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L Jetté

Publications and source records attributed to L Jetté.

15 recordsLinked to original sources
PubMed1997

P-glycoprotein is a dimer in the kidney and brain capillary membranes: effect of cyclosporin A and SDZ-PSC 833.

Radiation-inactivation studies were performed in order to elucidate the oligomeric nature of P-glycoprotein (P-gp) expressed in brain capillaries and renal brush border membranes (BBMs). Irradiation of renal BBMs resulted in a dose-dependent loss of P-gp, which corresponded to a target size (TS) of 255 and 211 kDa, as detected by Western blot and [125I]arylazidoprazosin labeling, respectively. Similar TSs were determined for P-gp expressed in brain capillaries. These TSs correspond to approximately twice the size (120 kDa) of deglycosylated P-gp. Furthermore, the estimated TS for P-gp was not significantly different when renal BBMs were incubated with SDZ-PSC 833 (PSC) prior and during exposure to ionizing radiation. To confirm these results, the size of P-gp was evaluated from its mobility on blue-native polyacrylamide gels followed by Western blot analysis. Using this method, an apparent molecular size of 334 and 264 kDa was determined for P-gp in brain capillaries and renal BBMs, respectively. This corresponds to approximately twice the size of the glycosylated monomeric subunit of P-gp in brain capillaries (162 kDa) or renal BBMs (140 kDa). P-gp expressed in renal BBMs isolated from rats which had been treated daily with cyclosporin A (CsA) or PSC also migrated as a 264 kDa protein. These results suggest that P-gp exists mainly as a dimer in normal tissues and that resistance modulators such as CsA and PSC do not alter its oligomeric state.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

HIV-1 genome dimerization: kissing-loop hairpin dictates whether nucleotides downstream of the 5' splice junction contribute to loose and tight dimerization of human immunodeficiency virus RNA.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Adjacent genomic RNAs from human immunodeficiency virus type 1 (HIV-1) can form loose or tight dimers depending on whether their respective kissing-loop hairpins (nts 248-270 in HIV-1Lai) bond via their autocomplementary sequences (ACS) or via the ACS and stem sequences [Laughrea, M., & Jetté, L. (1996a) Biochemistry 35, 1589-1598]. Loose dimers from HIV-1Mal, but not HIV-1Lai, are stabilized by a sequence (3'DLS) located downstream of the 5' splice junction [Laughrea, M., & Jetté, L. (1996b) Biochemistry 35; 9366-9374]. To understand the ACS-3'DLS interplay in the formation and stability of loose and tight HIV-1 RNA dimers, we replaced the ACS of HIV-1Lai (GCGCGC262) by GUGCAC, GUGCGC (two alternative HIV-1 ACS), or GAGCUC (a non-HIV ACS). For each mutant, RNAs truncated immediately upstream or downstream of the 3'DLS were prepared; their ability to dimerize and their thermal stabilities were compared. The results suggest that the ACS determines whether the 3'DLS participates in RNA dimerization: (1) GAGCUC262 led to poorly stable loose dimers due to the inability of the 3'DLS to stabilize them (the 3'DLS stabilized the GUGCAC and GUGCGC RNAs); (2) GAGCUC262 led to poor formation of tight dimers, due to an inhibitory effect of the 3'DLS (the 3'DLS had little effect on the tight dimerization of the GUGCAC, GUGCGC and GCGCGC RNAs). The results indicate that communication exists between HIV-1 RNA sequences respectively located upstream and downstream of the 5' splice junction; they are consistent with the idea that the 3'DLS plays two ACS-dependent roles in the dimerization process: loose dimer stabilization in HIV-1 RNAs bearing an HIV ACS (unless the ACS already conferred a thermostability equal or superior to that offered by the 3'DLS), and inhibition of tight dimer formation in an HIV-1 RNA bearing a non-HIV ACS.

Base Sequence↗
PubMed1997

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization.

