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Digital tomosynthesis (DTS) can enhance diagnostic accuracy by providing 3-D volume images with a remarkably low-X-ray dose. The aim of this study is to provide an initial assessment of the image quality and the X-ray dose for a mobile DTS system employing a moving carbon-nanotube (CNT)-based digital X-ray source array and a fixed detector for extremity scans. This design allows to reduce the source-to-detector distance (SDD) to only 400 mm, thereby enabling a compact and highly mobile system. We first measured the entrance surface dose (ESD), which is the sum of the X-ray dose irradiated from individual projections using a dosimeter placed at the center of the X-ray detector. The ESDs obtained for hand, foot, and knee scan configurations were 0.15, 0.22, and 0.43 mGy, respectively, which were comparable to those obtained from 2-D radiography exposures. For the evaluation of its reconstructed image quality, the in-plane modulation transfer function (MTF), Z-resolution, geometry distortion, and image homogeneity were assessed by utilizing a wire-phantom, sphere-phantom, and PMMA phantoms. The reconstructed images of hand, ankle and knee phantoms were evaluated qualitatively. The results of the evaluation demonstrate the successful development of the mobile DTS system proposed in this article.

The SAFIR collaboration is currently developing a high-rate positron emission tomography (PET) insert to study fast kinetic processes in small animals. Our insert is designed for simultaneous image acquisition with a preclinical 7 T magnetic resonance (MR) imaging device. In contrast to existing preclinical PET scanners and inserts, our hardware is optimized for high-rate measurements with source activities up to 500 MBq. As a first step, the SAFIR Prototype insert was constructed. This already incorporates the final components, but has a reduced axial field-of-view (35.6 mm). We use lutetium-yttrium oxyorthosilicate crystals (2.12 mm $\times $ 2.12 mm $\times $ 13 mm) one-to-one coupled to silicon photomultipliers. All analog signals are digitized within the insert. We use 49 MR-compatible dc–dc converters in the insert to provide the power to all readout electronics. After shimming, no degradation of the homogeneity of the static $B_{0}$ field in the MR scanner was observed. During full operation, we saw a minor reduction in the signal-to-noise ratio of the MR data of 4.9%. With a low activity point source (22Na 0.65 MBq) we obtained a coincidence energy resolution of 13.8% full width at half maximum (FWHM) and a coincidence timing resolution of 194 ps (FWHM). First PET/MR rat brain and high-rate mouse cardiac images (84.9 MBq) are shown in this article.

ABSTRACTRugosin G, an ellagitannin trimer, was isolated from the water-soluble fraction of red rose petals, and its inhibitory activity against recombinant human histidine decarboxylase was investigated. Rugosin G showed potent inhibition compared to ellagitannin monomers and a dimer with macrocyclic structure (oenothein B), suggesting the potent inhibition of rugosin G was attributed to its linear oligomeric conformation.Abbreviations:HDC, histidine decarboxylase; Me2CO, acetone; EtOAc, ethyl acetate

The small animal fast insert for mRi (SAFIR) positron emission tomography insert was proposed for quantitative dynamic acquisition inside a preclinical 7T magnetic resonance imaging scanner to study kinetics of short-lived tracers. For this purpose, the SAFIR readout should be capable of handling high count rates and achieving excellent timing performance. We evaluated one of the available application specific integrated circuits (ASICs) for SiPM readout, namely SiPM timing chip (STiC) version 3.1. In this paper, we show the performances of the SAFIR PET detector with the STiC ASIC readout. The SAFIR PET detector consists of an $8 \times 8$ array of lutetium yttrium oxyorthosilicate $2.1\,\,{\mathrm {mm}} \times 2.1\,\,{\mathrm {mm}} \times 12$ mm crystals coupled, with optical grease, to an $8 \times 8$ array of SiPMs with a $2.0\,\,{\mathrm {mm}} \times 2.0$ mm photo-sensitive area. Signals from the individual SiPM channels were digitized by the STiC ASIC. Hit’s arrival time and time-over-threshold (TOT) were recorded into time stamps with 50.2-ps least significant bit. We obtained an average energy resolution of 18.5% full width at half maximum (FWHM) at 511-keV photopeak after TOT nonlinearity correction and an average coincidence resolving time resolution of 244-ps FWHM with time walk correction that satisfy our requirements specification on the detector performance.

