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1) Bone marrow‑derived mononuclear cells (BMMNC include endothelial progenitor cells (EPC which are character‑ ized by their secretion of angiogenic factors such as vascular endothelial growth factor (VEGF to recruit local endo‑ thelial cells thereby enabling the establishment of new blood vessels. Implantation of BMMNC has been clinically used for therapeutic purposes in the treatment of critical limb ischemia (CLI; results showed that it was ineffective in a substantial number of cases. To evaluate the appropriate concentration of pyruvate to achieve the highest VEGF gene expression cells were cultured with pyruvate at final concentrations up to 20 mM in 5% CO2 for 2‑4 days. The intracellular concentration of pyruvate was measured enzymatically and cell number and viability were determined. Expression levels of VEGF genes and numbers of CD31 + CD34 + cells were evaluated. Finally VEGF levels in the con‑ ditioned medium were examined in each condition. Pyruvate supplementation in murine BMMNC cultures success‑ fully increased intracellular pyruvate levels in a concentration‑dependent manner and 5 mM pyruvate was found to be the most appropriate to maintain viable cell number and up‑regulate VEGF gene after 2‑day culture. In addition VEGF in the conditioned medium was significantly elevated by the use of 5 mM pyruvate after 4‑day culture. From these results we suggest that preconditioning of BMMNC with 5 mM pyruvate for 2 days may be a useful way to safely and inexpensively enhance the angiogenic properties of BMMNC and the therapeutic effectiveness of cellular therapy for CLI.

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.

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.

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.

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.

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.

Using a dialysis electrode, previous studies showed a clear biphasic release of glutamate during anoxia and ischemia. In this study, we examined two hypotheses: (1) glutamate is of vesicular origin and its release is thus Ca2+- and ATP-dependent in the first phase, while in the second phase glutamate is derived primarily from the metabolic pool, and (2) reversed glutamate uptake, due to electrogenic stoichiometry, produces the second phase during anoxic insult in the rat brain. A dialysis electrode continuously perfused with glutamate oxidase and ferrocene-conjugated bovine serum albumin (BSA) optimized the time resolution of monitoring, allowing quantitative oxygen-independent, real-time measurement of the extracellular glutamate concentration ([Glu]e) during anoxia. [Glu]e dynamics were analyzed during anoxia by combining the dialysis electrode with focal microinjection of substances inducing glutamate release. Following anoxia in the rat brain, a sharp and rapid [Glu]e elevation took place (first phase). The [Glu]e elevation then shifted, continuing a gently sloping rise throughout the anoxic period (second phase). This first phase disappeared with intracranial administration of either Co2+ or omega-conotoxin. The second phase rise increased with focal microinjection of KCl (300 mM, 1 microL) and decreased with NaCl (300 mM, 1 microL), ultimately reaching a plateau in both cases. Preloading with a novel glutamate transporter inhibitor (tPDC) decreased both the first and second phases of [Glu]e elevation. This dialysis electrode system provides data supporting in vivo evidence that the peak of the first phase of [Glu]e elevation is derived from the "neurotransmitter pool," while the second phase is derived from the neuronal and glial "metabolic pool," which is, at least, partly related to a "reversed uptake" mechanism in the anoxic rat brain.

The effects of the GABA A agonist, isoguvacine, on NMDA-induced burst firing of substantia nigra dopaminergic neurons were studied with intracellular and whole cell recordings in vitro. NMDA application caused the neurons to fire in rhythmic bursts. Although the NMDA-induced bursty firing pattern was insensitive to hyperpolarization by current injection, it was reversibly abolished by the selective GABA A agonist, isoguvacine. The block of the rhythmic burst pattern by isoguvacine application occurred regardless of whether the chloride reversal potential was hyperpolarizing ( E Cl − =−70 mV) or depolarizing ( E Cl − =−40 mV). In either case, the input resistance of the dopaminergic neurons was dramatically decreased by application of isoguvacine. It is concluded that GABA A receptor activation by isoguvacine disrupts NMDA receptor-mediated burst firing by increasing the input conductance and thereby shunting the effects of NMDA acting at a distally located generator of rhythmic burst firing.

