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P Bois

Publications and source records attributed to P Bois.

At least 19 recordsLinked to original sources
PubMed2000

Thyroid hormone increases the conductance density of f-channels in rabbit sino-atrial node cells.

In hyperthyroidism, the main cardiac manifestation is an increase in resting heart rate with a great degree of sinus tachycardia. As the pacemaker current, I(f), is reputed to be the main ionic mechanism involved in sinus rhythm, the objective of this study was to investigate the effect of triiodothyronine (T3) on I(f), in sino-atrial (SA) node cells isolated from rabbit heart, using the patch-clamp technique in whole-cell configuration. In cells incubated for 5-7 h in a medium containing 1 microM T3, the hormone increased I(f) by rising the maximal conductance without inducing shift of the activation curve or change in the reversal potential. The mean values of I(f) density (42.05+/-5.93 pA/pF, n = 13) and maximal conductance density (854.25+/-124.83 pS/pF, n = 16) were significantly increased compared with those measured in untreated cells (20.21+/-2.53 pA/pF, n = 14; 392.69+/-54.95; pS/pF, n = 16). In cells treated with T3, 0.1 microM isoprenaline, a beta-adrenergic agonist, was still able to induce a positive shift of the voltage dependence of I(f) activation. Reverse T3, the inactive form of T3, used in the same conditions, had no effect on I(f). According to the results, it was suggested that the acceleration of resting heart rate observed in hyperthyroidism, might be the consequence of a T3-induced stimulation of f-channel transcription in SA node myocytes.

Animals↗
PubMed1999

Human minisatellites, repeat DNA instability and meiotic recombination.

Minisatellites include some of the most variable loci in the human genome and are superb for dissecting processes of tandem repeat DNA instability. Single DNA molecule analysis has revealed different mutation processes operating in the soma and germline. Low-level somatic instability results in simple intra-allelic rearrangements. In contrast, high frequency germline instability involves complex gene conversions and is therefore recombinational in nature, almost certainly occurring at meiosis. To determine whether true meiotic crossovers occur at human minisatellites, we have used polymorphisms near the repeat array to recover recombinant DNA molecules directly from sperm DNA. Analysis of minisatellite MS32 has revealed an intense and highly localised meiotic crossover hotspot centred upstream of the array, the first example of a human hotspot defined at the molecular level. This hotspot extends into the beginning of the repeat array, resulting in unequal and equal crossovers. Array crossovers occur much less frequently than array conversions but appear to arise by a common process, most likely by alternative processing of a recombination initiation complex. The location of MS32 at the boundary of a recombination hotspot suggests that this locus has evolved as a by-product of localised meiotic recombination activity, and that minisatellites might in general mark recombinationally proficient hotspots or hot domains in the genome. Finally, sperm crossover analysis makes it possible to explore the molecular rules that govern human meiotic recombination, and to detect phenomena such as meiotic drive that could provide a possible connection between recombination and DNA sequence diversity itself.

DNA↗
PubMed1999

Minisatellite instability and germline mutation.

Tandem-repeat DNA actively turns over in the genome by a variety of poorly understood dynamic mechanisms. Minisatellites, a class of tandem repeats, have been shown to cause disease by influencing gene expression, modifying coding sequences within genes or generating fragile sites. There has been recent rapid progress towards understanding molecular turnover processes at human minisatellites. Instability at GC-rich minisatellites appears to involve distinct mutation processes operating in somatic and germline cells. In the germline, complex conversion-like events occur, probably during meiosis. Repeat turnover appears to be controlled by intense recombinational activity in DNA flanking the repeat array, suggesting that minisatellites might evolve as by-products of localised meiotic recombination in the human genome. In contrast, AT-rich minisatellites appear to evolve by intra-allelic processes such as replication slippage. Curiously, minisatellites in other organisms appear to be more stable than their human counterparts, suggesting species-specific differences in turnover processes. Some yeast models display human-like minisatellite turnover processes at meiosis. However, all attempts to transfer human germline instability to transgenic mice have failed. Finally, tandem repeat instability in various species appears to be extremely sensitive to environmental agents such as radiation via a mechanism which remains enigmatic.

Animals↗
PubMed1998

Isolation and characterization of mouse minisatellites.

