石嶺 久子

Toxic compounds from the mother's diet and medication in addition to genetic factors and infection during pregnancy remain risks for various congenital disorders and misbirth. To ensure the safety of food and drugs for pregnant women, establishment of an in vitro system that morphologically resembles human tissues has been long desired. In this study, we focused on dorsal mesoderm elongation, one of the critical early development events for trunk formation, and we established in vitro autonomous elongating tissues from human induced pluripotent stem cells (hiPSCs). This artificial tissue elongation is regulated by MYOSIN II and FGF signaling, and is diminished by methylmercury or retinoic acid (RA), similar to in vivo human developmental disabilities. Moreover, our method for differentiation of hiPSCs requires only a short culture period, and the elongation is cell number-independent. Therefore, our in vitro human tissue elongation system is a potential tool for risk assessment assays for identification of teratogenic chemicals via human tissue morphogenesis.

The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.

Esophageal cancer is one of the most frequent causes of cancer-related deaths worldwide. This is due to its asymptomatic nature or mild nonspecific symptoms. Most patients are diagnosed after appearance of prominent symptoms, and tumors are frequently accompanied by severe infiltration. Therefore, molecular biomarkers for the prognosis of early-stage esophageal cancer are desired. In this study, we examined the prognostic potential of lipase H (LIPH), a recently reported biomarker for lung adenocarcinoma and squamous carcinoma. We found that LIPH mRNA is also frequently upregulated in esophageal adenocarcinoma. Immunohistochemical analysis confirmed LIPH protein expression in various esophageal tumor tissue sections. Interestingly, higher expression of LIPH in esophageal adenocarcinoma showed a positive correlation with longer survival of patients. Our data suggest that LIPH may have prognostic value for esophageal cancer.

Three-dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose-derived stem/stromal cells (hASCs) in a non-cross-linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20-50 mu m) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX-2), and 40% of the cells were SSEA-3-positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia-reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate-buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage.

Stage-specific embryonic antigen-3 (SSEA-3)-positive multipotent mesenchymal cells (multilineage differentiating stress-enduring [Muse] cells) were isolated from cultured human adipose tissue-derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic-activated cell sorting into positive and negative fractions, a SSEA-3(+) cell-enriched fraction (Muse-rich) and the remaining fraction (Muse-poor). Muse-rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse-poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse-poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse-rich cells significantly accelerated wound healing compared with treatment with Muse-poor Cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell-depleted or ischemic conditions of various organs and tissues.

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells. Stem Cells2014;32:3099-3111

Background: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. Methodology/ Principal Findings: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. Conclusions/ Significance: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineagespecific differentiation of ES cells.

Ionizing radiation is often used to treat progressive neoplasms. However, the consequences of long-term radiation exposure to healthy skin tissue are poorly understood. We aimed to evaluate the short-and long-term radiation damage to healthy skin of the same irradiation given either as single or fractional doses. C57BL/J6 mice were randomly assigned to one of three groups: a control and two exposure groups (5 Gy x2 or 10 Gy x1). The inguinal area was irradiated (6-MeV beam) 1 week after depilation in the treatment groups. Skin samples were evaluated macroscopically and histologically for up to 6 months after the final exposure. After anagen hair follicle injury by irradiation, hair cycling resumed in both groups, but hair graying was observed in the 10 Gy x1 group but not in the 5 Gy x2 group, suggesting the dose of each fractional exposure is more relevant to melanocyte stem cell damage than the total dose. On the other hand, in the long term, the fractional double exposures induced more severe atrophy and capillary reduction in the dermis and subcutis, suggesting fractional exposure may cause more depletion of tissue stem cells and endothelial cells in the tissue. Thus, our results indicated that there were differences between the degrees of damage that occurred as a result of a single exposure compared with fractional exposures to ionizing radiation: the former induces more severe acute injury to the skin with irreversible depigmentation of hairs, while the latter induces long-term damage to the dermis and subcutis. (C) 2015 S. Karger AG, Basel

