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Biomedical subjects

M Vasseur

Publications and source records attributed to M Vasseur.

At least 19 recordsLinked to original sources
PubMed2000

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane.

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

Amino Acid Sequence↗
PubMed2000

Rotavirus infection impairs intestinal brush-border membrane Na(+)-solute cotransport activities in young rabbits.

The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both Na(+)-D-glucose (SGLT1) and Na(+)-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizin-binding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of Na(+)-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established.

Age Factors↗
PubMed1999

Heterogeneity of pig intestinal D-glucose transport systems.

Heterogeneity of intestinal D-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two D-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a D-glucose and D-mannose transporter exhibiting no preference for Na(+) over K(+). A nonsaturable D-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker L-glucose. It is also shown that, in this kinetic work, 1) use of D-glucose-contaminated D-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range (>/=50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion.

Animals↗
PubMed1998

Direct inhibitory effect of CCCP on the Cl(-)-H+ symporter of the guinea pig ileal brush-border membrane.

The effect of carbonyl cyanide-m-chlorophenylhydrazone (CCCP) on Cl- uptake across the brush-border membrane (BBM) was quantified using 36Cl and BBM vesicles from guinea pig ileum. CCCP inhibited only partially both the pH gradient-activated Cl- uptake and Cl-/Cl- exchange activities present in these vesicles. In contrast, CCCP had no effect on the initial (2-30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the main action of CCCP does not consist of dissipating any imposed pH gradient but rather in inhibiting directly the pH gradient-activated Cl- uptake and Cl-/Cl- exchange activities characterizing the intestinal BBM. Because these two activities can be explained in terms of a single (homogeneous) random, nonobligatory two-site Cl(-)-H+ symporter, in which Cl-/Cl- exchange occurs by counterflow [F. Alvarado and M. Vasseur. Am. J. Physiol. 271 (Cell Physiol. 40): C1612-C1628, 1996], we developed a new, more general three-site symport model that fully explains the Cl- uptake inhibitions caused by CCCP. This new model postulates the existence of a third, allosteric, inhibitory CCCP-binding site separate from either of the two substrate-binding sites of the Cl(-)-H+ symporter, the Cl(-)-binding and the H(+)-binding sites. Finally, we show that, to explain the partial inhibitions observed, it is necessary to postulate that all the substrate-bound carrier complexes, = C-S, I = C-S, A = C-S, and IA = C-S, where C is carrier, I is inhibitor, S is substrate, and A is activator, can form and be translocated.

Animals↗
PubMed1997

pH gradient effects on chloride transport across basolateral membrane vesicles from guinea-pig jejunum.

1. The effects of alkaline-inside pH gradients on 36Cl- uptake were quantified by using brush-border membrane (BBM) and basolateral membrane (BLM) vesicles from guinea-pig jejunum. 2. With BBM vesicles, a pHo/pHi gradient of 5.0/7.5 yielded fast overshoots involving a random, non-obligatory Cl(-)-H+ symport, strongly inhibited by CCCP. In contrast, BLM vesicles responded to similar pH gradients with much smaller, delayed overshoots, unaffected by CCCP. 3. The initial Cl- entry rates into BLM vesicles were a function of each pHo, pHi and delta pH value. They were stimulated by valinomycin in the presence of inward-directed K+ gradients. Short-circuiting the membrane potential with equilibrated K+ and valinomycin inhibited pH gradient-dependent Cl- uptake, but only partially. 4. Taken together, these results indicate that guinea-pig jejunal BLM vesicles possess both Cl- conductance and Cl(-)-H+ symport activities. 5. Even when different, the BBM and the BLM symporters are mechanistically similar. Neither of them involves a Cl(-)-OH- antiport, nor a simultaneous Cl(-)-anion exchange mechanism. Rather, for each membrane, all of these activities (symport, anion exchange) can be explained in terms of a single mobile carrier acting as a random, non-obligatory Cl(-)-H+ symporter where exchange occurs simply by counterflow. Net Cl- translocation via either the ternary (Cl(-)-C-H+) or the binary (Cl(-)-C) complexes accounts, respectively, for the existence of two, operationally distinct, electroneutral and rheogenic components. 6. The BBM symporter appears to involve an AE2 protein, but the molecular identity of the BLM one remains to be established.