Minisatellites provide the most informative system for analyzing processes of tandem repeat turnover in humans. However, little is known about minisatellites and the mechanisms by which they mutate in other species. To this end, we have isolated and characterized 76 endogenous mouse VNTRs. Fifty-one loci have been localized on mouse chromosomes and, unlike in humans, show no clustering in proterminal regions. Sequence analysis of 25 loci revealed the majority to be authentic minisatellites with GC-rich repeat units ranging from 14 to 47 bp in length. We have further characterized 3 of the most polymorphic loci both in Mus musculus subspecies and in inbred strains by using minisatellite variant repeat mapping (MVR) by PCR to gain insight into allelic diversity and turnover processes. MVR data suggest that mouse minisatellites mutate mainly by intra-allelic nonpolar events at a rate well below 10(-3) per gamete, in contrast to the high-frequency complex meiotic gene conversion-like events seen in humans. These results may indicate a fundamental difference in mechanisms of minisatellite mutation and genome turnover between mice and humans.

Alleles↗
PubMed1998

Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation.

Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose-response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.

Animals↗
PubMed1998

A novel unstable mouse VNTR family expanded from SINE B1 elements.

Human hypervariable minisatellites provide highly informative loci for analyzing processes of tandem repeat turnover, but little is known about mechanisms of instability in other species. We have therefore screened the mouse genome for analogous VNTR loci. One of the probes we isolated, MMS10, detects a highly variable rodent-specific family of multiple loci derived by expansion of a common GGCAGA repeat unit from within a subset of B1 short interspersed elements. The mean germline mutation rate for loci detected by MMS10 was estimated at 1.7% per offspring band, though analysis of individual loci showed substantial variation in germinal instability apparently related to repeat array size. This MMS10 family of expanded hexanucleotide repeat loci provides a novel resource for investigating mechanisms of tandem repeat turnover in the mouse and an efficient means for monitoring germline mutations induced by external agents such as ionizing radiation.

Animals↗
PubMed1998

Characterization of a hyperpolarization-activated current in dedifferentiated adult rat ventricular cells in primary culture.

1. The presence of a hyperpolarization-activated pacemaker (I(f)-like current was tested in dedifferentiated adult rat ventricular myocytes up to 12 days in primary culture with the whole-cell patch clamp technique. 2. An I(f)i-like current was found and characterized on freshly isolated and cultured ventricular cells. Both activation and density of the current varied in relation to the stage of dedifferentiation. The current was activated from -92.0 +/- 2.5 and -63 +/- 1.0 mV at the beginning (4-day-cultured cells) and end of the dedifferentiation process (12 days), respectively. Its density measured at 170 mV progressively increased from -2.34 +/- 0.36 to -6.12 +/- 0.64 pA pF-1 between the two farthest stages of cellular remodeling. In freshly isolated cells the current was activated at -108.0 +/- 1.5 mV and its current density measured at -170 mV was -1.97 +/- 0.56 pA pF-1. 3. The current was blocked by extracellular CsCl (3mM) in a voltage-dependent manner. Modification of reversal potentials obtained at various values of [K+]o ( 5.4 or 25 mM) and [Na+]o (140 or 30 mM) suggests that the current was carried by both K+ and Na+ ions. 4. It is concluded that the hyperpolarization-activated inward current, recorded in freshly isolated and in cultured ventricular cells has characteristics similar to those of I(f). In adult rat ventricular cells it is activated in a non-physiological potential range, but can be elicited in a more physiological range when the cells are remodelled through a dedifferentiated way. It is suggested that such a current could be implicated in ventricular arrhythmias developed in pathological events.

Animals↗
PubMed1998

Current and future contributions of transgenic mice to the analysis of germline toxicology.

Evermore sophisticated tests are need to study germline toxicology. The gene conversion-based systems developed in Leicester and in the USA are steps in the right direction, but a lot of validation both in vivo and in vitro is required. Transgenic technology can also be used to research the biology of testis, so that we know more how to make it more human-like. If you talk to toxicologists, they always complain: 'but it 's only a rat, it's only a mouse, it's not a man'. In future, once we understand more biology--it might be possible to make the toxicological response of a transgenic mouse more human-like. As we all know, the testis is a complex biological system and it is only when we get a better understanding of what is going on to the fundamental level are such developments possible. Indeed, it might be possible to do even more exciting things, such as taking mitotic human tissue culture cells and to inducing them to enter meiosis in vitro. Such a system would be a natural complement to the in vitro tests widely used in industry.