In Japan, about 80% of deaths occur in hospitals, especially long-term care beds. The purpose of this study was to clarify the nursing practices used for such older patients at the end-of-life stage in long-termcare wards via the modified grounded theory approach (M-GTA).

Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganiihistidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria(rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganiiHDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmariaexhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

A small-animal single photon emission computed tomography (SPECT) system having compact size and low cost was designed using monolithic LGSO scintillation crystals and micro parallel-hole collimators through Monte Carlo simulations. A spatial resolution of ~1 mm and a sensitivity of ~100 cps/MBq were achieved with a four-head SPECT system. A hot rod with a diameter of 1.0 mm was resolved in the SPECT image of the ultra-micro hot spot phantom. Using a thin monolithic crystal and micro collimator, we achieved high spatial resolution and high sensitivity.

Burning mouth syndrome (BMS) causes idiopathic pain or a burning sensation in clinically normal oral mucosa. Burning mouth syndrome is a chronic disease with an unknown etiology. Burning mouth syndrome is also idiopathic, and a consensus regarding diagnosis/treatment has not been reached yet. Recent studies have supported the suggestion that BMS is a neuropathic pain disorder in which both the peripheral and central nervous systems are involved. Tricyclic antidepressants (nortriptyline and amitriptyline), serotonin-noradrenaline reuptake inhibitors (SNRIs) (duloxetine and milnacipran), and antiepileptic drugs, potential-dependent calcium channel α2δ subunit ligands (gabapentine and pregabalin), are currently recommended as the first-choice drugs for neuropathic pain. In this study, we report 5 patients with BMS in whom there was no response to SNRI (milnacipran or duloxetine), or administration was discontinued because of adverse reactions, but in whom pregabalin therapy markedly reduced or led to the disappearance of pain in a short period. Pregabalin, whose mechanism of action differs from that of SNRIs, may become a treatment option for BMS patients who are not responsive to or are resistant to SNRIs.

Both organic and inorganic phosphorus (Pi) are present in regularly consumed foods, such as meats, eggs, and dairy products. Pi is often included in foods as an additive (as hidden phosphorus). The intestinal peptide transporter PepT1 mediates protein absorption, which is disturbed in renal insufficiency. Our aim was to determine the effects of dietary Pi content on the peptide transport activity and expression of PepT1.

We examined the pain-relieving effect of duloxetine on chronic nonorganic orofacial pain (burning mouth syndrome and atypical odontalgia), considering the influence of baseline depressive symptoms.

Abstract: Purpose: Adding time-of-flight (TOF) information to positron emission tomography (PET) can lead to an improvement in image quality because TOF information allows the estimation of gamma ray annihilation positions in three dimensions. For accurate position estimation, precise time resolution is the most important characteristic required of TOF-PET detectors. The aims of this study were to establish the optimal setup for timing measurements and to evaluate the timing performance of a fast photomultiplier tube (PMT) (Hamamatsu R9800) coupled with a lutetium yttrium orthosilicate (LYSO) scintillation crystal for TOF-PET detector development. Methods: Performance of a fast PMT (diameter, 25 mm; length, 55 mm) coupled with a LYSO crystal (4 × 4 × 10 mm3) was measured in coincidence experiments with 511 keV annihilation photons from a 22Na gamma source. Timing measurement optimization was carried out with a leading-edge discriminator (LED) and a constant fraction discriminator (CFD). Results: Results showed the optimal timing resolution could be obtained with a CFD time delay of 1 ns; timing resolutions (FWHM) of the LYSO crystal-coupled fast PMT were measured to be 198 and 254 ps for two different R9800 PMTs. The energy resolutions at 511 keV were 10.8% and 10.6%, respectively. Additional experiments to determine the position uncertainty were performed at three different source positions and the position uncertainty (FWHM) in the TOF measurement using these PMTs was estimated to be 4.50 cm. Conclusions: The performance of fast R9800 PMT coupled with a LYSO crystal showed good potential for development as a TOF-PET detector module.

Abstract: Because positron emission tomography (PET) provides biochemical information in vivo with the sensitivity at the sub-pico-molar level, pre-clinical research using PET plays an important role in biological and pharmaceutical sciences. However, small animal imaging by PET has been challenging with respect to spatial resolution and sensitivity due to the small volume of the imaging objects. A DOI-encoding technique allows for pre-clinical PET to simultaneously achieve high spatial resolution and high sensitivity. Thus many DOI-encoding methods have been proposed. In this paper we describe why DOI measurements are important, what is required in DOI-encoding designs, and how to extract DOI information in scintillator-based DOI detectors. Recently, there has been a growing interest in DOI measurements for TOF PET detectors to correct time walk as a function of DOI position. Thus, the DOI-encoding method with a high time performance suitable for TOF detectors is now required. The requirements to improve the time resolution in DOI detectors are discussed as well.

