Improvement of comparative map between porcine chromosomes 1 and 7 and human chromosomes 6, 14, and 15 by using human YACs.
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Animals↗
Biomedical subjects
P Pinton
Publications and source records attributed to P Pinton.
Recombinant aequorin as tool for monitoring calcium concentration in subcellular compartments.
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Aequorin↗
Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming.
In recent years, mitochondria have emerged as important targets of agonist-dependent increases in cytosolic Ca(2+) concentration. Here, we analyzed the significance of Ca(2+) signals for the modulation of organelle function by directly measuring mitochondrial and cytosolic ATP levels ([ATP](m) and [ATP](c), respectively) with specifically targeted chimeras of the ATP-dependent photoprotein luciferase. In both HeLa cells and primary cultures of skeletal myotubes, stimulation with agonists evoking cytosolic and mitochondrial Ca(2+) signals caused increases in [ATP](m) and [ATP](c) that depended on two parameters: (i) the amplitude of the Ca(2+) rise in the mitochondrial matrix, and (ii) the availability of mitochondrial substrates. Moreover, the Ca(2+) elevation induced a long-lasting priming that persisted long after agonist washout and caused a major increase in [ATP](m) upon addition of oxidative substrates. These results demonstrate a direct role of mitochondrial Ca(2+) in driving ATP production and unravel a form of cellular memory that allows a prolonged metabolic activation in stimulated cells.
Adenosine Triphosphate↗
Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell.
In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.
Aequorin↗
Targeting GFP to organelles.
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Cells, Cultured↗
A calcium signaling defect in the pathogenesis of a mitochondrial DNA inherited oxidative phosphorylation deficiency.
In recent years, genetic defects of the mitochondrial genome (mtDNA) were shown to be associated with a heterogeneous group of disorders, known as mitochondrial diseases, but the cellular events deriving from the molecular lesions and the mechanistic basis of the specificity of the syndromes are still incompletely understood. Mitochondrial calcium (Ca2+) homeostasis depends on close contacts with the endoplasmic reticulum and is essential in modulating organelle function. Given the strong dependence of mitochondrial Ca2+ uptake on the membrane potential and the intracellular distribution of the organelle, both of which may be altered in mitochondrial diseases, we investigated the occurrence of defects in mitochondrial Ca2+ handling in living cells with either the tRNALys mutation of MERRF (myoclonic epilepsy with ragged-red fibers) or the ATPase mutation of NARP (neurogenic muscle weakness, ataxia and retinitis pigmentosa). There was a derangement of mitochondrial Ca2+ homeostasis in MERRF, but not in NARP cells, whereas cytosolic Ca2+ responses were normal in both cell types. Treatment of MERRF cells with drugs affecting organellar Ca2+ transport mostly restored both the agonist-dependent mitochondrial Ca2+ uptake and the ensuing stimulation of ATP production. These results emphasize the differences in the cellular pathogenesis of the various mtDNA defects and indicate specific pharmacological approaches to the treatment of some mitochondrial diseases.
Adenosine Triphosphatases↗
Physical assignments of human chromosome 13 genes on pig chromosome 11 demonstrate extensive synteny and gene order conservation between pig and human.
Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.
Animals↗
Mitochondria as biosensors of calcium microdomains.
The notion that the agonist-dependent increases in intracellular Ca2+ concentration, on ubiquitous signalling mechanism, occur with a tightly regulated spatio-temporal pattern has become an established concept in modern cell biology. As a consequence, the concept is emerging that the recruitment of specific intracellular targets and effector system mechanisms depends on exposure to local [Ca2+] that differs substantially from the mean [Ca2+]. A striking example is provided by mitochondria, intracellular organelles that have been overlooked for a long time in the field of calcium signalling due to the low affinity of their Ca(2+)-uptake pathways. We will summarize here some of the evidence indicating that these organelles actively participate in Ca2+ homeostasis in physiological conditions (with consequences not only for the control of their function, but also for the modulation of the complexity of calcium signals) because they have the capability to respond to microdomains of high [Ca2+] transiently generated in their proximity by the opening of Ca2+ channels.
Aequorin↗
Contribution to the comparative map between humans and pigs.
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Animals↗
Human chromosome 3 and pig chromosome 13 show complete synteny conservation but extensive gene-order differences.