Animals↗
PubMed1998

Nucleotide modulation of f-channel activity in rabbit sino-atrial node myocytes.

f-channel nucleotide modulation was investigated in sino-atrial (SA) node cells isolated from rabbit hearts, using an inside-out macropatch configuration. Saturating doses (30 microM) of phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS, were tested on the cAMP-induced shift of I(f) activation. Responses were not altered when Sp-cAMPS was combined with cAMP. When Rp-cAMPS was superfused with subsaturating cAMP concentrations (1-10 microM), it inhibited cAMP-induced I(f) activation shift. cGMP and cIMP reversibly shifted the I(f) conductance-voltage curve to more positive values; however they had a lesser specificity than that of cAMP. The efficacy ranking for I(f) activation by cyclic nucleotides was: cAMP > cGMP > cIMP. Non cyclic nucleotides (ATP, ADP and AMP) failed to change I(f) activation, indicating that the cyclic nature of nucleotides seems to be essential to f-channel modulation. Similarities between f-channels and cyclic nucleotide-gated (CNG) channels are discussed.

Animals↗
PubMed1997

Activation of f-channels by cAMP analogues in macropatches from rabbit sino-atrial node myocytes.

1. The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp-cAMPs and Sp-cAMPs, was investigated on hyperpolarization-activated 'pacemaker' current (i(f)) recorded in inside-out macropatches from rabbit sino-atrial (SA) node myocytes. 2. When superfused on the intracellular side of f-channels at the concentration of 10 microM, both cAMP derivatives accelerated i(f) activation; their action was moderately less pronounced than that due to the same concentration of cAMP. 3. The measurement of the i(f) conductance-voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of i(f) to more positive voltages with no change in maximal (fully activated) conductance. 4. Dose-response relationships of the shift of the i(f) activation curve showed that both Rp-cAMPs and Sp-cAMPs act as agonists in the cAMP-dependent direct f-channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 microM, and Hill coefficients of 0.82 and 1.12 for Sp-cAMPs and Rp-cAMPs, respectively. 5. The activating action of Rp-cAMPs, a known antagonist of cAMP in the activation of cAMP-dependent protein kinase, confirms previously established evidence that f-channel activation does not involve phosphorylation. These results also suggest that the cAMP binding site of f-channels may be structurally similar to the cyclic nucleotide binding site of olfactory receptor channels.

Animals↗
PubMed1997

Acetylcholine modulates I(f) and IK(ACh) via different pathways in rabbit sino-atrial node cells.

The aim of the present study was to examine whether a direct G-protein pathway was necessary to explain the depression of the cardiac pacemaker current (I(f)) by acetylcholine (ACh). Pacemaker current and spontaneous fluctuations of muscarinic K+ current (IK(ACh)) were simultaneously measured in sino-atrial (SA) node cells isolated from rabbit hearts, using cell-attached and outside-out macro-patches. The effects of 1 microM ACh, added to the bathing solution, were compared on I(f) and on IK(ACh), known to be activated via a direct G-protein mechanism. In a cell-attached configuration where cytoplasmic substrates were present, ACh depressed I(f) by a negatively directed shift of the open probability curve of 7.6 +/- 0.3 mV. ACh never induced modifications in spontaneous openings of muscarinic K+ channels. These results indicate that ACh added to the external solution is unable to modulate IK(ACh) recorded in the membrane-delimited mode, via a G-protein pathway. The ACh depressing effect on I(f) is related to changes in second messenger activity. In outside-out conditions, with guanosine triphosphate (GTP) added to the pipette solution. ACh increased the variance of IK(ACh) fluctuations from 1.66 +/- 0.50 pA2 to 6.60 +/- 2.05 pA2 (at -120 mV). indicating direct G-protein action. ACh had no effect on I(f). It is concluded that in SA node cells, the regulation of I(f) by muscarinic receptors does not involve a direct G-protein pathway. ACh depresses I(f) by a mechanism that probably implicates reduction of intracellular cAMP production.

Acetylcholine↗
PubMed1997

Human minisatellite MS32 (D1S8) displays somatic but not germline instability in transgenic mice.

Human minisatellite MS32 (D1S8) shows instability both in the germline and, at much lower levels, in somatic DNA. To investigate factors that influence somatic and germline mutation, large cosmid-based constructs containing MS32 were introduced into mice, bred to homozygosity and tested for instability in blood and sperm. Analysis of single copy and multicopy transgenic lines revealed somatic mutants occurring at a frequency comparable with that seen in man. As in humans, these mutants arose mainly by simple intra-allelic duplications and deletions. In contrast, analysis of sperm DNA from four different transgenic lines showed no trace of the complex recombination-based germline instability seen in man, even using PCR-based approaches capable of detecting very rare mutants. These data provide further evidence that germline and somatic mutation at human minisatellite MS32 occur via distinct pathways, that a major barrier exists to the transfer of germline instability from humans to mice and that the mouse germline appears to be protected from mitotic instability of the type seen in blood.

