neurons discharge phasically at a relatively low frequency. Such a pattern of glycine liberation from presynaptic terminals may affect the kinetics of post-synaptic glycine receptors. To examine this influence, we have analyzed the behavior of glycine receptors in response to repetitive stimulation at frequencies at which consecutive outside-out currents did not superimpose (0.5-4 Hz). Neurotransmitter release was mimicked on outside-out patches from zebrafish hindbrain Mauthner cells using fast flow application techniques. The amplitude of outside-out currents evoked by short (1 ms) repetitive applications of a saturating concentration (3 mM) of glycine remained unchanged for application frequencies 1 Hz. Glycine-evoked current simulations using a simple Markov model describing zebrafish glycine receptor kinetic behavior, indicates that this down-regulation of glycine receptor efficacy is due to a progressive accumulation of the receptors in a long lasting desensitization state. Our simulations suggest that this down-regulation can occur even when spontaneous inhibitory currents were generated randomly at a frequency>1 Hz.

Animals↗
PubMed2006

CHO cells expressing the high affinity alpha(IIb)beta3 T562N integrin demonstrate enhanced adhesion under shear.

BACKGROUND: Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. OBJECTIVES: To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor. MATERIALS AND METHODS: We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. RESULTS: At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N. CONCLUSIONS: Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.

Animals↗
PubMed2003

Kinetic properties of the alpha2 homo-oligomeric glycine receptor impairs a proper synaptic functioning.

Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express alpha2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly alpha1/beta heteromeres). Here, the kinetics properties of GlyRs at the single-channel level have been recorded in real-time by means of the patch-clamp technique in the outside-out configuration coupled with an ultra-fast flow application system (< 100 micros). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the alpha2 GlyR subunit. We show that the onset, the relaxation and the desensitisation of alpha2 homomeric GlyR-mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand-gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mM glycine; 1 ms pulse duration), the opening probability of alpha2 homomeric GlyRs was low (P(o) approximately = 0.1) when compared to mature synaptic GlyRs (Po = 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of alpha2 homomeric GlyRs when compared to the activation kinetics of mature alpha1/beta GlyRs. Such slow kinetics suggest that embryonic alpha2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non-synaptic paracrine-like release of agonist, which is known to occur in the embryo.

Algorithms↗
PubMed2002

Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra.

The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single-channel and whole-cell patch-clamp recordings in brain slices from rats aged 7-21 postnatal days (P). In neonatal rats (P7-P10), GlyRs exhibited a main conductance state of 100-110 pS with a mean open time of 16 ms. In juvenile rats (P19-P22), both the GlyR main conductance state (46-55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 microM picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine-evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to alpha(2) homomeric GlyRs in neonatal rats and alpha(1)/beta heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch from the homomeric state to the heteromeric state. This age corresponds to a maturation phase for DA neurones. The application of 1 microM gabazine blocked spontaneous or evoked inhibitory synaptic current, while the addition of 1 microM strychnine had no effect, suggesting a lack of functional glycinergic synapses on DA neurones. Although it has been proposed that taurine is co-released with GABA at GABAergic synapses on DA neurones, in the present study the stimulation of GABAergic fibres failed to activate GlyRs. Blockade of taurine transporters and applications of high K(+) and hyposmotic solutions were also unable to induce any strychnine-sensitive current. We conclude that functional maturation of GlyRs can occur in the absence of any detectable GlyR activation in DA neurones of the SNc.

Animals↗
PubMed2001

The glycinergic inhibitory synapse.

Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has yielded new insights on glycine neurotransmission. Glycinergic synapses are now known not to be restricted to the spinal cord and the brainstem. Presynaptic machinery for glycine release and uptake, the structure and function of postsynaptic receptors and the factors (both pre- and postsynaptic) which control the strength of glycinergic inhibition have been extensively studied. It is now established that glycinergic synapses can be excitatory in the immature brain and that some inhibitory synapses can corelease gamma-aminobutyric acid (GABA) and glycine. Moreover, the presence of glycine transporters on glial cells and the capacity of these cells to release glycine suggest that glycine may also act as a neuromodulator. Extensive molecular studies have revealed the presence of distinct subtypes of postsynaptic glycine receptors with different functional properties. Mechanisms of glycine receptors aggregation at postsynaptic sites during development are better understood and functional implications of variation in receptor number between postsynaptic sites are partly elucidated. Mutations of glycine receptor subunits have been shown to underly some human locomotor disorders, including the startle disease. Clearly, recent work on glycine receptor channels and the synapses at which they mediate inhibitory signalling in both young and adult animals necessitates an update of our vision of glycinergic inhibitory transmission.