Glutamate has been proposed to play a critical role in acute ischemic pathophysiology in the brain. In this study, glutamate was monitored by the dialysis electrode technique, in which glutamate is oxidized by glutamate oxidase producing hydrogen peroxide which is then amperometrically detected on a platinum electrode set at +650 mV vs Ag/AgCl. A dialysis electrode, which consists of a microdialysis probe with a built-in platinum electrode, provides a continuous glutamate oxidase perfusion inside of the probe. Perfusion with this solution allows real-time monitoring of glutamate dynamics in the extracellular space during ischemia. This study was designed to collect detailed information on rapid changes in the extracellular glutamate concentration of the rat striatum and demonstrated two distinct phases of glutamate release during early severe brain ischemia.

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.

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.

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.

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.

In in vivo extracellular recordings from anesthetized adult rats, midbrain dopaminergic neurons fire spontaneously at low rates, averaging around 4 spikes/second (Bunney et al., 1973; Guyenet and Aghajanian, 1978; Deniau et al., 1978; Tepper et al., 1982). Under these conditions, the neurons exhibit 3 distinct patterns or modes of firing. The first is that of pacemaker-like firing, characterized by very regular interspike intervals (Wilson et al., 1977; Tepper et al., 1995). The second, and most common pattern of activity in vivo is a random, or occasional mode (Wilson et al., 1977), characterized by long post-firing inhibition which rises smoothly into a flat autocorrelation function indicating that the remaining interspike intervals are distributed randomly, best characterized by a Poisson distribution. The third, least common mode of firing, is burst firing, in which the neurons exhibit stereotyped bursts of 2–8 action potentials in which the first intraburst interspike interval is around 60 ms, followed by progressively increasing interspike intervals and progressively decreasing spike amplitudes (Grace and Burney, 1984). The bursts are not usually rhythmic or continuous, and occur embedded in background random single-spike firing activity. Both in anesthetized and unanesthetized rats (Freeman et al., 1985), dopaminergic neurons often switch between different firing modes, and the three firing patterns are best thought of as a continuum, with the pacemaker-like firing on one end and burst firing on the other.

The subthalamic nucleus (STN) is one of the principal sources of excitatory glutamatergic input to dopaminergic neurons of the substantia nigra, yet stimulation of the STN produces both excitatory and inhibitory effects on nigral dopaminergic neurons recorded extracellularly in vivo. The present experiments were designed to determine the sources of the excitatory and inhibitory effects. Synaptic potentials were recorded intracellularly from substantia nigra pars compacta dopaminergic neurons in parasagittal slices in response to stimulation of the STN. Synaptic potentials were analyzed for onset latency, amplitude, duration, and reversal potential in the presence and absence of GABA and glutamate receptor antagonists. STN-evoked depolarizing synaptic responses in dopaminergic neurons reversed at approximately −31 mV, intermediate between the expected reversal potential for an excitatory and an inhibitory postsynaptic potential (EPSP and IPSP). Blockade of GABAA receptors with bicuculline caused a positive shift in the reversal potential to near 0 mV, suggesting that STN stimulation evoked a near simultaneous EPSP and IPSP. Both synaptic responses were blocked by application of the glutamate receptor antagonist, 6-cyano-7-nitroquinoxalene-2,3-dione. The confounding influence of inhibitory fibers of passage from globus pallidus and/or striatum by STN stimulation was eliminated by unilaterally transecting striatonigral and pallidonigral fibers 3 days before recording. The reversal potential of STN-evoked synaptic responses in dopaminergic neurons in slices from transected animals was approximately −30 mV. Bath application of bicuculline shifted the reversal potential to ∼5 mV as it did in intact animals, suggesting that the source of the IPSP was within substantia nigra. These data indicate that electrical stimulation of the STN elicits a mixed EPSP-IPSP in nigral dopaminergic neurons due to the coactivation of an excitatory monosynaptic and an inhibitory polysynaptic connection between the STN and the dopaminergic neurons of substantia nigra pars compacta. The EPSP arises from a direct monosynaptic excitatory glutamatergic input from the STN. The IPSP arises polysynaptically, most likely through STN-evoked excitation of GABAergic neurons in substantia nigra pars reticulata, which produces feed-forward GABAA-mediated inhibition of dopaminergic neurons through inhibitory intranigral axon collaterals.

