Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia.

Ninein is expressed in the cytoplasm of angiogenic tip-cells and regulates tubular morphogenesis of endothelial cells.

OBJECTIVE: Angiogenesis is an integral part of many physiological processes but may also aggravate pathological conditions such as cancer. Development of effective angiogenesis inhibitors requires a thorough understanding of the molecular mechanisms regulating vessel formation. The aim of this project was to identify proteins that regulate tubular morphogenesis of endothelial cells. METHODS AND RESULTS: Phosphotyrosine-dependent affinity-purification and mass spectrometry showed tyrosine phosphorylation of ninein during tubular morphogenesis of endothelial cells. Ninein was recently identified as a centrosomal microtubule-anchoring protein. Our results show that ninein is localized in the cytoplasm in endothelial cells, and that it is highly expressed in the vasculature in normal and pathological human tissues. Using embryoid bodies as a model of vascular development, we found that ninein is abundantly expressed in the cytoplasm of endothelial cells during sprouting angiogenesis, in particular in the sprouting tip-cell. In accordance, siRNA-dependent silencing of ninein in endothelial cells inhibited tubular morphogenesis. CONCLUSIONS: In this study, we show that ninein is expressed in developing vessels and in endothelial tip cells, and that ninein is critical for formation of the vascular tube. These data strongly implicate ninein as an important new regulator of angiogenesis.

Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.

Gene expression profiles in T24 human bladder carcinoma cells by inhibiting an L-type amino acid transporter, LAT1.

Inhibition of LAT1 (L-type amino acid transporter 1) activity in tumor cells could be effective in the inhibition of tumor cell growth by depriving tumor cells of essential amino acids. Because of the high level of expression of LAT1 in tumor cells, LAT1 inhibitors would be useful for anticancer therapy in suppressing tumor growth without affecting normal tissues. In recent years, cDNA microarray technique is useful technology for anticancer drug development. It allows identifying and characterizing new targets for developments in cancer drug therapy through the understanding genes involved in drug action. The present study was designed to investigate gene expression profile induced by LAT1 inhibitor using gene chip technology. Human bladder carcinoma cells (T24 cells) were treated with classical system L inhibitor 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid (BCH). Gene chip experiment was applied for treated and untreated cells after 3 and 12 h. Two independent experiments with a high degree of concordance identified the altered expression of 151 and 200 genes after 3 and 12 h BCH treatment. Among these genes, 132 and 13 were up-regulated and 19 and 187 were down-regulated by 3 and 12 h BCH treatment respectively. We found that BCH affected the expression of a large number of genes that are related to the control of cell survival and physiologic behaviors. These data are useful for understanding of intracellular signaling of cell growth inhibition induced by LAT1 inhibitors as candidate for anticancer drug therapy.

Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells.

System L is a major nutrient transport system responsible for the Na(+)-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LAT1) is up-regulated to support tumor cell growth. LAT1 is also essential for the permeation of amino acids and amino acid-related drugs through the blood-brain barrier. To search for in vitro assay systems to examine the interaction of chemical compounds with LAT1, we have investigated the expression of system L transporters and the properties of [14C]L-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immunofluorescence analyses have reveled that T24 cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas T24 cells do not express the other system L isoform LAT2. The uptake of [14C]L-leucine by T24 cells is Na(+)-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [14C]L-leucine uptake by amino acids and amino acid-related compounds in T24 cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C]L-leucine uptake is, therefore, mediated by LAT1 in T24 cells. Consistent with LAT1 in Xenopus oocytes, the efflux of preloaded [14C]L-leucine is induced by extracellularly applied substrates of LAT1 in T24 cells. This efflux measurement has been proven to be more sensitive than that in Xenopus oocytes, because triiodothyronine, thyroxine and melphalan were able to induce the efflux of preloaded [14C]L-leucine in T24 cells, which was not detected for Xenopus oocyte expression system. T24 cell is, therefore, proposed to be an excellent tool to examine the interaction of chemical compounds with LAT1.

Moderate low temperature preserves the stemness of neural stem cells and suppresses apoptosis of the cells via activation of the cold-inducible RNA binding protein.

We hypothesized that one of the mechanisms underlying the protection of brain injury by therapeutic hypothermia is associated with preservation of neural stem cells. We investigated effects of moderate low temperature and the contribution of a cold-inducible molecule for the stemness of neural stem cells. The MEB5 mouse neural stem cell line was cultured in the presence or absence of EGF, and apoptosis, mRNA expression, and immunocytochemistry of the differentiation markers nestin and GFAP were evaluated at 37 or 32°C. We investigated the contribution of the cold-inducible RNA binding protein (CIRP) on apoptosis and differentiation of MEB5 cells at 32°C. EGF deprivation increased the number of apoptotic cells, decreased expression of nestin, and increased expression of GFAP. The moderate low temperature prevented apoptosis and decreases in expression of GFAP in MEB5 by EGF deprivation. The moderate low temperature significantly increased expression of CIRP. siRNA against CIRP significantly increased the apoptotic cell population of MEB5 cells via EGF deprivation at 32°C. These findings suggest that moderate low temperature preserved stemness of neural stem cells and prevented cell apoptosis via the stimulation of CIRP, and one of the mechanisms of rescue of brain injury by the moderate hypothermia is associated with preservation of neural stem cells.

Mouse embryonic stem cells give rise to gut-like morphogenesis, including intestinal stem cells, in the embryoid body model.

Embryonic stem (ES) cells have been proposed as candidates for cell replacement therapy in patients with intestinal failure because these cells can be expanded indefinitely without losing their pluripotent phenotype. We investigated the differentiation capacity of mouse ES cells into gut-like structures, including intestinal stem cells, and defined culture conditions for efficient induction of formation of these structures. ES cell-derived gut-like structures (ES-guts) were reproducibly induced in developing embryoid bodies (EBs) by day 21 of differentiation culture. ES-guts contained an endodermal epithelium, a smooth muscle layer, interstitial cells of Cajal, and enteric neurons and showed spontaneous contraction. Transplantation of ES-guts under the kidney capsules of immunodeficient mice induced formation of highly differentiated epithelium composed of absorptive cells and goblet cells in the grafts. Immunoreactivity for Musashi-1 (Msi-1), a marker of intestinal stem cells, was detected in 1.9% of the columnar epithelial cells in the graft. Culture with 0.1% dimethyl sulfoxide increased the numbers of ES-guts in EBs, and serum-replacement (SR) culture, in comparison to standard ES culture containing 15% serum, increased the area ratio of ES-guts to EBs. SR culture also promoted maturation of epithelium to form a single layer of columnar epithelial cells, including absorptive cells and goblet cells. Expression of Msi-1 mRNA and protein was significantly enhanced when EBs were cultured under SR conditions. In conclusion, SR conditions efficiently induce formation of ES-guts and promote differentiation of epithelium, including intestinal stem cells. These results suggest the feasibility of cell-based therapy for intestinal failure based on ES cell culture systems.

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule.

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.

Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters.

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.