Ablation of vitamin D signaling rescues bone, mineral, and glucose homeostasis in Fgf-23 deficient mice.

To explore further the role of the vitamin D axis for fibroblast growth factor-23 (FGF23) signaling, we mated Fgf-23 deficient (Fgf-23(-/-)) mice and vitamin D receptor (VDR) mutant mice with a non-functioning VDR. To prevent secondary hyperparathyroidism in VDR and compound mutant mice, all mice were kept on a rescue diet enriched with calcium, phosphorus, and lactose. Consistent with previous findings, Fgf-23(-/-) animals showed hypercalcemia, hyperphosphatemia, growth retardation, ectopic calcifications, severe osteoidosis, skin atrophy, and renal dysfunction. In addition, here we describe that Fgf-23(-/-) mice are hypoglycemic, and have profoundly increased peripheral insulin sensitivity and improved subcutaneous glucose tolerance, but normal renal expression of the aging suppressor gene Klotho. Although VDR and double mutants on the rescue diet still had moderately elevated parathyroid hormone serum levels and lower bone mineral density compared to wild-type mice, double mutant mice were normocalcemic and normophosphatemic, and had normal body weight, normal renal function, and no ectopic calcifications. Ablation of vitamin D signaling in compound mutants also normalized subcutaneous glucose tolerance tests and insulin secretory response. In conclusion, our results indicate that the alterations in mineral and carbohydrate metabolism present in Fgf-23(-/-) mice require an intact vitamin D signaling pathway.     

Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1⁺ cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies.     

The elongated first fibronectin type III domain of collagen XIV is an inducer of quiescence and differentiation in fibroblasts and preadipocytes

Collagen XIV (CXIV) is a fibril-associated collagen that is mainly expressed in well differentiated tissues and in late embryonic development. Because CXIV is almost absent in proliferating and/or dedifferentiated tissues, a functional role in maintaining cell differentiation is suspected. We demonstrate antiproliferative, quiescence- and differentiation-inducing effects of human CXIV and its recombinant fragments on mesenchymal cells. In primary human fibroblasts, in mouse 3T3 fibroblasts and in 3T3-L1 preadipocytes, CXIV reduced de novo DNA synthesis by 75%, whereas cell numbers and viability remained unaltered. Cells showed no signs of apoptosis, and maximal proliferation was restored when serum was supplemented, thus indicating that CXIV induced reversible cellular quiescence. Exposure of fibroblasts to CXIV in vitro led to cellular bundles and clusters. CXIV also triggered trans-differentiation of 3T3-L1 preadipocytes into adipocytes, as could be shown by lipid accumulation and by expression of the glucose transporter Glut4. These effects were also observed with the amino-terminal recombinant fragment Gln(29)-Pro(154) that harbors the first fibronectin type III domain and a 39-amino-acid extension, whereas no activity was found for all other recombinant CXIV fragments. Based on these finding the development of small molecular analogs that modulate fibroblast cell growth and differentiation, e.g. in wound healing and fibrosis, seems feasible.     

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.     

Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

Background

Several recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.

Results

We describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.

Conclusion

TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1236-7) contains supplementary material, which is available to authorized users.     

T cell differentiation antigens on lymphocytes in the human intestinal lamina propria.

Recently, T cell subpopulations presumably representing memory T lymphocytes have been described in vitro. Intestinal lamina propria T cells (LP-T) have characteristics resembling those of memory cells. We therefore investigated the expression of surface Ag associated with memory phenotype in vitro on lamina propria lymphocytes (LPL) and PBL and on the T cell subpopulations defined by the bright expression of CD45R0 by flow cytometric analysis of isolated cell populations. LPL had significantly increased percentages of CD45R0 and CD58 positive cells compared with PBL. Whereas PBL showed bimodal expression profiles of CD45R0, CD58, and CD2, the vast majority of LPL was bright for these Ag. Expression of CD45RA was significantly reduced in both frequency and intensity in LPL, and LPL had significantly reduced percentages of CD11a/CD18 and CD29 positive cells compared with PBL. The CD45R0 bright T cell subpopulations of both PBL and LPL were characterized by a lack of CD45RA. CD45R0 bright T cells from the peripheral blood (PB-T) were predominantly bright for CD2, CD58, CD29, and CD11a/CD18 whereas CD45R0 dim PB-T had bimodal expression profiles and CD45R0 negative PB-T were dim or even negative for these Ag. CD45R0 bright LP-T were also bright for CD2 and CD58 but had significantly reduced surface densities of CD11a/CD18 and CD29 compared with CD45R0 bright PB-T. The surface density of CD29 on CD45R0 bright LP-T corresponded to that of CD45R0 negative PB-T, and a significant proportion of CD45R0 bright LP-T was even negative for CD11a/CD18 and CD29. Additionally, CD45R0 bright LP-T in contrast to PB-T were characterized by a lack of 1-selectin and the expression of CDw49a and the mucosa-specific T cell Ag HML-1 on high percentages of cells. Our results show that the phenotype of CD45R0 bright T cells from the lamina propria clearly deviates from that of memory T cells in vitro and of CD45R0 bright T cells in the peripheral blood. We conclude that memory T cell populations in vivo undergo specific differentiation depending on their tissue localization, leading to unique phenotypic and presumably functional features.