A comparative map of human chromosome 3 (HSA 3) and pig chromosome 13 (SSC 13) was constructed using physically assigned pig sequence-tagged sites (STSs). Pig STSs representing 11 HSA 3 genes, including v-Raf-1 murine leukemia viral oncogene homolog 1 (RAF1), retinoic acid beta receptor (RARB), cholecystokinin (CCK), pituitary transcription factor 1 (POU1F1), ceruloplasmin (CP), guanine nucleotide binding protein, alpha-inhibiting polypeptide 2 (GNAI2), sucrase-isomaltase (SI), rhodopsin (RHO), dopamine receptor D3 (DRD3), growth-associated protein 43 (GAP43), and somatostatin (SST), were developed. Ten pig STSs were regionally mapped using a somatic cell hybrid panel (SCHP) to SSC 13 with 80-100% concordance. Large-insert probes were obtained by screening a pig yeast artificial chromosome (YAC) library with primers for each STS. Several YACs were identified for DRD3, GAP43, POU1F1, RHO, SI, and SST for fluorescence in situ hybridization (FISH) mapping. Single gene and bi-color FISH with each pairwise combination were used to further define the gene order on SSC 13. While these data confirm chromosome painting results showing that HSA 3 probes hybridize to a major portion of SSC 13, they also demonstrate extensive gene-order differences between man and pig within this large conserved synteny group. Interestingly, several conserved chromosomal regions have been detected between pig and mouse that are not conserved between man and mouse, suggesting that the SSC 13 gene arrangement may be the closest to that of the ancestral eutherian chromosome.
Animals↗
[Medico-economic evaluation of abciximab].
Abciximab (RéoPro) is a chimeric monoclonal antibody directed against platelet glycoprotein IIb-IIIa. It reduces the frequency of major cardiac ischaemic events after percutaneous transluminal coronary angioplasty (PTCA). This treatment is more expensive than the commonly used treatment (aspirin and heparin). However, a treatment can only be validly assessed in term of its cost-benefit ratio. The authors present the results of a medico-economic evaluation of abciximab in the French institutional context, based on the three-year clinical results of the EPIC study (Evaluation of abciximab for the Prevention of Ischemic Complications). The efficacy of treatment was expressed by means of two quantitative indicators: the number of patients with no major ischaemic event (MIE) and the number of years of life without myocardial infarction or revascularization. According to French good practice recommendations for economic evaluation of therapeutic strategies, major ischaemic events were assessed by using the complete cost of the corresponding homogeneous groups of diseases (GHM). Medico-economic evaluation of abciximab showed that the net excess cost per patient for national health insurance was 395 Francs, while the cost of the product is 5,300 Francs. The incremental cost per additional patient with no major ischaemic event is 6,585 Francs and the incremental cost per additional year of life without myocardial infarction or revascularization is 2,469 F. From a public health point of view, abciximab administered by bolus injection plus a 12-hour infusion, presents a good cost-effectiveness ratio in the prevention of cardiac ischaemic complications in high-risk patients after PTCA.
Abciximab↗
The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum.
In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.
Aequorin↗
Analysis of distribution in the human, pig, and rat genomes points toward a general subtelomeric origin of minisatellite structures.
We have developed approaches for the cloning of minisatellites from total genomic libraries and applied these approaches to the human, rat, and pig genomes. The chromosomal distribution of minisatellites in the three genomes is strikingly different, with clustering at chromosome ends in human, a seemingly almost even distribution in rat, and an intermediate situation in pig. A closer analysis, however, reveals that interstitial sites in pig and rat often correspond to terminal cytogenetic bands in human. This observation suggests that minisatellites are created toward chromosome ends and their internalization represents secondary events resulting from rearrangements involving chromosome ends.
Animals↗
Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses.
The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.
Adenosine Triphosphate↗
New light on mitochondrial calcium.
The possibility of specifically addressing recombinant probes to mitochondria is a novel, powerful way to study these organelles within living cells. We first showed that the Ca(2+)-sensitive photoprotein aequorin, modified by the addition of a mitochondrial targeting sequence, allows to monitor specifically the Ca2+ concentration in the mitochondrial matrix ([Ca2+]m) of living cells. With this tool, we could show that, upon physiological stimulation, mitochondria undergo a major rise in [Ca2+]m, well in the range of the Ca2+ sensitivity of the matrix dehydrogenases, in a wide variety of cell types, ranging from non excitable, e.g., HeLa and CHO, and excitable, e.g., cell lines to primary cultures of various embryological origin, such as myocytes and neurons. This phenomenon, while providing an obvious mechanism for tuning mitochondrial activity to cell needs, appeared at first in striking contrast with the low affinity of mitochondrial Ca2+ uptake mechanisms. Based on indirect evidence, we proposed that the mitochondria might be close to the source of the Ca2+ signal and thus exposed to microdomains of high [Ca2+], hence allowing the rapid accumulation of Ca2+ into the organelle. In order to verify this intriguing possibility, we followed two approaches. In the first, we constructed a novel aequorin chimera, targeted to the mitochondrial intermembrane space (MIMS), i.e., the region sensed by the low-affinity Ca2+ uptake systems of the inner mitochondrial membrane. With this probe, we observed that, upon agonist stimulation, a portion of the MIMS is exposed to saturating Ca2+ concentrations, thus confirming the occurrence of microdomains of high [Ca2+] next to mitochondria. In the second approach, we directly investigated the spatial relationship of the mitochondria and the ER, the source of agonist-releasable Ca2+ in non-excitable cells. For this purpose, we constructed GFP-based probes of organelle structure; namely, by targeting to these organelles GFP mutants with different spectral properties, we could label them simultaneously in living cells. By using an imaging system endowed with high speed and sensitivity, which allows to obtain high-resolution 3D images, we could demonstrate that close contacts (< 80 nm) occur in vivo between mitochondria and the ER.