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Animals↗
PubMed1996

HIV-1 genome dimerization: formation kinetics and thermal stability of dimeric HIV-1Lai RNAs are not improved by the 1-232 and 296-790 regions flanking the kissing-loop domain.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization posits that the 233-285 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is at least the core dimerization domain of HIV-1 RNA. This region is called the kissing-loop domain. In addition, it can be argued that sequences within the 296-401 region [Paillart et al. (1994) J. Biol. Chem. 269, 27486-27493] or 5' of the primer binding site [Laughrea & Jetté (1996) Biochemistry 35, 1589-1598] might play some role in the dimerization process. Accordingly, we have studied the effect of regions 1-232 and 296-790 on the dimerization kinetics and thermal stability of HIV-1Lai RNAs containing the kissing-loop domain (HIV-1Lai is a typical representative of North American and European HIV-1 viruses). Experiments conducted at high and low ionic strength indicate that these regions have no strongly positive effect on the dimerization process. Our experiments also indicate that the kissing-loop domain of HIV-1Lai has an apparent dissociation temperature 13 degrees C higher than that of the HIV-1Mal kissing-loop domain (HIV-1Mal is a Central African virus whose kissing-loop domain has a "weak" GUGCAC autocomplementary sequence). Because the 296-401 region of HIV-1Mal RNA stabilizes dimeric RNAs by < or = 12 degrees C (Paillart et al., 1994), we infer that the contributions of sequences downstream of U295 are (at best) concealed in HIV-1Lai and in most American and European HIV-1 viruses, i.e., in viruses whose kissing-loop domain is characterized by a "strong" GCGCGC autocomplementary sequence.

Base Sequence↗
PubMed1996

Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248-271 are dispensable for dimer formation.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization [Laughrea, M., & Jetté, L. (1994) Biochemistry 33, 13464-13474; Skripkin et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4945-4949] posits that the 248-270 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is the full or at least the core dimerization domain of HIV-1 RNA. Here, we show by nested deletion analysis that the 3' boundary of the HIV-1 dimerization domain is immediately downstream of hairpin 248-270 and that the isolated region 248-271 dimerizes at least as readily as longer RNAs. Among various HIV-1Lai RNA transcripts containing hairpin 248-270, all form two types of dimer, as is implicit in the kissing-loop model. The high-stability dimer resists semidenaturing conditions and the low-stability dimer cannot, which is consistent with the model. At physiological temperatures, low-stability dimers are usually formed, as if dimerization without nucleocapsid proteins corresponded to loop-loop interaction without switching from intra- to interstrand hydrogen bonding. Our results show that the 3' DLS (a sequence immediately 3' from the 5' splice junction and originally thought to be the dimerization domain of the HIV-1 genome) and adjacent nucleotides are not necessary for efficient dimerization of HIV-1Lai RNA at low and high ionic strength. Upstream of hairpin 248-270 exists another "DLS-like" sequence that we name 5' DLS: like the isolated 3' DLS, the isolated 5' DLS forms an apparently nonphysiological structure that can become substantially dimeric at high ionic strength.

Base Sequence↗
PubMed1996

Cyclosporin A treatment induces overexpression of P-glycoprotein in the kidney and other tissues.

To see whether P-glycoprotein (PGP) expressed in renal brush-border membranes (BBM) could interact with compounds known as modulators of multidrug resistance (MDR), photoaffinity-labeling experiments were performed. A 145k-Da protein was photolabeled with [125I] iodoarylazidoprazosin, and this labeling was reduced in the presence of cyclosporin A (CsA) and PSC-833 (PSC). Interaction of CsA with PGP was further investigated by treating rats with daily subcutaneous injections of CsA (10 mg.kg-1.day-1). After this treatment, PGP expression levels were dramatically increased in renal BBM, intestine, liver, and many other tissues except the brain. This induction was a reversible process, since after cessation of CsA administration PGP levels declined to reach values similar to those of the control groups. The increase in PGP expression in the kidney was also detected in photolabeling experiments, suggesting the induction of a functional PGP. A higher dose of CsA (50 mg/kg) given as a bolus injection did not modify PGP expression] in renal BBM. These results demonstrate that CsA induces reversible overexpression of PGP in the rat. This may present significant relevance in the design of clinical trials using CsA as a chemosensitizing agent.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Interaction of drugs with P-glycoprotein in brain capillaries.