Animals↗
PubMed1997

Solubilized and cleaved VP7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles.

It has been previously shown that rotavirus triple-layered particles induce permeabilization of liposomes and membrane vesicles. These effects were mediated by one or both of the solubilized outer-capsid proteins, VP4 and VP7. Permeabilization was dependent on trypsin treatment of the viral particles, suggesting that VP4 was involved. To analyse the respective roles of the outer-capsid proteins in this permeabilization process, we have used membrane vesicles loaded with carboxyfluorescein and virus-like particles derived from insect cells co-expressing various sets of capsid proteins. Virus-like particles containing VP2, VP6 and VP7 (VLP2/6/7) are as efficient in permeabilizing vesicles as triple-layered particles. As with double-layered particles, virus-like particles made of VP2 and VP6 had no effect on vesicle permeabilization. Permeabilization of membrane vesicles required trypsinization of the VP7 solubilized from VLP2/6/7. These results show that solubilized and trypsinized VP7 is able to induce membrane permeabilization, independently of the presence of VP4.

Antigens, Viral↗
PubMed1996

Crosslinking of double-stranded oligonucleotides containing O-methyl-substituted pyrophosphate groups to the HNF1 transcription factor in nuclear cell extract.

Probing of the HNF1 (hepatocyte nuclear factor I) DNA-binding region using a set of DNA duplexes containing pyrophosphate or O-methyl-substituted pyrophosphate internucleotide groups at different positions of the HNF1 recognition sequence was performed. The histidine-tagged HNF1/1-281 DNA binding domain and nuclear extract from rat liver were used. We showed that HNF1 from these species specifically binds to modified DNA duplexes. A correlation in binding affinity of both types of duplexes was detected. Crosslinking of the HNF1 DNA-binding domain and HNF1 in nuclear liver extract to DNA duplexes carrying O-methyl-substituted pyrophosphate groups was observed. The crosslinking efficiency of HNF1 in liver extract to substituted pyrophosphate-modified DNA duplex, containing a reactive internucleotide group between nucleotides G and T of the GT dinucleotide immediately 5' to the TAAT recognition sequence, amounts to 40% of the efficiency of non-covalent association. Nonspecific crosslinking of the reactive DNA duplexes to other components of nuclear extract was not observed. These results indicate that DNA duplexes carrying substituted pyrophosphate internucleotide groups can specifically bind and crosslink with DNA-binding proteins, especially transcription factors in crude preparations and could constitute a potential tool to control the expression of disease-causing genes.

Animals↗
PubMed1996

Theoretical and experimental discrimination between Cl(-)-H+ symporters and Cl-/OH- antiporters.

A Cl(-)-H+ symport and a Cl-/OH- antiport cannot be readily distinguished physicochemically, but a kinetic distinction is theoretically possible, because a Cl(-)-H+ symporter involves a two-site carrier whereas a Cl-/OH- antiporter involves a single-site carrier. Accordingly, we have developed kinetic models and equations that we have tested by studying Cl- uptake by isolated guinea pig ileal brush-border membrane vesicles as a function of Cl- or H+ concentration. We conclude that a two-site Cl(-)-H+ symporter with a 1:1 stoichiometry explains the pH-dependent Cl- uptake and Cl-/Cl- exchange activities of the brush-border membrane in terms of a single random nonobligatory mobile carrier where exchange occurs by counterflow. This symport, probably involving an anion exchanger (AE 2) protein, differs, therefore, functionally from the erythrocyte's band 3 AE1, which involves an antiport. The question is whether members of the AE gene family can be functionally diverse, even when their primary structures exhibit up to 50% overall homologies.