We developed a method for in vivo real-time monitoring of the concentration of extracellular glutamate ([Glu]e) in the brain under anoxic conditions. A dialysis electrode (Sycopel Int., UK) was employed as a sensing device to measure the concentration of glutamate by enzyme amperometry, and an electron mediator, ferrocene, was introduced into the electrode together with glutamate oxidase. The ferrocene was covalently conjugated with a high molecular weight molecule, bovine serum albumin, to avoid outward diffusion through the dialysis membrane. With this set-up, the amperometric response was independent of the pO2 around the electrode in vitro up to 400 microM glutamate. Using this method, we investigated the dynamics of [Glu]e in the rat striatum during anoxia. [Glu]e increased rapidly at 102+/-5.4s (n = 6) after the start of nitrogen inhalation. The increase continued for about 30 s, and then [Glu]e decreased. The peak value of delta[Glu]e was 141+/-37 micro M. [Glu]e subsequently underwent another gradual increase, reaching 213+/-69 microM at 15 min after the start of nitrogen inhalation. This distinct biphasic profile was reproducible. We conclude that this method is very useful for monitoring [Glu]e in the brain under low pO2 conditions.

Abstract 984 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Approximately 90% of patients present during the first year of life or in early childhood. About 40–50% of DBA cases are familial with autosomal dominant, while the remainder is sporadic cases whose mode of inheritance is largely unknown. Although anemia is the most prominent feature of DBA, up to 40% of patients also accompany other symptoms including growth retardation and/or a variety of congenital malformations. Recent studies have shown that the disease could be associated with heterozygous mutations in ribosomal protein (RP) genes, including six small subunit RP genes RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26 as well as four large subunit RP genes RPL5, RPL11, RPL26, and RPL35A, which collectively account for about 50% of patients with DBA. In addition, germline mutations in the GATA1 gene encoding a hematopoietic transcription factor, have been also reported in two DBA families. However, it is clear that the molecular etiology of many DBA cases remains to be covered. To identify new mutations that are responsible for DBA, we performed whole-exome sequencing on 40 DBA patients with no documented mutations/deletions involving known DBA genes. After excluding all variants registered in the 1000 Genomes Project, or dbSNP131, or found in our inhouse SNP database, we searched for non-synonymous mutations involving RP genes as possible candidate for novel DBA genes. In this study, we identified probable pathogenic mutations in two novel RP genes, RPS27 and RPL27 in two patients. The first case was a 1-year-old girl who harbored a single nucleotide substitution at the splice acceptor site in intron 1 of RPL27 (c.-2–1G>A), which results in splicing error. She had atrial septal defect and pulmonary stenosis, and responded to steroid treatment. The second case was a 2-year-old girl carrying a frameshift deletion of RPS27 (c.90delC, p.Tyr31ThrfsX5), leading to a premature truncation. This patient had no abnormalities and responded to steroid treatment. An additional five missense SNVs affecting single cases was identified in five genes, including RPL3L, RPL8, RPL13, RPL18A, and RPL31, together with two in-frame deletions of RPL6 and RPL14 in two patients, which cause deletion of a single amino-acid. However, the pathological significance in these 7 cases is uncertain. In the remaining 31 patients, no mutations were detected in RP genes. In conclusion, we identified novel germline mutations of RP genes that could be responsible for DBA, further confirming the concept that the RP genes are common targets of germline mutations in DBA patients and also suggested the presence of non-RP gene targets for DNA. Disclosures: No relevant conflicts of interest to declare.