Allosteric Regulation↗
PubMed2001

High-affinity zinc potentiation of inhibitory postsynaptic glycinergic currents in the zebrafish hindbrain.

Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC(50), 0.5-1 microM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR response in control conditions. Using tricine- (10 mM) buffered zinc solution, we then showed that zinc increases the amplitude of outside-out responses evoked by 0.1-0.5 mM glycine with an EC(50) of 15 nM. In contrast zinc had no effect on the amplitude of currents evoked by a saturating concentration (3-10 mM) of glycine. This suggests that zinc enhances GlyR apparent affinity for glycine. The study of the effects of zinc on the kinetics of the response indicates that this increase of apparent affinity is due to a decrease of the glycine dissociation rate constant. We then analyzed the effects of zinc on postsynaptic GlyRs in whole cell recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Chelation of contaminant heavy metals decreased the amplitude and the duration of the mIPSCs; inverse effects were observed by adding zinc in buffered solutions containing nanomolar free zinc concentrations. Zinc plus tricine or tricine alone did not change the coefficient of variation ( approximately 0.85) of the mIPSC amplitude distributions. These results suggest that postsynaptic GlyRs are not saturated after the release of one vesicle.

Animals↗
PubMed2001

Limits to the development of fast neuromuscular transmission in zebrafish.

Zebrafish embryos have small and slow miniature end-plate currents (mEPCs), whereas only a few days later larval mEPCs are an order of magnitude larger and faster, being among the fastest of all neuromuscular synapses. To identify the bases for these changes we compared, in embryos and larvae, the properties and distributions of acetylcholine (ACh) receptors (AChRs) and acetylcholinesterase (AChE) as well as the ultrastructure of the developing neuromuscular junctions (NMJs). To mimic synaptic release, patches of muscle membrane were exposed briefly (for 1 ms) to a saturating concentration (10 mM) of ACh. The AChR deactivation kinetics were twice as slow in embryos compared with larvae. In both embryos and larvae, AChRs demonstrated open channel block by millimolar ACh, and this was detected during mEPCs, indicating that a high concentration of ACh is released at immature and mature NMJs. AChR and AChE distributions were compared using the selective fluorescently conjugated labels alpha-bungarotoxin and fasciculin 2, respectively. In larvae, punctate AChR clusters were detected whereas junctional AChE staining was less intense than that found at adult NMJs. Transmission electron microscopy revealed immature nerve endings in embryos that were closely juxtaposed to the surrounding muscle cells, whereas mature larval NMJs had a wider synaptic cleft with a conspicuous basal lamina over a limited region of synaptic contact. Our results indicate that ACh is released at high concentrations at immature NMJs, but its clearance is prolonged and the AChRs are dispersed, resulting in a slow mEPC time course until a mature cleft appears with densely packed faster AChRs and abundant AChE.

Acetylcholine↗
PubMed2001

Clinical and laboratory findings of cytomegalovirus infection in 115 hospitalized non-immunocompromised adults.

We report a retrospective study of 115 hospitalized non-immunocompromised adults with proved or presumed diagnosis of cytomegalovirus infection. Clinical symptoms were fever (95%), constitutive symptoms (80%), joint and muscle pain (41%), shivering (32%), abdominal pain (26%), non-productive cough (20%), cutaneous eruption (20%), and diarrhea (10%). Examination found hepatomegaly (25%), splenomegaly (23%), cutaneous rash (20%), adenopathy (19%), pharyngitis (9%), jaundice (3%) or signs of meningeal irritation (1%). Seventeen patients had a gastrointestinal form (hepatitis, jaundice, colitis, antral gastritis or cholecystitis), eight had a pattern of hemopathy, two interstitial pneumonitis, two pericarditis, two immune thrombocytopenic purpura, two a polymyalgia rheumatica-like pattern, one thrombotic thrombocytopenic purpura, one cutaneous vasculitis and one meningoencephalitis. Sixty-four percent of the patients had atypical lymphocytosis. Hepatocellular injury occurred in 90% of the patients. Nineteen of the patients had biological immune abnormalities. Cytomegalovirus infection should be mainly suspected in any patient with persistent fever, isolated or associated with signs of poor specificity, or in some patients with visceral manifestations of initially unknown origin.

Adolescent↗
PubMed2001

Defect of heparin binding in plasma and recombinant von Willebrand factor with type 2 von Willebrand disease mutations.