Animals↗
PubMed1995

Inhibition of HSV-1 proliferation by decoy phosphodiester oligonucleotides containing ICP4 recognition sequences.

Transcriptional control in eukaryotes results from the interplay between DNA sequences in promoters, enhancers, or silencers and transcription factors. Selective control of gene expression can thus be achieved by inhibiting specific transcription factor/DNA interactions. Transcriptional activity of DNA binding transcription factors can be inhibited by competition with double-stranded oligonucleotides (decoys) that contain their specific recognition sequences. The immediate early protein ICP4 of herpes simplex virus type 1 (HSV-1) is a sequence-specific DNA binding protein that is essential for viral replication. We synthesized double-stranded hairpin phosphodiester oligonucleotides carrying ICP4 sites and demonstrated their ability to specifically titrate ICP4. Upon addition to Vero cells, ICP4 hairpin decoys significantly reduced HSV-1 titers (IC50 = 0.3 microM), whereas a control hairpin oligonucleotide had no activity. Antiviral activity of ICP4 hairpin decoys was correlated to their relative binding affinities. These results show that phosphodiester oligonucleotides can compete for binding of specific transcription factors within cells, thus providing a potential therapeutic tool to control disease-causing genes.

Animals↗
PubMed1994

Effects of repeated treatments with an extract of Ginkgo biloba (EGb 761), bilobalide and ginkgolide B on the electrical activity of pancreatic beta cells of normal or alloxan-diabetic mice: an ex vivo study with intracellular microelectrodes.

1. The effects of repeated (5-day) treatments with an extract of Ginkgo biloba leaves (EGb 761), bilobalide, and ginkgolide B on the in vitro electrical activity of insulin-secreting pancreatic beta cells of mice have been examined using intracellular microelectrodes. 2. EGb 761 (200 mg/kg/day, p.o.) protected beta cells against the toxic effects of alloxan (50 mg/kg, i.v.), an effect characterized by a restoration of membrane potential (Vr) and an increase in spike frequency (Fs/30), an indicator of insulin secretion. 3. Treatment of non-diabetic mice with EGb 761 (200 mg/kg/day, p.o.) increased Fs/30 of their beta cells, as tested by in vitro exposure of the cells to 11.1 mM glucose, an effect that also occurred with bilobalide (8 mg/kg/day, i.p.) but not with ginkgolide B (4 mg/kg/day, i.p.). 4. Since bilobalide and ginkgolide B caused opposite effects on the sensitivity of beta cells to glucose, the stimulatory effect of EGb 761 on Fs/30 may be attributed to its content of bilobalide. 5. In contrast to its ex vivo effect, the direct in vitro effect of EGb 761 (10 and 25 micrograms/ml) on beta cells favors a decrease in electrical activity, indicating that its in vivo action might be indirect (e.g. via the formation of an active metabolite).

Action Potentials↗
PubMed1994

Improved anti-herpes simplex virus type 1 activity of a phosphodiester antisense oligonucleotide containing a 3'-terminal hairpin-like structure.

We synthesized a series of 20-mer antisense phosphodiester oligonucleotides constituting of a 5'-dodecameric sequence, complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) pre-mRNAs IE4 and IE5, flanked in 3' by octameric sequences adopting hairpin-like structures of different stabilities. The presence of the minihairpins in 3' protected the 20-mer phosphodiester oligonucleotides against serum nuclease degradation, this protection being well correlated to the reported melting temperatures of the minihairpins, and to the gel mobilities of the 20-mer oligonucleotides. While no protection was observed using a linear 8-mer, the addition in 3' of the most stable minihairpin--H8--increased more than eightfold the nuclease resistance of the linear antisense dodecamer. We analyzed the effect of such a protection on the anti-HSV-1 antisense activities of the oligonucleotides. When bearing H8 in 3', the antisense dodecamer was 10 times more active than in the absence of 3'-flanking sequence, while a linear 20-mer control containing the antisense sequence was only 3 times more active. This work provides the basis for a further rational design of phosphodiester antisense oligonucleotides, taking advantage of the specific properties conferred by their conformations.

