Levels of neutral genetic diversity within and between populations were compared between outcrossing (self-incompatible) and inbreeding populations in the annual plant genus Leavenworthia. Two taxonomically independent comparisons are possible, since self-incompatibility has been lost twice in the group of species studied. Within inbred populations of L. uni£ora and L. crassa, no DNA sequence variants were seen among the alleles sampled, but high diversity was seen in alleles from populations of the outcrosser L. stylosa, and in self-incompatible L. crassa populations. Diversity between populations was seen in all species. Although total diversity values were lower in the sets of inbreeding populations, between-population values were as high, or higher, than those in the outcrossing taxa. Possible reasons for these diversity patterns are discussed. As the e¡ect of inbreeding appears to be a greater than twofold reduction in diversity, we argue that some process such as selection for advantageous mutations, or against deleterious mutations, or bottlenecks occurring predominantly in the inbreeders, appears necessary to account for the ¢ndings. If selection for advantageous mutations is responsible, it appears that it must be some form of local adaptive selection, rather than substitution of alleles that are advantageous throughout the species. This is consistent with the ¢nding of high between-population diversity in the inbreeding taxa.
To test the theoretical prediction that highly inbreeding populations should have low neutral genetic diversity relative to closely related outcrossing populations, we sequenced portions of the cytosolic phosphoglucose isomerase (PgiC) gene in the plant genus Leavenworthia, which includes both self-incompatible and inbreeding taxa. On the basis of sequences of intron 12 of this gene, the expected low diversity was seen in both populations of the selfers Leavenworthia uniflora and L. torulosa and in three highly inbreeding populations of L. crassa, while high diversity was found in self-incompatible L. stylosa, and moderate diversity in L. crassa populations with partial or complete self-incompatibility. In L. stylosa, the nucleotide diversity was strongly structured into three haplotypic classes, differing by several insertion/deletion sequences, with linkage disequilibrium between sequences of the three types in intron 12, but not in the adjacent regions. Differences between the three kinds of haplotypes are larger than between sequences of this gene region from different species. The haplotype divergence suggests the presence of a balanced polymorphism at this locus, possibly predating the split between L. stylosa and its two inbreeding sister taxa, L. uniflora and L. torulosa. It is therefore difficult to distinguish between different potential causes of the much lower sequence diversity at this locus in inbreeding than outcrossing populations. Selective sweeps during the evolution of these populations are possible, or background selection, or merely loss of a balanced polymorphism maintained by overdominance in the populations that evolved high selfing rates.
Proper functioning of intracellular membrane trafficking in endothelial cells is indispensable for maintaining endothelial barrier, as well as for regulated secretion of a variety of pro-and anticoagulants and inflammatory proteins. While the involvement of several heterotrimeric G proteins in regulation of membrane trafficking has been documented, there is only one work published on the possible role of G␣12 in this respect in PC12 cells, where G␣12 has been reported to inhibit exocytosis. In the present study, we addressed possible roles of G␣12 in membrane trafficking in endothelial cells, using human umbilical vein endothelial cells (HUVEC) as a model. Using confocal microscopy, ECIS measurements, and velocity sedimentation assays, we examined the effects of overexpression of wild-type or constitutively active G␣12, as well as siRNA-mediated depletion of endogenous G␣12, on several markers in resting or thrombin-stimulated HUVEC. Our data indicate that G␣12 has stimulatory effect on exocytosis in HUVEC. Moreover, we were able to detect macromolecular complexes of G␣12 with transported proteins, suggesting its direct involvement in membrane trafficking.
We have previously shown that treatment of bovine endothelial cell monolayers with phorbol myristate acetate leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. Here we demonstrate that phorbol myristate acetate induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of actin and myosin redistribution is not followed by caldesmon. Coimmunoprecipitation experiments revealed that short-term (5 minutes) treatment with phorbol ester leads to the weakening of caldesmon ability to bind actin and myosin. Prolonged incubation with phorbol myristate acetate, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. Phorbol ester stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing weakening of the cortical actin ring, whereas inhibition of protein kinase C reverses phorbol ester-induced cytoskeletal rearrangement. Our results suggest that protein kinase C-dependent phosphorylation of caldesmon is involved in phorbol ester-mediated complex cytoskeletal changes leading to the endothelial cell barrier compromise.
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