Lung cancer is one of the most frequent causes of cancer-related death worldwide. However, molecular markers for lung cancer have not been well established. To identify novel genes related to lung cancer development, we surveyed publicly available DNA microarray data on lung cancer tissues. We identified lipase member H (LIPH, also known as mPA-PLA1) as one of the significantly upregulated genes in lung adenocarcinoma. LIPH was expressed in several adenocarcinoma cell lines when they were analyzed by quantitative real-time polymerase chain reaction (qPCR), western blotting, and sandwich enzyme-linked immunosorbent assay (ELISA). Immunohistochemical analysis detected LIPH expression in most of the adenocarcinomas and bronchioloalveolar carcinomas tissue sections obtained from lung cancer patients. LIPH expression was also observed less frequently in the squamous lung cancer tissue samples. Furthermore, LIPH protein was upregulated in the serum of early- and late-phase lung cancer patients when they were analyzed by ELISA. Interestingly, high serum level of LIPH was correlated with better survival in early phase lung cancer patients after surgery. Thus, LIPH may be a novel molecular biomarker for lung cancer, especially for adenocarcinoma and bronchioloalveolar carcinoma. (C) 2013 Elsevier Inc. All rights reserved.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as alpha-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation. (C) 2013 Elsevier Inc. All rights reserved.

Adipose tissue (AT) is composed of mature adipocytes and stromal vascular fraction (SVF) cells, including adipose stem/stromal cells (ASCs). We characterized hematopoietic cells residing in human nonobese AT by analyzing the SVF isolated from human lipoaspirates and peripheral blood (PB). Flow cytometry revealed that AT-resident hematopoietic cells consisted of AT-resident macrophages (ATMs) or lymphocytes with a negligible number of granulocytes. AT-resident lymphocytes were composed of helper T cells and natural killer cells. Almost no B cells and few cytotoxic T cells were observed in nonobese AT. More than 90% of ATMs were M2 state CD206+ macrophages (CD45 +/CD14+) that were located in the periendothelium or interstitial spaces between adipocytes. We also discovered a novel subpopulation of CD34+/CD206+ ATMs (11.1% of CD206+ATMs) that localized in the perivascular region. Microarray of noncultured CD34 +/CD206+ ATMs, CD34-/CD206+ ATMs, CD45-/CD31-/CD34+ ASCs, and PB-derived circulating monocytes revealed that CD34+/CD206+ ATMs shared characteristics with ASCs and circulating monocytes. Unlike CD34 -/CD206+ ATMs, CD34+/CD206+ ATMs could grow in adherent culture and were capable of differentiating into multiple mesenchymal (adipogenic, osteogenic, and chondrogenic) lineages, similar to ASCs. CD34+/CD206+ ATMs grew rapidly and lost expression of CD45, CD14, and CD206 by passage 3, which resulted in a similar expression profile to ASCs. Thus, this novel ATM subpopulation (CD45+/CD14 +/CD34+/CD206+) showed distinct biological properties from other ATMs and circulating monocytes/macrophages. The CD34 +/CD206+ ATMs possessed characteristics similar to ASCs, including adherence, localization, morphology, and mesenchymal multipotency. This AT-resident subpopulation may have migrated from the bone marrow and may be important to tissue maintenance and remolding. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

The mechanisms of gastrointestinal morphogenesis in mammals are not well understood. This is partly due to the lack of appropriate markers that are expressed with spatiotemporal specificity in the gastrointestinal tract during development. Using mouse embryos, we surveyed markers of the prospective stomach region during gastrointestinal morphogenesis. The initiation of organ bud formation occurs at E10.5 in mice. These primordia for the digestive organs protrude from a tube-like structured endoderm and have their own distinct morphogenesis. We identified 3 cell surface genes - Adra2a, Fzd5, and Trpv6 - that are expressed in the developing stomach region during gastrointestinal morphogenesis using a microarray-based screening. These novel genes will be useful in expanding our understanding of the mechanisms of gastrointestinal development. (c) 2012 Elsevier B.V. All rights reserved.

Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in Delta UA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in Delta UA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in Delta UA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.