Antiviral Agents↗
PubMed1994

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

Animals↗
PubMed1994

Rheogenic Cl- conductance and Cl(-)-Cl(-)-exchange activities in guinea pig jejunal basolateral membrane vesicles.

We describe a method for the simultaneous purification of apical (brush-border membrane) and basolateral membrane (BLM) vesicles from the same sample of guinea pig jejunum. We applied functional tests to demonstrate the absence of reciprocal cross contamination between the two vesicle preparations. By using the BLM vesicles and a rapid filtration technique, we quantified 36Cl uptake under conditions of equilibrated pH (pHout = pHin = 7.5). The presence of 200 mM cis of either Na+ or K+, or an equimolar mixture of both, significantly increased the initial Cl- entry rate. In the presence of K+, valinomycin further increased Cl- uptake, but no Cl- uphill transport was ever observed under any of the conditions. All the increases were abolished by voltage clamping, indicating that the alkali-metal ions act by creating an inside-positive membrane potential capable of stimulating a Cl(-)-conductance pathway. In the absence of K+, BLM vesicle preloading to obtain a [Cl-]out/[Cl-]in = 16/200 mM gradient (delta Cl-) resulted in a 500% increase in the initial 36Cl- entry rate, accompanied by a transient Cl- accumulation, with an overshoot at approximately 5 min. In the presence of both a positive-inside electrical gradient (delta psi) and a delta Cl-, the initial Cl- uptake rate was increased by 800%, indicating that the effects of delta psi and of delta Cl- are additive. The delta Cl- effect was blocked, but only partially, by short-circuiting the membrane potential with equilibrated K+ and valinomycin, thus indicating that it has both rheogenic and electroneutral components. We conclude that Cl- influx across the guinea pig intestinal BLM involves a Cl(-)-conductance pathway plus a distinct Cl(-)-Cl(-)-exchange system, exhibiting both electroneutral and rheogenic components. Alternatively, the possibility can also be entertained that the conductance and the exchange pathways share a common molecular basis, e.g., a nonobligatory Cl(-)-Cl- exchanger or rheogenic uniport.

Animals↗
PubMed1994

[Synthesis of cyclic oligodeoxyribonucleotides with non-nucleotide inserts].

An effective method for the oligonucleotide cyclization using BrCN-induced chemical ligation was developed. The novel idea to incorporate non-nucleotide inserts makes the process of cyclizations independent of the inner secondary structure of the linear precursor. An set of assays were developed to confirm the cyclic structure of the compounds obtained.

Base Sequence↗
PubMed1993

Oligonucleotide circularization by template-directed chemical ligation.

An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpin-like structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.

Base Sequence↗
PubMed1993

Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.

Albumins↗
PubMed1993

Structural and sequence elements required for the self-cleaving activity of the hepatitis delta virus ribozyme.

The hepatitis delta virus (HDV) is a subviral RNA that contains a self-cleaving activity that is similar to the ribozyme activity found in certain plant pathogens. However, the sequences surrounding the cleavage site are unrelated to the hammerhead or hairpin ribozyme motifs, and it is considered to be a distinct ribozyme type. We made site-specific changes within two regions of the smallest contiguous HDV sequence that has optimal activity and kinetically analyzed the data at different temperatures to determine the potential roles of the residues. We distinguish between those changes that affect the rate of catalysis and those that promote the formation of inactive structures. We find that nucleotides +45 to +72 downstream from the cleavage site, which can form a hairpin structure, are dispensable for catalytic activity but that they enhance the cleavage efficiency. Nucleotides +17 to +19 and +28 to +30 form Watson and Crick base pairs that are important for activity, but the actual sequence is not critical. In contrast, the nucleotides between +21 and +26 are important for activity, and they may be involved in significant tertiary interactions.

Base Sequence↗