Elevated PTH induces endothelial-to-chondrogenic transition in aortic endothelial cells

Previous studies have shown that increased parathyroid hormone (PTH) attributable to secondary hyperparathyroidism in chronic kidney disease accelerates the arteriosclerotic fibrosis and calcification. Although the underlying mechanisms remain largely unknown, endothelial cells (ECs) have recently been demonstrated to participate in calcification in part by providing chondrogenic cells via the endothelial-to-mesenchymal transition (EndMT). Therefore, this study aimed to investigate whether elevated PTH could induce endothelial-to-chondrogenic transition in aortic ECs and to determine the possible underlying signaling pathway. We found that treatment of ECs with PTH significantly upregulated the expression of EndMT-related markers. Accordingly, ECs treated with PTH exhibited chondrogenic potential. In vivo, lineage-tracing model-subjected mice with endothelial-specific green fluorescent protein fluorescence to chronic PTH infusion showed a marked increase in the aortic expression of chondrocyte markers, and confocal microscopy revealed the endothelial origin of cells expressing chondrocyte markers in the aorta after PTH infusion. Furthermore, this in vitro study showed that PTH enhanced the nuclear localization of β-catenin in ECs, whereas β-catenin siRNA or DKK1, an inhibitor of β-catenin nuclear translocation, attenuated the upregulation of EndMT-associated and chondrogenic markers induced by PTH. In summary, our study demonstrated that elevated PTH could induce the transition of ECs to chondrogenic cells via EndMT, possibly mediated by the nuclear translocation of β-catenin.

19 DIRECT INTRODUCTION OF GENE CONSTRUCTS INTO THE PRONUCLEUS-LIKE STRUCTURE OF CLONED EMBRYOS: A NEW STRATEGY FOR THE GENERATION OF GENETICALLY MODIFIED PIGS

Because of a rising demand for complex porcine disease models for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a new strategy for the generation of genetically modified pigs, termed “nuclear injection.” This new strategy could allow adding large constructs into cloned embryos with a genetically modified background. Moreover, the generation of multiple transgenic pigs based on already existing transgenic cells could be facilitated due to a reduction of recloning steps. To evaluate the reliability of this approach, developmental ability of the embryos in vitro or in vivo and integration or expression efficiency of the transgene were examined. Somatic cell NT using in vitro matured oocytes was performed. Wild-type cells were used as nuclear donors. Centrifugation was done 10 h after activation for visualisation of a PN-like structure. Subsequently, linearized pmaxGFP (10 ng μL–1; Amaxa Biosystems) was directly injected into the PN-like structure of the cloned embryos. Expression efficiency in blastocysts generated by nuclear injection was compared to blastocysts generated by the classical PN injection using in vitro-produced zygotes. Injected embryos were transferred to recipient pigs without green fluorescent protein (GFP) selection, and fetuses collected at Day 68 were characterised for their integration and expression pattern of the transgene. Eighty percent of the reconstructed embryos (633/787) exhibited a PN-like structure, which made them available for the method. Green fluorescent protein fluorescence was observed in about half of total blastocysts (52.5%, 21/40), which was comparable to classical PN injection (68.4%, 28/41). Green fluorescent protein fluorescence of blastocysts ranged from mosaic to uniform patterns. In total, 478 pmaxGFP-injected embryos were transferred into 4 recipients, 4 fetuses were collected from one of them. In one of the fetuses that developed normally, the integration of the transgene was confirmed by PCR in different major organs from all 3 primary germ layers and placenta. The integration pattern of the transgene was mosaic (43 out of 84 single-cell colonies established from kidney were positive for GFP DNA by PCR). However, the proportion of GFP-expressing cells was very low (5 out of 84 colonies expressed GFP), which might indicate silencing of transgene expression. Our pilot study demonstrated that the direct introduction of gene constructs into cloned embryos could be a new strategy for the generation of genetically modified pigs. This approach could also be applied to rescue embryos with lethal knockouts by transfer of corresponding human genes, to generate pigs as bioreactors, e.g. for antibodies. This work was supported by the German Research Council – Transregio Collaborative Research Center 127 “Xenotransplantation.”

Diverse Microhabitats Experienced by Halomonas variabilis on Salt-Secreting Leaves

ABSTRACTThe leaf surfaces of the salt-excreting treeTamarix aphyllaharbor a wide diversity of halophilic microorganisms, includingHalomonassp., but little is known of the factors that shape community composition in this extreme habitat. We isolated a strain ofHalomonas variabilisfrom the leaf surface ofT. aphyllaand used it to determine the heterogeneity of salt concentrations experienced by bacteria in this environment. This halophilic strain was transformed with aproU::gfpreporter gene fusion, the fluorescence of which was responsive to NaCl concentrations up to 200 g liter−1. These bioreporting cells were applied toT. aphyllaleaves and were subsequently recovered from dew droplets adhering to the leaf surface. Although cells from within a given dew droplet exhibited similar green fluorescent protein fluorescence, the fluorescence intensity varied between droplets and was correlated with the salt concentration measured in each drop. Growth ofH. variabiliswas observed in all droplets, regardless of the salt concentration. However, cells found in desiccated microniches between dew drops were low in abundance and generally dead. Other bacteria recovered fromT. aphylladisplayed higher desiccation tolerance thanH. variabilis, both in culture and on inoculated plants, despite having lower osmotic tolerance. Thus, theTamarixleaf surface can be described as a salty desert with occasional oases where water droplets form under humid conditions. While halotolerant bacteria such asHalomonasgrow in high concentrations of salt in such wet microniches, other organisms are better suited to survive desiccation in sites that are not wetted.

Sigma Factor Genes sigC, sigE, and sigG Are Upregulated in Heterocysts of the Cyanobacterium Anabaena sp. Strain PCC 7120

ABSTRACT We used gfp transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Reporter strains containing gfp fusions with the upstream regions of sigB2, sigD, sigI, and sigJ did not show developmental regulation. Time-lapse microscopy of sigC, sigE, and sigG reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.

Ghrelin Amplifies Dopamine Signaling by Cross Talk Involving Formation of Growth Hormone Secretagogue Receptor/Dopamine Receptor Subtype 1 Heterodimers

Our objective is to determine the neuromodulatory role of ghrelin in the brain. To identify neurons that express the ghrelin receptor [GH secretagogue receptor (GHS-R)], we generated GHS-R-IRES-tauGFP mice by gene targeting. Neurons expressing the GHS-R exhibit green fluorescence and are clearly evident in the hypothalamus, hippocampus, cortex, and midbrain. Using immunohistochemistry in combination with green fluorescent protein fluorescence, we identified neurons that coexpress the dopamine receptor subtype 1 (D1R) and GHS-R. The potential physiological relevance of coexpression of these two receptors and the direct effect of ghrelin on dopamine signaling was investigated in vitro. Activation of GHS-R by ghrelin amplifies dopamine/D1R-induced cAMP accumulation. Intriguingly, amplification involves a switch in G protein coupling of the GHS-R from Gα11/q to Gαi/o by a mechanism consistent with agonist-dependent formation of GHS-R/D1R heterodimers. Most importantly, these results indicate that ghrelin has the potential to amplify dopamine signaling selectively in neurons that coexpress D1R and GHS-R.