P-glycoprotein (P-gp) is expressed at high levels in a variety of non-cancerous tissues such as the endothelial cells of the blood-brain barrier (BBB) capillaries. These thin capillaries tightly regulate the movement of substrates from the circulating blood into the brain. P-gp may be involved in the exclusion of various drugs from the capillary endothelial cells, blocking their entry into the brain. However, interactions of drugs with P-gp expressed in brain capillaries remain to be characterized. We have performed photoaffinity labeling studies using [125I]arylazidoprazosin (IAAP) to evaluate the inhibitory efficiency of various compounds. Cyclosporin A (CsA) and its derivative PSC 833 (PSC) were the most effective inhibitors of IAAP binding among the drugs tested. The magnitude of inhibition was: PSC > CsA > quinidine > vinblastine > verapamil < actinomycin D > colchicine > reserpine > bilirubin > doxorubicin > progesterone. Cremophor El, the vehicle used to administer CsA and PSC intravenously, was also able to inhibit IAAP photolabeling of P-gp. Labeling experiments were also performed using a photoactivatable [3H]CsA derivative. Photolabeling of P-gp with this compound was abolished almost completely by CsA and PSC. In vivo studies were also performed by treating rats with CsA [10 mg/(kg.day) for 10 days]. Following this treatment, no alteration in the level of P-gp expression in brain capillaries was observed. These results suggest that, at the proper dosage, administration of CsA to cancer patients could help to enhance the response of brain tumors to chemotherapeutic agents without modifying the intrinsic level of P-gp expression in this tissue.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein.

P-glycoprotein (P-gp) is expressed in various non-cancerous tissues such as the endothelial cells of the blood-brain barrier. We used several monoclonal antibodies (mAbs) and isoform-specific polyclonal antibodies to establish which P-gp isoforms are expressed in isolated mouse brain capillaries. P-gp class I isoform was detected in capillaries with a Western immunoblotting procedure using a specific antiserum. No immunoreactivity was observed with either class II- or class III-specific antisera. Immunoreactivity was observed with mAb C219. However, this antibody detected two distinct immunoreactive proteins (155 and 190 kDa) in the isolated brain capillaries. These two proteins comigrated as a broad band when the samples were submitted to heat prior to gel electrophoresis. The glycoprotein nature of these two antigens was evaluated by their sensitivity to N-glycanase treatment. Following this treatment, the size of the proteins was reduced from 190 and 155 kDa to 180 and 120 kDa, respectively. Triton X-114 phase-partitioning studies showed that the 190 kDa immunoreactive protein was poorly solubilized by Triton X-114, while the 155 kDa protein was partitioned in the detergent-rich phase. In labelling experiments, only the 155 kDa protein was photolabelled with [125I]iodoarylazidoprazosin. These results show that a 190 kDa protein detected by antibody C219 is an antigen unrelated to the three P-gp isoforms presently known. Cross-reactivity of C219 with an unrelated protein emphasizes the fact that more than one antibody should be used in the assessment of P-gp expression in cell lines and tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA.

The genome of all retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. We report the results of experiments designed to identify the 5' and 3' boundaries of the dimerization domain of the HIV-1 genome: (1) An HIV-1 RNA starting at nucleotide 252 or at other downstream positions (four tested) does not dimerize despite the inclusion of the whole of a previously proposed dimerization domain (nucleotides 295-401); (2) an RNA starting between nucleotides 242 and 249 (five positions tested) dimerizes to a variable extent depending on the starting position; (3) an RNA starting at nucleotide 233 or at other upstream positions (five tested) is fully or > 80% dimeric; (4) an RNA starting at nucleotide 1 but lacking the 233-251 or the 242-251 region is, respectively, fully monomeric or about 50% monomeric; (5) the 343-401 region contains two strings of G's (GGGGG367 and GGG384) that had been postulated to promote genome dimerization through the formation of guanine quartets. We have deleted the 379-401, 358-401, and 343-401 regions from otherwise dimeric RNAs without changing their ability to dimerize. We reach three conclusions: (1) a dimerization signal exists upstream of the major 5' splice donor (nucleotide 290); (2) the previously proposed downstream dimerization domain is insufficient to promote dimerization and has a 3' half that is not necessary to obtain fully dimeric RNAs; (3) the 5' boundary of the HIV-1 dimerization domain is located somewhere between nucleotides 233 and 242, and the 3' boundary is located no farther than at nucleotide 342, making it possible that the 5' and 3' boundaries of the HIV-1 dimerization domain are both located within the leader sequence. We speculate that the 248-270 or 233-285 region forms a hairpin that is the core dimerization domain of HIV-1 RNA.

Base Sequence↗
PubMed1994

Intracoronary infusion of bradykinin: effects on noradrenaline overflow following reperfusion of ischemic myocardium in the anesthetized dog.