The aim of our study was to characterise heparin-binding properties of mutated von Willebrand factor (VWF) in 24 patients plasmas with type 2 von Willebrand disease (VWD). and in 15 recombinant VWF (rVWF) with the corresponding mutations. Binding of mutated rVWF or plasma VWF was compared to that of WT-rVWF or normal pool plasma VWF. Four mutations, at positions C509, V551, R552 and R611 lead to significantly decreased binding to heparin in both plasma and rVWF. Interestingly, whereas these four residues are distant in the primary structure of VWF-A1domain, they are close to each other in its three-dimensional structure. Structural analysis suggested how folding problems and destabilisation due to these mutations could induce reorganisation of surface regions involved in heparin binding. In contrast, no heparin-binding defect was found associated with different type 2 VWF mutants, at positions G561, E596, I662, R543, R545, V553, R578 or L697.

Amino Acid Substitution↗
PubMed2001

Neuromuscular transmission on the rebound.

Recent work at the zebrafish neuromuscular junction (NMJ) has shown that positively charged acetylcholine (ACh), at the high concentrations reached in the cleft during neuromuscular transmission, blocks acetylcholine receptors (AChRs) as soon as they open. Thus after two ACh molecules bind and open the channel, a third molecule enters and blocks the pore at a site resembling that for block by local anesthetics, suggesting that ACh is the endogenous anesthetic of the NMJ. Recovery from open channel block results in a rebound synaptic current only after ACh is cleared from the cleft. Kinetic modeling of other AChRs suggests that a rebound current is generated at all vertebrate NMJs, from fish to frogs to mammals. Open channel block prolongs the current at fast zebrafish NMJs in order to more effectively spread charge along the fibers, akin to multiple central synapses spread over dendrites. Together these findings indicate the need for a fundamental revision of current thinking about neuromuscular transmission at many levels, including channel structure, function and pharmacology.

Acetylcholine↗
PubMed2000

Recovery from open channel block by acetylcholine during neuromuscular transmission in zebrafish.

At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recorded in vivo have an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect of synaptic release onto small numbers of ACh receptor channels (AChRs). Positively charged ACh acted at hyperpolarized potentials and at millimolar concentrations as a fast ("flickering") open channel blocker of AChRs. Because of filtering, the open channel block resulted in reduced amplitude of single channel currents. Immediately after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound current depended on the ACh concentration and resembled in time course the mEPC. A simple kinetic model of the AChR that includes an open channel-blocking step accounted for our single channel results, as well as the experimentally observed slowing of the time course of mEPCs recorded at a hyperpolarized compared with a depolarized potential. Recovery from AChR block is a novel mechanism of synaptic transmission that may contribute in part at all NMJs.

Acetylcholine↗
PubMed2000

[Hematologic and immunologic manifestations of primary cytomegalovirus infections in non-immunocompromised hospitalized adults].

PURPOSE: Cytomegalovirus (CMV) infection in non-immunocompromised adults can sometimes induce hematological and immunological disorders that may mislead diagnosis. METHODS: Case reports of hospitalized non-immunocompromised adults with positive serology for CMV including the presence of immunoglobulin M or seroconversion were assessed in a retrospective study (1981-1998). We focused on clinical and biological abnormalities showing the role of CMV in disruption of functioning of hematological and immunological systems. RESULTS: Among 115 patients, lymphoma-like syndrome with large adenopathies and/or splenomegaly was diagnosed in eight patients, uncovering underlying CMV infection. Lymphoma was accompanied by hematoma in two patients. Three patients presented leg purpura (with thrombotic thrombocytopenic purpura in one case), one patient had cutaneous vasculitis and on other a Still's disease. Blood abnormalities were mononucleosis (64%), anemia (20%), and thrombopenia (25%) often of peripheral or hemolytic origin or due to hypersplenia. Electrophoresis of serum proteins showed an increase in immune globulins in 56% of the cases and monoclonal abnormality in nine cases. Immunological assessment was conducted in 18 patients. At least one abnormality was depicted in ten patients, consisting of either antinuclear, anti-platelet or anti smooth muscle antibodies, cryoglobulinemia, rheumatoid factor, or reduced complement fixation. CONCLUSION: Testing for CMV infection can be of value in case of blood or immunological disorders associated with clinical or biological signs. Although hematological disorders occur early, they are rarely severe. Immunological disorders are rarely symptomatic, but often raise issues regarding the potential genesis of immune diseases in at-risk patients.

Adolescent↗
PubMed2000

von Willebrand factor binding to heparin in various types of von Willebrand disease.

