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Ingestion of amino acids is fundamental for cellular activity. Amino acids are important components for protein synthesis but are also crucial for intracellular metabolic reactions and signal transduction. Following activation, immune cells induce metabolic reprogramming to generate adequate energy and constitutive substances. Hence, the delivery of amino acids by transporters is necessary for the progression of metabolic rewiring. In this review, we discuss how amino acids and their transporters regulate immune cell functions, with emphasis on LAT1, a transporter of large neutral amino acids. Furthermore, we explore the possibility of targeting amino acid transporters to improve immune disorders and cancer immune therapies.

Background/aim 18F-fluorodeoxyglucose (FDG) uptake measurement on positron emission tomography/computed tomography (PET/CT) is difficult in renal tumors because of the nearby renal parenchyma and urinary tract, which excrete FDG. We carefully examined the maximum standardized uptake value (SUVmax) on FDG-PET/CT and investigated the relationship between major glucose transporters in the kidney and clear cell renal cell carcinoma (ccRCC) progression. Patients and methods Forty-five patients with ccRCC underwent FDG-PET/CT for staging and nephrectomy. Glucose transporter mRNA expression was examined in the removed kidney. Results SUVmax was increased in high-stage and high-grade tumors. Glucose transporter 1 (GLUT1) mRNA expression was higher in tumor tissues, in contrast to other glucose transporters. SUVmax was not correlated with GLUT1 mRNA expression. Kaplan-Meier analysis showed reduced overall and recurrence-free survival in the high SUVmax group. Conclusion Primary ccRCC lesions show a high SUVmax and GLUT1 mRNA over-expression. SUVmax increases with tumor upstaging and upgrading.

Bronchial asthma (asthma) is characterized by chronic airway inflammation, reversible obstruction, and hyperresponsive conditions. Although most asthma patients have been becoming controllable by virtue of inhaled corticosteroid (ICS), substantial number of patients still do not respond to the steroid-based therapy. Mast cells, eosinophils, and helper T (Th) 2 cells have been considered as key players in asthma pathogenesis. However, emerging studies have revealed that Th subsets other than Th2, as well as various other immune cells, significantly contribute to the development of steroid-resistant intractable asthma. T cells and other inflammatory cells require incorporating a large amount of nutrients such as amino acids and glucose to exhibit their full function following activation. Based on this remarkable character, it has recently been suggested that the pharmacological inhibition of amino acid transporters is promising for treating immunological and inflammatory disorders through the suppression of inflammatory cell activation. In this review, we explore the possible management of intractable asthma by developing a selective inhibitor for L-type amino acid transporter (LAT) 1.

Previous studies demonstrated that hamster sperm hyperactivation is suppressed by extracellular Na+ by lowering intracellular Ca2+ levels, and Na+/Ca2+-exchanger (NCX) specific inhibitors canceled the suppressive effects of extracellular Na+. These results suggest the involvement of NCX in the regulation of hyperactivation. However, direct evidence of the presence and functionality of NCX in hamster spermatozoa is still lacking. This study aimed to reveal that NCX is present and is functional in hamster spermatozoa. First, NCX1 and NCX2 transcripts were detected via RNA-seq analyses of hamster testis mRNAs, but only the NCX1 protein was detected. Next, NCX activity was determined by measuring the Na+-dependent Ca2+ influx using the Ca2+ indicator Fura-2. The Na+-dependent Ca2+ influx was detected in hamster spermatozoa, notably in the tail region. The Na+-dependent Ca2+ influx was inhibited by the NCX inhibitor SEA0400 at NCX1-specific concentrations. NCX1 activity was reduced after 3 h of incubation in capacitating conditions. These results, together with authors’ previous study, showed that hamster spermatozoa possesses functional NCX1 and that its activity was downregulated upon capacitation to trigger hyperactivation. This is the first study to successfully reveal the presence of NCX1 and its physiological function as a hyperactivation brake.

