Mutation Rate Switch inside Eurasian Mitochondrial Haplogroups: Impact of Selection and Consequences for Dating Settlement in Europe

R-lineage mitochondrial DNA represents over 90% of the European population and is significantly present all around the planet (North Africa, Asia, Oceania, and America). This lineage played a major role in migration “out of Africa” and colonization in Europe. In order to determine an accurate dating of the R lineage and its sublineages, we analyzed 1173 individuals and complete mtDNA sequences from Mitomap. This analysis revealed a new coalescence age for R at 54.500 years, as well as several limitations of standard dating methods, likely to lead to false interpretations. These findings highlight the association of a striking under-accumulation of synonymous mutations, an over-accumulation of non-synonymous mutations, and the phenotypic effect on haplogroup J. Consequently, haplogroup J is apparently not a Neolithic group but an older haplogroup (Paleolithic) that was subjected to an underestimated selective force. These findings also indicated an under-accumulation of synonymous and non-synonymous mutations localized on coding and non-coding (HVS1) sequences for haplogroup R0, which contains the major haplogroups H and V. These new dates are likely to impact the present colonization model for Europe and confirm the late glacial resettlement scenario

Recherche des signaux effecteurs dans l'exposition membranaire de la phosphatidylsérine procoagulante (exploration des voies MAPK/ERK et pro-apoptotique mitochondriale)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Platelet specific promoters are insufficient to express protease activated receptor 1 (PAR1) transgene in mouse platelets.

The in vivo study of protease activated receptors (PARs) in platelets is complicated due to species specific expression profiles. Human platelets express PAR1 and PAR4 whereas mouse platelets express PAR3 and PAR4. Further, PAR subtypes interact with one another to influence activation and signaling. The goal of the current study was to generate mice expressing PAR1 on their platelets using transgenic approaches to mimic PAR expression found in human platelets. This system would allow us to examine specific signaling from PAR1 and the PAR1-PAR4 heterodimer in vivo. Our first approach used the mouse GPIbα promoter to drive expression of mouse PAR1 in platelets (GPIbα-Tg-mPAR1). We obtained the expected frequency of founders carrying the transgene and had the expected Mendelian distribution of the transgene in multiple founders. However, we did not observe expression or a functional response of PAR1. As a second approach, we targeted human PAR1 with the same promoter (GPIbα-Tg-hPAR1). Once again we observed the expected frequency and distributing of the transgene. Human PAR1 expression was detected in platelets from the GPIbα-Tg-hPAR1 mice by flow cytometry, however, at a lower level than for human platelets. Despite a low level of PAR1 expression, platelets from the GPIbα-Tg-hPAR1 mice did not respond to the PAR1 agonist peptide (SFLLRN). In addition, they did not respond to thrombin when crossed to the PAR4-/- mice. Finally, we used an alternative platelet specific promoter, human αIIb, to express human PAR1 (αIIb-Tg-hPAR1). Similar to our previous attempts, we obtained the expected number of founders but did not detect PAR1 expression or response in platelets from αIIb-Tg-hPAR1 mice. Although unsuccessful, the experiments described in this report provide a resource for future efforts in generating mice expressing PAR1 on their platelets. We provide an experimental framework and offer considerations that will save time and research funds

Correction: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30-100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3(-/-) mice had increased G(q) signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca(2+)) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca(2+) release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3(+/-)) had an intermediate increase in maximum Ca(2+) mobilization. Treatment of PAR3(-/-) mice platelets with P2Y(12) antagonist (2MeSAMP) did not affect Ca(2+) mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G(12/13) signaling in response to thrombin was not significantly different between wild type and PAR3(-/-) mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca(2+) mobilization and PKC activation in mouse platelets by physical interaction

Effect of 2MeSAMP on PAR4 enhancing intracellular Ca²⁺ mobilization in mouse platelets.

<p>Fura 2-loaded wild type (black) and PAR3^−/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin (<b>A</b>,) or 2 mM AYPGKF (<b>B</b>) for 10 min at 37°C in the presence of 2 mM of CaCl₂. The difference between the maximum increase and the basal intracellular Ca²⁺ mobilization was measured. The results are the mean (± SD) of three independent experiments (* <i>p</i><0.05).</p

Dose response curve of Ca²⁺ mobilization in the absence of extracellular Ca²⁺ in mouse platelets.

<p>Fura 2-loaded wild type (black line) and PAR3^−/− (gray line) platelets were resuspended in Ca²⁺-free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: (<b>A</b>) thrombin (1–100 nM), (<b>C</b>) AYPGKF (0.15–2 mM), or (<b>E</b>) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca²⁺ mobilization in platelets stimulated with: (<b>B</b>) thrombin, (<b>D</b>) AYPGKF, or (<b>F</b>) thapsigargin. The results are the mean (± SD) of three independent experiments (* <i>p</i><0.05).</p

PAR4 expression on mouse platelets.

<p>Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3^−/− (gray line), and PAR4^−/− (shaded) mice platelets using anti-PAR4-FITC antibodies.</p

α_IIb-Tg-hPAR1 transgenic mice on a PAR4^−/− background do not respond to thrombin.

<p>(<b>A</b>) Intracellular calcium mobilization was measured in platelets from α_IIb-Tg-hPAR1-PAR4^−/− mice in response to thrombin (10 nM). The tracing is representative of three independent experiments. (<b>B</b>) P-selectin expression was measured in the surface of platelets from wild type (wt) (gray bars) or α_IIb-Tg-hPAR1-PAR4^−/− (white bars) mice by flow cytometry using FITC conjugated P-selectin antibody in response to thrombin (10 nM) or SFLLRN (30 µM). (<b>C</b>) Platelets were treated as in (B) and integrin αIIbβ3 activation was measured using PE conjugated JON/A antibody. The results are the mean of three independent experiments.</p

Generation and characterization of transgenic mice expressing mouse PAR1 transgene under control of mouse GPIbα promoter (GPIbα-Tg-mPAR1).

<p>(<b>A</b>) Schematic representation of the transgene construct. The cDNA for mouse PAR1 was inserted into a vector containing the mouse GPIbα, promoter small-t intron of simian virus 40 (SV40) in the 5′-untranslated region (UTR) and SV40 polyadenylation (polyA) sequence in the 3′ -UTR. (<b>B</b>) Representative genotyping from GPIbα-Tg-mPAR1. The control PCR reactions used primers specific for the bradykinin B2 receptor (BkB2). (<b>C</b>) Platelet aggregation in response to thrombin (100 nM) or SFLLRN (50 µM) expressed as a percentage of the maximal light transmission. The results are the mean of six independent experiments.</p