The type IIc Na+-dependent phosphate cotransporter (NaPi-IIc) is specifically targeted to, and expressed on, the apical membrane of renal proximal tubular cells and mediates phosphate transport. In the present study, we investigated the signals that determine apical expression of NaPi-IIc with a focus on the role of the N- and the C-terminal tails of mouse NaPi-IIc in renal epithelial cells [opossum kidney (OK) and Madin-Darby canine kidney cells]. Wild-type NaPi-IIc, the cotransporter NaPi-IIa, as well as several IIa-IIc chimeras and deletion mutants, were fused to enhanced green fluorescent protein (EGFP), and their cellular localization was analyzed in polarized renal epithelial cells by confocal microscopy and by cell-surface biotinylation. Fluorescent EGFP-fused NaPi-IIc transporter proteins are correctly expressed in the apical membrane of OK cells. The apical expression of N-terminal deletion mutants (deletion of N-terminal 25, 50, or 69 amino acids) was not affected by truncation. In contrast, C-terminal deletion mutants (deletion of C-terminal 45, 50, or 62 amino acids) did not have correct apical expression. A more detailed mutational analysis indicated that a domain (amino acids WLHSL) in the cytoplasmic C terminus is required for apical expression of NaPi-IIc in renal epithelial cells. We conclude that targeting of NaPi-IIc to the apical cell surface is regulated by a unique amino acid motif in the cytoplasmic C-terminal domain.

In the present study, we evaluated the roles of type II and type III sodium-dependent Picotransporters in fibroblast growth factor 23 (FGF23) activity by administering a vector encoding FGF23 with the R179Q mutation (FGF23M) to wild-type (WT) mice, Npt2a knockout (KO) mice, Npt2c KO mice, and Npt2a−/−Npt2c−/−mice (DKO mice). In Npt2a KO mice, FGF23M induced severe hypophosphatemia and markedly decreased the levels of Npt2c, type III Na-dependent Pitransporter (PiT2) protein, and renal Na/Pitransport activity. In contrast, in Npt2c KO mice, FGF23M decreased plasma phosphate levels comparable to those in FGF23M-injected WT mice. In DKO mice with severe hypophosphatemia, FGF23M administration did not induce an additional increase in urinary phosphate excretion. FGF23 administration significantly decreased intestinal Npt2b protein levels in WT mice but had no effect in Npt2a, Npt2c, and DKO mice, despite marked suppression of plasma 1,25(OH)2D3levels in all the mutant mice. The main findings were as follow: 1) FGF23-dependent phosphaturic activity in Npt2a KO mice is dependent on renal Npt2c and PiT-2 protein; 2) in DKO mice, renal Pireabsorption is not further decreased by FGF23M, but renal vitamin D synthesis is suppressed; and 3) downregulation of intestinal Npt2b may be mediated by a factor(s) other than 1,25(OH)2D3. These findings suggest that Npt2a, Npt2c, and PiT-2 are necessary for the phosphaturic activity of FGF23. Thus complementary regulation of Npt2 family proteins may be involved in systemic Pihomeostasis.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodium-dependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (Pi) reabsorption in adult mice. In fact, mice that are null for Npt2c (Npt2c−/−) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insights and the relative importance of Npt2a and Npt2c, we now studied Npt2a−/−Npt2c+/+, Npt2a+/−Npt2c−/−, and Npt2a−/−Npt2c−/−double-knockout (DKO). DKO mice exhibited severe hypophosphatemia, hypercalciuria, and rickets. These findings are different from those in Npt2a KO mice that show only a mild phosphate and bone phenotype that improve over time and from the findings in Npt2c KO mice that show no apparent abnormality in the regulation of phosphate homeostasis. Because of the nonreddundant roles of Npt2a and Npt2c, DKO animals showed a more pronounced reduction in Pitransport activity in the brush-border membrane of renal tubular cells than that in the mice with the single-gene ablations. A high-Pidiet after weaning rescued plasma phosphate levels and the bone phenotype in DKO mice. Our findings thus showed in mice that Npt2a and Npt2c have independent roles in the regulation of plasma Piand bone mineralization.