In order to avoid the secondary exposure of medical personnel to toxic materials under biochemical hazard conditions, we have reported a method for non-contact monitoring of heart and respiratory rates, using microwave radar or laser irradiation. In large-scale disasters, it is important to be able to diagnose shock without touching patients. We evaluated a non-contact method of monitoring arterial blood pressure alterations of New Zealand rabbits induced by blood loss, using He-Ne laser reflection on the common carotid artery. PVR was significantly correlated with systolic blood pressure (r = 0.95, p &lt 0.01), where PV = peak voltage of reflected laser amplitude, and PVR = PV(present moment state)/PV(normal state). The following formula was derived using the least-squares linear fitting: SBP = 69.6 PVR + 8.2, in which SBP is the systolic blood pressure. Before blood withdrawal, the mean blood pressure, heart rate and haematocrit were 68 ± 3 mmHg, 154 ± 10 bpm and 40 ± 2%, respectively. After intervention, the mean blood pressure, heart rate and haematocrit were 38 ± 5 mmHg, 197 ± 25 bpm and 30 ± 2%, respectively. The proposed non-contact method appears promising for future clinical application in determining arterial blood pressure alterations. It is likely to be useful in reducing the risk of secondary exposure to toxic chemicals or infectious organisms in the case of large-scale disasters. © 2008 Informa UK Ltd.

Paroxysmal dystonic choreoathetosis (PDC) is thought to be a hereditary channelopathy mapped to chromosome 2q32-36. By means of linkage analysis on a large Japanese family, we have narrowed the PDC locus that contains 32 candidate genes. Here, we report that a heterozygous mutation (A7V) in one of such genes, myofibrillogenesis regulator 1 (MR-1), is responsible for PDC in the Japanese family. This is consistent with the finding in American PDC families. We further report that there are several other polymorphisms in MR-1 in the Japanese PDC family. To characterize MR-1, we generated specific antibodies against MR-1 and performed the immunohistochemical analysis in rat brain. The results of the MR-1 localization will be discussed. Similar to other channelopathies such as epilepsy and migraine, PDC is characterized by involuntary movement attacks, and is presumed to be induced by abnormalities of ion channels. Although MR-1 may be associated with some ion channels, its physiological functions remain unclear. Further characterization of MR-1 including its molecular function and relationship to ion channels, may facilitate not only to understand pathophysiology of PDC, but also to develop effective therapies for paroxysmal neurological disorders. [J Physiol Sci. 2006;56 Suppl:S157]

文字認識やパターン認識などに用いられる画像処理技術である二値化処理と細線化処理を適用することで、分散培養・組織培養における、枝分かれし、曲線的で複雑に伸長する神経突起長をより正確に計算する方法を組み込んだソフトウェア(Mneurites)を創作した。後根神経節の分散培養を用いてこの方法を従来の方法と比較し、より正確な結果を得ることができた。らせん神経節や後根神経節の小片培養における神経突起の計測を行った。従来困難であった小片培養においてもこの方法が適用できた。神経の発生、再生などの仕組みを解析する上で非常に有用であることが示唆された。