INTRODUCTION: The purpose was to study von Willebrand factor (vWF) binding to heparin in different types of von Willebrand disease (vWD). MATERIALS AND METHODS: Plasma samples from 92 patients were representative of most vWD subtypes as they included 13 type 1, ten type 2N, 27 type 2A, 23 type 2B, and 19 type 2M patients. We selected assay conditions suitable for the screening of plasma vWF concentrations as low as 15 U/dl vWF:Ag. We determined the range of vWF concentrations in plasma where the percentage of (125)I-MAb/vWF complexes bound to heparin-agarose beads was constant. This range of dilution allowed circumvention of potential competition by other plasma heparin-binding proteins. RESULTS: The multimeric composition of vWF had hardly any influence on the ability of vWF to bind to heparin. Results were expressed as the ratio of heparin-binding capacity of patients' plasma to that of normal pool plasma. We found a ratio of 0.99+/-0.004 (mean+/-s.e.m.) for 23 normal individual donors. Furthermore, when comparing the mean values of plasma vWF-heparin binding ratios by ANOVA F-test in the six groups (one normal and five vWD), we found significant differences between them (P<0.0001). Pairwise comparison of multiples by the Scheffe's test indicated that the mean values of ratios in type 2A on the one hand and type 2M on the other, were significantly lower than in normal plasma, type 2N, type 2B and type 1. CONCLUSION: Our data suggest a relationship between the ability of vWF to bind to heparin and to the platelet GPIb receptor, since type 2B and 2N patients have an increased or normal ability to bind to GPIb whereas type 2A and 2M patients have an impaired interaction with that receptor.

Antibodies, Monoclonal↗
PubMed2000

Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo.

We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 microM), kynurenic acid (1 mM), and bicuculline (10 microM) and were found to be sensitive to strychnine (1 microM). The mIPSCs were first observed in 26-29 h postfertilization (hpf) embryos at a very low frequency of approximately 0.04 Hz, which increased to approximately 0.5 Hz by 30-40 hpf, and was approximately 10 Hz in newly hatched (>50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar ( approximately 100 pA), suggesting rapid formation of the postsynaptic matrix. The 20-80% rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in larvae (approximately 0.3 ms), likely due to slower diffusion of glycine at the younger, immature synapses. The mIPSCs decayed with biexponential (tau(off1) and tau(off2)) time courses with a half-width in 26-29 hpf embryos that was longer and more variable than in older embryos and larvae. In 26- to 29-hpf embryos, tau(off1) was approximately 15 ms and tau(off2) was approximately 60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where tau(off1) was approximately 40 ms and tau(off2) was approximately 160 ms. In 30-40 hpf embryos, the events were faster, with tau(off1) approximately 9 ms and tau(off2) approximately 40 ms, and in larvae, events declined somewhat further to tau(off1) approximately 4 ms and tau(off2) approximately 30 ms. Point-per-point amplitude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as approximately 45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) from the onset of synaptogenesis. Fast-flow (1 ms) application of a saturating concentration of glycine (3-10 mM) to outside-out patches obtained at 26-29 hpf revealed GlyR currents that decayed biexponentially with time constants resembling the values found for intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.

Animals↗
PubMed1999

Modulation by heparin of the interaction of the A1 domain of Von Willebrand factor with glycoprotein Ib.

The conformation of the A1 domain of von Willebrand factor (vWF) is a critical determinant of its interaction with the glycoprotein (GP) Ib/V/IX complex. To better define the regulatory mechanisms of vWF A1 domain binding to the GPIb/V/IX complex, we studied vWF-dependent aggregation properties of a cell line overexpressing the GPIbalpha, GPIbbeta, and GPIX subunits (CHO-GPIbalphabeta/IX cells). We found that CHO-GPIbalphabeta/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbalphabeta/IX cell aggregation was completely inhibited by the recombinant VCL fragment of vWF that contains the A1 domain. Surprisingly, the substitution of heparin for ristocetin resulted in the formation of CHO-GPIbalphabeta/IX cell aggregates. Using monoclonal antibodies blocking vWF interaction with GPIb/V/IX or mocarhagin, a venom metalloproteinase that removes the amino-terminal fragment of GPIbalpha extending from aa 1 to 282, we demonstrated that both ristocetin- and heparin-induced aggregations involved an interaction between the A1 domain of vWF and the GPIbalpha subunit of the GPIb/V/IX complex. The involvement of heparin in cell aggregation was also demonstrated after treatment of heparin with heparinase that abolished CHO-GPIbalphabeta/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbalphabeta/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.

Animals↗