Recent studies have demonstrated that klotho protein plays a role in calcium/phosphate homeostasis. The goal of the present study was to investigate the regulation of Na-Picotransporters in klotho mutant (kl/kl) mice. The kl/kl mice displayed hyperphosphatemia, high plasma 1,25(OH)2D3levels, increased activity of the renal and intestinal sodium-dependent Picotransporters, and increased levels of the type IIa, type IIb, and type IIc transporter proteins compared with wild-type mice. Interestingly, transcript levels of the type IIa/type IIc transporter mRNA abundance, but not transcripts levels of type IIb transporter mRNA, were markedly decreased in kl/kl mice compared with wild-type mice. Furthermore, plasma fibroblast growth factor 23 (FGF23) levels were 150-fold higher in kl/kl mice than in wild-type mice. Feeding of a low-Pidiet induced the expression of klotho protein and decreased plasma FGF23 levels in kl/kl mice, whereas colchicine treatment experiments revealed evidence of abnormal membrane trafficking of the type IIa transporter in kl/kl mice. Finally, feeding of a low-Pidiet resulted in increased type IIa Na-Picotransporter protein in the apical membrane in the wild-type mice, but not in kl/kl mice. These results indicate that hyperphosphatemia in klotho mice is due to dysregulation of expression and trafficking of the renal type IIa/IIc transporters rather than to intestinal Piuptake.

Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-Picotransporter gene, suggesting that the type IIc transporter plays a prominent role in renal phosphate handling. The goal of the present study was to investigate the regulation of the type IIc Na-Picotransporter by parathyroid hormone (PTH). Type IIc Na-Picotransporter levels were markedly increased in thyroparathyroidectomized (TPTX) rats. Four hours after administration of PTH, type IIc transporter protein levels were markedly decreased in the apical membrane fraction but recovered to baseline levels at 24 h. Immunohistochemical analyses demonstrated the presence of the type IIc transporter in the apical membrane and subapical compartments in the proximal tubular cells in TPTX animals. After administration of PTH, the intensity of immunoreactive signals in apical and subapical type IIc transporter decreased in the renal proximal tubular cells in TPTX rats. Colchicine completely blocked the internalization of the type IIc transporter. In addition, leupeptin prevented the PTH-mediated degradation of the type IIa transporter in lysosomes but had no effect on PTH-mediated degradation of the lysosomal type IIc transporter. In PTH-treated TPTX rats, the internalization of the type IIc transporter occurred after administration of PTH(1–34) (PKA and PKC activator) or PTH(3–34) (PKC activator). Thus the present study demonstrated that PTH is a major hormonal regulator of the type IIc Na-Picotransporter in renal proximal tubules.

AbstractIn general, twins have delayed language development early in childhood compared with singletons. The purpose of this study was to clarify the overall linguistic features of twins. A Japanese version of the Illinois Test of Psycholinguistic Abilities (ITPA) was administered in 24 twin pairs (aged 3 to 4 years) at their own homes. The overall language abilities of the twins were in the normal range (based on ITPA normative data: mean scale score 36.0 ? 6.0 points), and for the ITPA subtests only, Auditory Reception fell within the range of language disorder (mean scale score 24.9 ? 5.1 points). The findings suggest that in 3- to 4-year-old Japanese twins, overall language abilities are not delayed. However, there may be specific difficulties with auditory reception skills.