System asc transporter Asc-1, expressed in the brain, transports D- and L-serine with high affinity. To determine the localization of Asc-1 in the rat brain, we isolated a cDNA for the rat orthologue of Asc-1. The encoded protein designated as rAsc-1 (rat Asc-1) exhibited 98% sequence identity to mouse Asc-1 (mAsc-1). Based on amino acid sequences of rAsc-1 and mAsc-1, twopolyclonal antibodies against Asc-1 were generated and used for the immunohistochemical analysis on the cerebral and cerebellar cortices of rats and mice. Asc-1 immunoreactivity was detected in neurons, including cerebellar Purkinje neurons and pyramidal neurons in the neocortex and hippocampus. It was clearly localized in dendrites as well as somata. The localization of Asc-1 in brain suggests the significant contribution of Asc-1 to amino acid mobilization in brains including the synaptic clearance of D-serine and the neuronal uptake of L-serine that is essential for survival and dendrite growth of Purkinje neurons in particular. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Cochlear hair cells are presumed to live in culture for many days, yet they are difficult to identify in cultured tissues. We stained hair cells in cochlear sections with FM1-43 and cultured them in collagen matrix. Three rows of outer hair cells and a single row of inner ones were distinguished by staining with FM1-43. Fixation of the sections with paraformaldehyde caused loss of the FM1-43 fluorescence, indicating that FM1-43 stained only live hair cells. In sections cultured for 48 h, almost all hair cells were still positive with FM1-43. Culture with gentamycin caused loss of FM1-43-positive cells. h. serum-free, long-term cultures (15 days) performed without antibiotics or neurotrophins, the row alignment of FM1-43-positive hair cells was still maintained. Membranous labyrinth-like vacuoles enveloping hair cells were formed in the collagen matrix. Accordingly, FM1-43 is an efficient marker for identifying live hair cells in cultured tissues. Moreover, cochlear hair cells are revealed to live for weeks in serum-free culture without exogenous neurotrophins. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Live Merkel cells in the skin and hair follicles are known to incorporate a fluorescence dye, quinacrine, which has been utilized to identify and dissect the cells for experiments. Quinacrine fluorescence of the cells is, however, quickly lost and quinacrine-stained Merkel cells soon become difficult to identify in tissue culture. To find dyes that remain in the cells for a long period of time, we tested many fluorescence dyes and found that FM dyes (such as FM1-43) are useful markers for live Merkel cells. In the rat footpad skin, FM1-43 was shown to stain 95% of live Merkel cells that were already stained with quinacrine. FM4-64 stained 98% of quinacrine-stained Merkel cells. Merkel cells in sinus hair follicles were also stained with FM dyes. The fluorescence intensity of FM dyes was stronger than that of quinacrine, and the shape of the cells was more distinct in the FM-dye-stained cells. To test how long FM dyes remain in live cells, FM-dye-stained Merkel cells in hair follicles were embedded in collagen gel and were cultured in a serum-free medium. FM-dye-stained cells were easily identified even after 7 days of culture. During the culture, Merkel cells changed their shape, moved in the preparation and tended to aggregate on the surface. We conclude that FM dyes are powerful tools for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate FM dyes suggests that vesicles in the cells are likely to have mechanisms of recycling in a manner similar to those in neurons and secretory cells.

Merkel cells have long been assumed to exhibit a function of guiding sensory nerve fibers to the skin and hairs. However, there has been little experimental evidence that supports this hypothesis. In order to test this possibility, we performed co-culture of sinus hair follicles and sensory ganglia in a collagen gel, intending to examine how sensory nerve fibers grow to the Merkel cell-enriched regions of sinus hair follicles. During co-culture with a serum-free medium supplemented with NGF and NT3, nerve fibers from sensory ganglia grew in a manner making convergence to the superior enlargement of hair follicles at which Merkel cells were enriched. Live Merkel cells stained with FM1-43 dye were demonstrated to be present in co-culture preparations. The presence of Merkel cells in this region of hair follicles was also demonstrated by immunostaining against cytokeratin 20. Immunostaining of a co-culture preparation against synaptophysin I showed that the hair follicle was surrounded tightly with synaptophysin I-enriched newly grown sensory nerve fibers. We therefore considered that Merkel cells are likely to have a capability of attracting sensory nerve fibers.

Live Merkel cells are known to incorporate a fluorescent dye, quinacrine (3,3',4',5,7-pentahydroxyflavone). Quinacrine fluorescence in the cells is, however, quickly lost and quinacrine-stained Merkel cells become difficult to identify in tissue culture. To find dyes that stay in the cells for a long period of time, we tested many fluorescent dyes and found that FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)-styryl)pyridinium dibromide) is a useful marker for live Merkel cells. In the rat footpad skin, FM1-43 was revealed to stain most of the live Merkel cells that were already stained with quinacrine. Merkel cells in sinus hair follicles were also stained with FM1-43 dye. The fluorescence intensity of the FM dye was stronger than that of quinacrine, and the shape of the cells was more distinct in the FM1-43-stained cells. We thus conclude that the FM dye is a powerful tool for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate the FM dye suggests that vesicles in Merkel cells are likely to represent recycling in a manner similar to those in neurons and secretory cells.

We identified a novel Na+-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na+-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1 Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc ((4F2) under bar heavy chain) or rBAT related to (b) under bar (0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na+-independent transport activity for acidic amino acids. Distinct from the Na+-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and L-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.