Aequorin↗
Characterization of reciprocal translocations in pigs using dual-colour chromosome painting and primed in situ DNA labelling.
We report the use of dual-colour chromosome painting to determine the exact nature of certain chromosome rearrangements observed in the pig (Sus scrofa domestica). The chromosomal abnormalities were detected by GTG- and RBG-banding techniques. The initially proposed interpretations were: (1) rcp(6;13)(p1.5;q4.1); (2) rcp(11;16)(p1.4;q1.4); (3) rcp(6;16)(p1.1;q1.1); (4) rcp(13;17)(q4.1;q1.1); (5) rcp(6;14)(q2.7;q2.1); (6) rcp(3;5)(p1.3;q2.3); (7) rcp(2; 14)(q1.3;q2.7); (8) rcp(15;17)(q1.3;q2.1). Hybridizations were carried out with biotin- and digoxigenin-labelled probes obtained by priming authorizing random mismatches polymerase chain reaction (PARM-PCR) amplification of porcine flow-sorted chromosomes. In some cases, i.e. (1), (4), (5), (6), (7) and (8), the fluorescence in situ hybridization (FISH) results allowed confirmation of the interpretations proposed with classical cytogenetic methods. Chromosome painting proved the reciprocity of the translocation in cases (1), (6) and (8), whereas modifications of the formula were proposed for case (2). Primed in situ DNA labelling (PRINS) experiments have also been carried out in case (3) using a primer specific for the centromeres of acrocentric chromosomes (first experiment) or a primer specific for the centromeres of a subset of meta- and submetacentric chromosomes including chromosome 6 (second experiment). It allowed us to demonstrate that the breakpoints occurred in the centromeric region of chromosome 16 and in the p. arm of chromosome 6, just above the centromere.
Animals↗
Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.
We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.
Animals↗
[Abciximab-induced thrombopenia during treatment of acute coronary syndromes by angioplasty].
ReoPro (abciximab) is the Fab fragment of a chimeric monoclonal antibody directed against platelet glycoprotein IIb-IIIa. Its efficacy to prevent ischaemic complications after PTCA has been demonstrated in 3 studies: EPIC, EPILOG, UPTAKE. One hundred and sixty five cases of thrombocytopenia (< 100,000/microliter) were reported in a series of 5461 patients randomized in these 3 studies (i.e. 3.0%), including 46 (2.03%) with placebo and 119 (3.73% with abciximab. Among the 2270 patients randomized to receive placebo, 11 (0.48%) cases of severe thrombocytopenia (< 50,000/microliter) were observed versus 34 (1.07%) with abciximab. Major acute thrombocytopenia (< 20,000/microliter and < 24 hours) occurred in 0.60% (20 patients) of cases with abciximab. Their mechanism remains unknown. A therapeutic challenge did not modify either their incidence, or their severity. The development of thrombocytopenia did not worsen the patient's prognosis and course was always favourable. Twenty five cases of thrombocytopenia (0.60%), including 3 cases of acute major thrombocytopenia (0.08%) were spontaneously reported in France among the first 4000 patients treated with abciximab post-marketing. All patients treated with abciximab must be monitored by platelet count, 2 to 4 hours after the bolus administration, then 12 and 24 hours later. These platelet counts should be performed on 3 tubes (EDTA, citrate, heparin) in order to eliminate pseudothrombocytopenia and a differential diagnosis. In the case of true thrombocytopenia (< 10,000/l), treatment should be suspended and the platelet count should be repeated daily until return to normal. In the case of thrombocytopenia less than 60,000/microliter, heparin and aspirin should also be systematically discontinued and, below 50,000/microliter, platelet transfusion is justified.
Abciximab↗