FGF23 (fibroblast growth factor 23) is a novel phosphaturic factor that influences vitamin D metabolism and renal re-absorption of Pi. The goal of the present study was to characterize the role of the VDR (vitamin D receptor) in FGF23 action using VDR(−/−) (VDR null) mice. Injection of FGF23M (naked DNA encoding the R179Q mutant of human FGF23) into VDR(−/−) and wildtype VDR(+/+) mice resulted in an elevation in serum FGF23 levels, but had no effect on serum calcium or parathyroid hormone levels. In contrast, injection of FGF23M resulted in significant decreases in serum Pi levels, renal Na/Pi co-transport activity and type II transporter protein levels in both groups when compared with controls injected with mock vector or with FGFWT (naked DNA encoding wild-type human FGF23). Injection of FGF23M resulted in a decrease in 25-hydroxyvitamin D 1α-hydroxylase mRNA levels in VDR(−/−) and VDR(+/+) mice, while 25-hydroxyvitamin D 24-hydroxylase mRNA levels were significantly increased in FGF23M-treated animals compared with mock vector control- or FGF23WT-treated animals. The degree of 24-hydroxylase induction by FGF23M was dependent on the VDR, since FGF23M significantly reduced the levels of serum 1,25(OH)2D3 [1,25-hydroxyvitamin D3] in VDR(+/+) mice, but not in VDR(−/−) mice. We conclude that FGF23 reduces renal Pi transport and 25-hydroxyvitamin D 1α-hydroxylase levels by a mechanism that is independent of the VDR. In contrast, the induction of 25-hydroxyvitamin D 24-hydroxylase and the reduction of serum 1,25(OH)2D3 levels induced by FGF23 are dependent on the VDR.

Dietary phosphate levels regulate the renal brush-border type IIa Na-Picotransporter. Another Na-Picotransporter, type IIc, colocalizes with type IIa Na-Picotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-Picotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-Pidiet compared with those fed a normal-Pidiet. Two hours after beginning a high-Pidiet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-Pidiet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-Picotransporter level is rapidly regulated by rapid adaptation to dietary Piin a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.

Recent studies suggest that vitamin D may play a role in intestinal Na+-dependent phosphate transport adaptation to variable levels of dietary Pi. Therefore, the goal of the current study was to assess Na+-dependent Picotransport activity in transgenic mice to determine whether vitamin D is an essential mediator of this process. Intestinal brush-border membrane (BBM), Na+-dependent Picotransport activity was significantly decreased in vitamin D receptor (VDR) null [VDR (−/−)] mice compared with wild-type (VDR+/+) mice. While intestinal Na-Picotransporter (type IIb) mRNA levels were similar in VDR (−/−) and VDR (+/+) mice, type IIb Na-Picotransporter protein expression was markedly suppressed in VDR (−/−) mice compared with VDR (+/+) mice. Furthermore, Na-Picotransport activity in renal BBM was similar in VDR (−/−) and VDR (+/+) mice, but type IIa Na-Picotransporter protein expression was decreased in VDR (−/−) mice. After administration of a low-Pidiet, type IIb protein expression was significantly increased in VDR (+/+) and VDR (−/−) mice, and type IIb protein expression was present in the intestinal BBM of VDR (−/−) mice. These data demonstrate that intestinal Na-Picotransport adaptation to a low-Pidiet occurs independently of vitamin D.

Treatment with PTH (parathyroid hormone) or a high-Pi diet causes internalization of the type IIa sodium-dependent phosphate (Na/Pi IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/Pi IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/Pi IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/Pi IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/Pi IIa co-transporter.

Inactivating mutations and/or deletions of PHEX ( Phosphate-regulating gene with Homologies to Endopeptidase on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans. In the present study, three Drosophila PHEX homologues (dPHEX-1, -2, -3) were isolated by the screening of a Drosophila cDNA library and expressed sequence tag (EST) database. The structural region involving motif II: 456WMXXXTKXXAXXK 468 (numbered according to human PHEX), motif VI: 602WW 603, and motif VIII: 746CXLW 749 was conserved in the dPHEX family. Zinc-coordinating motifs (HEFTH and GENIADNGG) were also conserved in the dPHEX family. All three dPHEX genes were expressed during all stages of Drosophila development. The expression of dPHEX-1 was suppressed by dietary phosphate deprivation, but the expression of dPHEX-2 and that of dPHEX-3 were not affected. In-situ hybridization showed a ubiquitous distribution of dPHEX-1 and dPHEX-2, while dPHEX-3 was highly expressed in the larval brain. In an analysis of subcellular localization, dPHEX-1 was localized to intracellular organelles and dPHEX-3 was localized predominately in the plasma membrane of Drosophila embryonic S2 cells. Homozygosity of a dPHEX-1 mutation, a transposon insertion in the dPHEX-1 promoter region, was completely lethal at an early stage of embryonic development. The present study indicates that three homologues are likely involved in the phosphate homeostasis of Drosophila.