Recently, it was reported that hamster sperm hyperactivation is regulated by extracellular Na+. Two types of catalytic Na+/K+-ATPase (NKA) α subunits (α1 and α4) are present in spermatozoa. In this work, the contribution of these NKA subunits to the regulation of hamster sperm hyperactivation was investigated using the specific inhibitor ouabain. When 10−6 M ouabain was added to the modified Tyrode’s albumin lactate pyruvate medium (mTALP) medium, hyperactivation was significantly inhibited, whereas 10−5-10−4 M ouabain was needed to significantly reduce the amount of motile spermatozoa. When a more detailed analysis of flagellar movement was performed, 10−6 M ouabain suppressed the hyperactivation-associated change in the patterns of flagellar motion without affecting the sliding velocity of microtubules. Since 10−6 M ouabain specifically inhibits the α4 subunit while 10−5-10−4 M ouabain inhibits both the α1 and α4 subunits, these results suggest the α1 subunit is necessary for the maintenance of motility while the α4 subunit is necessary for the hyperactivation-associated change in flagellar movement. Ouabain did not inhibit tyrosine phosphorylation, and activation of tyrosine phosphorylation-dependent signaling had no effect on the inhibition of hyperactivation by ouabain. The immediate recovery of hyperactivation was observed when ouabain was washed out after a 3-h incubation. whereas the administration of ouabain after the onset of hyperactivation significantly inhibited hyperactivation. These results suggest ouabain inhibited hyperactivation in a manner that was independent of time-requiring phosphorylation-mediated signaling.

Introduction Vascular endothelial cell disorders are closely related to cardiovascular disease (CVD) and pulmonary diseases. Abnormal lipid metabolism in the endothelium leads to changes in cell signalling, and the expression of genes related to immunity and inflammation. It is therefore important to investigate the pathophysiology of vascular endothelial disorders in terms of lipid metabolism, using a disease model of endothelium. Methods Human induced pluripotent stem cell-derived endothelial cells (iECs) were cultured on a matrigel to form an iEC network. Lipids in the iEC network were investigated by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) analysis. Ion fragments obtained by mass spectrometry were analysed using an infusion method, involving precursor ion scanning with fragment ion. Results The MALDI TOF IMS analysis revealed co-localized intensity of peaks at m/z 592.1 and 593.1 in the iEC network. Tandem mass spectrometry (MS/MS) analysis by MALDI-imaging, in conjunction with precursor ion scanning using an infusion method with lipid extracts, identified that these precursor ions were lysophosphatidylcholine (LPC) (22:5) and its isotype. Conclusion The MALDI-imaging analysis showed that LPC (22:5) was abundant in an iEC network. As an in vitro test model for disease and potential therapy, present analysis methods using MALDI-imaging combined with, for example, mesenchymal stem cells (MSC) to a disease derived iEC network may be useful in revealing the changes in the amount and distribution of lipids under various stimuli.

Background/aim We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. However, secretion of trypsin, another pancreatic enzyme, interferes with insulin production in such systems. We aimed to ascertain the minimum trypsin inhibitor (TI), dose for obtaining a sustained, stable rate of insulin secretion. Materials and methods The action of TI (1-10 μg/ml) on pancreatic preparations of male Wistar-Imamichi rats in organ bath experiments was assessed by measuring insulin, amylase, and trypsin activity. Results The level of insulin outflow remained steady in the TI-treated samples, in contrast to that in the untreated control, where insulin secretion decreased over time. The level of amylase outflow did not change significantly. Trypsin activity was significantly lower in the TI-treated samples than in the control. Conclusion Even low concentrations of TI can maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.

Organ bath experiments are conventionally used to investigate the physiological actions and effects of hormones and drugs on organ responses. We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations, to investigate substances that promote insulin secretion ex vivo. 1,5-anhydro-D-glucitol (1,5-AG) is found in foods, and exists in humans and rodents; however, whether 1,5-AG stimulates insulin secretion remains unclear. This study aimed to assess the effects of short-term 1,5-AG stimulation on insulin secretion in both ex vivo and in INS-1E (rat-derived) cells in vitro. Our results indicated that 1,5-AG had no potency to increase the proportion of insulin outflow both in ex vivo and in vitro experiments. Insulin outflow significantly increased upon stimulation with 10 μM glimepiride, a member of the sulfonylurea class of drugs, ex vivo. Glucose-stimulated insulin secretion was observed not only in INS-1E cells but also in rat pancreatic preparations. Our findings demonstrated that short-term exposure to 1,5-AG had no effect on insulin secretion in rats.