Alleles↗
PubMed1996

Instability of long inverted repeats within mouse transgenes.

Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.

Animals↗
PubMed1996

Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells.

1. The effect of the bradycardic agent S 16257 on the main ionic mechanisms of diastolic depolarization in sinoatrial node cells isolated from rabbit heart, was investigated by the patch-clamp technique in whole-cell and macro-patch recordings. 2. In whole-cell conditions, S 16257 induced a marked exponential use-dependent blockade of the hyperpolarization-activated I(f) current, without shift of the voltage range of its activation curve. The rate of block increased with the drug concentration. The IC50 for the block of I(f) was 2.8 x 10(-6) M. 3. A similar use-dependent decline of I(f) was obtained with 3 microM S 16257, in cell-attached and in inside out macro-patch configurations, suggesting that the bradycardic agent interacts with I(f) channels from the inside of the cell. 4. A high concentration of S 16257 (10 microM) had no detectable effect on T-type calcium current and slightly decreased L-type calcium current (-18.12 +/- 0.66%), without significant use-dependent blockade. 5. S 16257 had no effect on the delayed outward potassium current Ik at 3 microM and slightly decreased it only at high concentrations, -16.3 +/- 1.2% at 10 microM. In contrast, zatebradine, another bradycardic agent, reduced I k by 20.3 +/- 2.5% at 3 microM. 6. In conclusion, S 16257 may lower heart rate without significant negative inotropic action. In comparison with zatebradine, S 16257 had less effect on Ik suggesting less prolongation of repolarization time.

Animals↗
PubMed1995

Effect of human chorionic gonadotrophin on chloride current in human syncytiotrophoblasts in culture.

Human trophoblast differentiates in vivo and in vitro by the fusion of cytotrophoblastic cells to form syncytiotrophoblasts. A large amount of human chorionic gonadotrophin (hCG) is produced by the syncytiotrophoblasts, which express hCG luteinizing hormone (LH) receptors. Since recent investigations with electrophysiological techniques support the conclusion that hormonal effects can be mediated by modulations of the membrane ionic conductances of the cells, a perforated patch-clamp technique was used to investigate the possible presence of a chloride current evoked by hCG. The perifusion of hCG (500 mIU/ml) activated a time-independent current, which presents a linear current-voltage (I/V) relationship in symmetrical chloride concentrations. The reversal potential was -1.8 mV with 142 mM Cl- external solution and 134 mM cl- internal solution. This reversal potential shifted with changes in the transmembrane Cl- gradient. Moreover, this hCG-induced current was sensitive to 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50 microM), to diphenylalamine-2-carboxylic acid (DPC) (0.5 mM) and to 9-AC (1 mM), three known chloride channel blockers. These results confirm the autocrine action of hCG in the physiology of the trophoblast.

Cell Differentiation↗
PubMed1994

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single-copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.

Animals↗
PubMed1994

Effects of two chemically related new Ca2+ channel antagonists, SR33557 (fantofarone) and SR33805, on the L-type cardiac channel.

Fantofarone (SR33557) is a substituted indolizine and SR33805 is a substituted indole. These drugs have been shown to specifically bind to the alpha 1 subunit of the L-type Ca2+ channel at the same site, distinct from those of the classical 1,4-dihydropyridine, phenylalkylamine or benzothiazepine Ca2+ antagonists, but in negative allosteric interaction with them. The present work shows that fantofarone and SR33805 block L-type but not T-type Ca2+ channels in mouse cardiac cells in primary culture. This block is voltage-dependent. Fantofarone and SR33805 are potent Ca2+ channel blockers in depolarized conditions (i.e. at a holding potential of -40 mV) with an EC50 = 1.4 and 4.1 nM, respectively. In polarized conditions (i.e. at a holding potential of -80 mV), SR33805 is a better Ca2+ channel blocker (EC50 = 33 nM) than fantofarone (EC50 = 0.15 microM). Therefore differences in their chemical structures make the blocking action of fantofarone more sensitive to voltage than that of SR33805.

Animals↗