Growth is critically dependent on the retention of a variety of nutrients. The kidney contributes to this positive external balance. In the present study, we isolated a cDNA from the human and rat kidney that encodes a growth-related Na+-dependent inorganic phosphate (Pi) cotransporter (type IIc). Microinjection of type IIc cRNA into Xenopusoocytes demonstrated sodium-dependent Picotransport activity. Affinity for Piwas 0.07 mmin 100 mmNa+. The transport activity was dependent on extracellular pH. In electrophysiological studies, type IIc Na/Picotransport was electroneutral, whereas type IIa was highly electrogenic. In Northern blotting analysis, the type IIc transcript was only expressed in the kidney and highly in weaning animals. In immunohistochemical analysis, the type IIc protein was shown to be localized at the apical membrane of the proximal tubular cells in superficial and midcortical nephrons of weaning rat kidney. Hybrid depletion experiments suggested that type IIc could function as a Na/Picotransporter in weaning animals, but its role is reduced in adults. The finding of the present study suggest that the type IIc is a growth-related renal Na/Picotransporter, which has a high affinity for Piand is electroneutral.

The Notch pathway is an evolutionarily conserved signaling mechanism that is essential for cell-cell interactions. The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, we have analyzed the functional involvement of these Deltex domains in Notch signaling. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain was indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaved as a dominant-negative form. This dominant-negative Deltex inhibited Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting the dominant-negative Deltex might prevent the activation of the Notch receptor. We found that Deltex formed a homo-multimer, and mutations in the RING-H2 finger domain abolished this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupted the function of Deltex in vivo. However, when the same mutant was fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein had normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex.

We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, l-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.

Notch signaling plays an important role in cell-fate specification in multicellular organisms by regulating cell–cell communication. The Drosophila deltexgene encodes a modulator of the Notch pathway that has been shown to interact physically with the Ankyrin repeats of Notch. We isolated four distinct cDNAs corresponding to mouse homologs of deltex— mouse Deltex1(MDTX1), mouse Deltex2(MDTX2), mouse Deltex2?E(MDTX2?E), and mouse Deltex3(MDTX3). Deduced amino acid sequences of these four cDNAs showed a high degree of similarity to DrosophilaDeltex and its human homolog, DTX1 throughout their lengths, even though they possess distinct structural features. MDTX proteins formed homotypic and heterotypic multimers. We found that these genes were expressed in the central, peripheral nervous system and in the thymus, overlapping with those of mouse Notch1. In mammalian tissue culture cells, overexpression of any of the four mouse deltex homologs suppressed the transcriptional activity of E47, a basic helix-loop-helix (bHLH) protein, in a manner similar to suppression by an activated form of human Notch1 or human DTX1. In addition, overexpression of MDTX2 and MDTX2?E in C2C12 cells under differentiation-inducing conditions suppressed the expression of myogenin, one of the myogenic transcriptional factors; this was also similar to a previously reported activity of constitutively activated Notch. Furthermore, misexpression of any of the MDTXgenes in Xenopusembryos resulted in an expansion of the region expressing the neural cell adhesion molecule (N-CAM) gene, a marker for the neuroepithelium. Collectively, our results suggest that these mouse deltex homologs are involved in vertebrate Notch signaling and regulation of neurogenesis.

ABSTRACTDrosophilaArgos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the DrosophilaEGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor a-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

DrosophilaArgos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the DrosophilaEGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor α-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

Monoclonal antibodies were prepared against the high mobility group (HMG) protein 2b from chick liver chromatin and a monoclonal-antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for chick HMG 2b. The sensitivity of the assay is about ten times that of the previously described radioimmunoassay and solid-phase enzyme immunoassay for HMG proteins. With the use of ELISA technique, the amount of HMG 2b (μg protein/mg DNA) in the livers of 1-day old and 70-day old chicks was found to be 2.56 ± 0.4, and 1.20 ± 0.2, respectively. The age-dependent change in the level of HMG proteins probably reflects changes in the functional state of chromatin during ageing.

Dietary phosphate restriction can prevent or retard the progress of chronic renal failure (CRF) and secondary hyperparathyroidism. The klotho gene is involved in the development of a syndrome resembling human ageing, and klotho mutant mice show abnormal calcium/vitamin D metabolism, developing hyperphosphataemia and vascular calcification. Phosphate retention rescues the phenotype of klotho mice. The level of expression of klotho RNA was greatly reduced in the kidneys of all CRF patients. Dietary P<inf>i</inf> restriction induced klotho expression, which enhances the beneficial effect of P<inf>i</inf> restriction in patients with CRF and/or on haemodialysis.