OBJECTIVE: The aim of this study was to investigate the effects of intracoronary bradykinin (BK) infusion on noradrenaline release and ventricular arrhythmias induced by coronary occlusion and reperfusion in the anesthetized dog. METHODS: 14 anesthetized adult mongrel dogs of either sex underwent a 60 min occlusion of the left anterior descending coronary artery (LAD) followed by a 30-min reperfusion period. BK (1 ng.kg-1.min-1, n = 7), or its vehicle (Lactate Ringer, n = 7), infusions just distal to the left coronary ostium started 15 min before the LAD occlusion and were maintained throughout the experimental period. An epicardial vein, running parallel to the LAD was cannulated to enable the biochemical determinations. The effects of BK on ventricular arrhythmias, cardiac noradrenaline and lactate releases and creatine kinase activity were assessed. RESULTS: BK significantly reduced the amount of noradrenaline released at reperfusion by ischemic myocardium (from 82.1 +/- 31.7 to 11.9 +/- 9.6 ng.min-1), as well as plasma creatine kinase activity at 30 min of reperfusion. This is accompanied by a significant reduction in the incidence of reperfusion-induced sustained ventricular tachycardia. CONCLUSION: This suggests that the protective effect of bradykinin against reperfusion-induced sustained ventricular tachycardia could be associated with a reduction in cardiac noradrenaline release.

Animals↗
PubMed1993

High levels of P-glycoprotein detected in isolated brain capillaries.

P-glycoprotein (P-gp) is a highly-conserved membrane protein expressed in various multidrug-resistant cell lines. P-glycoprotein was detected in capillaries isolated from human, beef and rat brains with a Western immunoblotting procedure using the monoclonal antibody C219 (mAb C219) specific for P-gp. The mAb C219 detected a 180 kDa protein in brain capillaries isolated from all three species. The largest amount of antigen was detected in capillaries isolated from human brain. Specific binding was assessed by competitive inhibition of mAb C219 binding by the synthetic epitope VQEALD. The glycoprotein nature of the brain capillary proteins was confirmed by its sensitivity to N-glycanase treatment, which reduced their apparent molecular mass by 5 to 10 kDa. In addition, immunohistochemical studies using the antibodies C219, JSB-1 and C494 were performed. Bovine and rat capillaries showed reactivity only with the mAb C219. Heavy staining of human brain capillaries was observed with both antibodies C219 and JSB-1, while only weak staining was observed with antibody C494. These results clearly show that P-glycoprotein is strongly expressed at the blood-brain barrier (BBB) site and suggest that this protein may play a physiological role in regulating the access of certain molecules to the central nervous system, or in the secretory functions of the BBB.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Intracoronary administration of saralasin: effects on cardiac arrhythmias induced by ischaemia and reperfusion in the anaesthetised dog.

OBJECTIVE: The aim was to study (1) the effects of intracoronary saralasin, an angiotensin II receptor antagonist, on ischaemia induced and reperfusion induced regional cardiac noradrenaline release and ventricular arrhythmias; and (2) the implication of angiotensin II in coronary constriction during myocardial ischaemia. METHODS: Eighteen adult mongrel dogs, weight 22.6(SD 1.1) kg, anaesthetised with sodium pentobarbitone, were used for the study. The left anterior descending coronary artery was ligated for 60 min and then reperfused for 30 min. Saralasin (60 micrograms.kg-1, n = 9) or its vehicle (Ringer lactate, n = 9) was injected into the artery at the beginning of the occlusion period. Two epicardial veins, one running parallel to the left anterior descending coronary artery and the other parallel to the circumflex coronary artery, were cannulated for the measurement of their respective blood flows and of noradrenaline, lactate, and creatine kinase release. RESULTS: Saralasin decreased the incidence of ventricular fibrillation during coronary occlusion (from 44% in the vehicle treated group to 0% in the saralasin treated group, p = 0.0412). This effect was accompanied by significant vasodilatation in both epicardial veins during myocardial ischaemia. Neither the increases in noradrenaline, lactate, and creatine kinase release nor the incidence and duration of the ventricular arrhythmias following reperfusion were modified by the administration of saralasin. CONCLUSIONS: Intracoronary saralasin in the early phase of myocardial ischaemia increases the epicardial venous blood flow significantly, suggesting that angiotensin II is implicated in coronary constriction during ischaemia. This haemodynamic effect is accompanied by a significant decrease in the incidence of ventricular fibrillation. However, the renin-angiotensin system does not appear to be implicated in the reperfusion induced noradrenaline release nor in the incidence of the ventricular arrhythmias.

Animals↗
PubMedDate not supplied

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