Forty-five patients with ccRCC underwent FDG-PET/CT for staging and nephrectomy. Glucose transporter mRNA expression was examined in the removed kidney.SUVPrimary ccRCC lesions show a high SUV

Pituitary tumors (PTs) can cause significant mortality and morbidity due to limited therapeutic options. L-type amino acid transporters (LATs), in particular, the LAT1 isoform, is expressed in a variety of tumor cells. Pharmacological inhibition or genetic ablation of LAT1 can suppress leucine transport into cancer cells, resulting in suppression of cancer cell growth. However, roles of LAT1 in PTs have not been elucidated. Therefore, we assessed LAT1 expression in PTs and evaluated a LAT1-specific inhibitor, JPH203, on rat somatomammotroph tumor cells, GH4 cells. GH4 cells dominantly express LAT1 mRNA rather than other LAT isoforms, whereas LAT2 transcripts were most abundant in normal rat pituitary tissues. JPH203 inhibited leucine uptake and cell growth in GH4 cells in a concentration-dependent manner, and appeared to be independent of the mechanistic target, the rapamycin pathway. Although JPH203 did not induce apoptosis, it suppressed growth hormone production in GH4 cells. Also, genetic downregulation of LAT1 showed similar effects on cell growth and hormone production. These results indicated that restriction of LAT1 substrates by JPH203 modulated both cell growth and hormone production. In conclusion, LAT1 may be a new therapeutic target for PTs because its inhibition leads to suppression of cell growth as well as hormone production. JPH203 may represent a promising drug for clinical use in patients with PTs, with the potential of hormonal control and tumor suppression.

Recently, it was reported that hamster sperm hyperactivation is regulated by extracellular Na

This study was designed to investigate the effects of uric acid on vascular endothelial function in measurements carried out either at the bedside or the laboratory bench. First, we performed reactive hyperemia peripheral arterial tonometry using an EndoPAT 2000 device and measured serum uric acid levels in 92 outpatients with hypertension. The reactive hyperemia index (RHI) showed no correlation with serum uric acid level (R = -0.125, P = 0.235) in either overall patients or in a high-risk group of 51 patients with complications such as cardiovascular and cerebrovascular diseases, chronic kidney disease, and/or diabetes (R = -0.025, P = 0.860). However, in the remaining 41 patients in the low-risk group, RHI correlated negatively with serum uric acid level (R = -0.335, P = 0.032). Multiple regression analysis showed that serum uric acid level predicted RHI (R = -0.321, P = 0.043) in the low-risk group independent of age, body mass index, systolic blood pressure, and low density lipoprotein-cholesterol level. We then performed an in-vitro study using the WST-8 assay in human umbilical vein endothelial cells, which showed that hypoxic conditions reduced cell viability. Treatment with uric acid caused a further reduction in cell viability, while ascorbic acid improved viability. Using Western blot analysis, we observed that uric acid reduced endothelial nitric oxide synthase phosphorylation during hypoxic conditions. Serum uric acid level is associated with peripheral vascular endothelial function in patients with low-risk hypertension and uric acid could directly impair endothelial function under hypoxic conditions. These results are relevant to the interventional studies examining the cardiovascular protective effect of hypouricemic agents.

In cells that proliferate abundantly, such as cancer cells, the expressions of many nutrient transporters increase in order to drive the uptake of required nutrients. The system L transporters are the main sodium-independent neutral amino acid transporters. They have four isoforms：L-type amino acid transporter （LAT） 1, 2, 3, and 4. The expression of LAT1 has been reported in lines of cultured choriocarcinoma cells. However, the role of LAT1 in choriocarcinomas has not been adequately clarified. This study examined the role of LAT1 in these cell lines JAR and JEG-3. Based on an examination of mRNA and protein expression, it was determined that both JAR and JEG-3 cells express LAT1. The uptake of leucine, a substrate of LATs, by JAR and JEG-3 cells showed that there are sodium-independent leucine transport. To examine whether leucine transport is mediated by LAT1, a leucine uptake was examined with loading of JPH203, a LAT1 specific inhibitor, and transport inhibition was clearly seen. Cell counts were measured to examine whether cell proliferation was decreased by JPH203 treatment. Inhibition of JEG-3 cell proliferation was seen on day 4 of the treatment. No inhibition of JAR cell proliferation was seen by low concentrations of JPH203, however inhibition was seen on a higher concentration of JPH203. JPH203 decreased cell activityconcentration-dependently in JEG-3 cells. JAR cell, however, showed an increased activity following JPH203 treatment at low concentrations. Thus, the study found that LAT1-mediated amino acid transport occurs in these two different lines of cultured choriocarcinoma cells. Moreover, it showed that the two types of cells responded differently with respect to inhibition of cell proliferation and changes in cell activity as a result of inhibition of LAT1-mediated amino acid transport. The results showed that LAT1-mediated amino acid transport in choriocarcinoma is complex and has a variety of functions.

An integrated analysis was performed with data from 4 phase 2 and phase 3 studies of tofogliflozin in which patients with type 2 diabetes mellitus received the sodium‐glucose cotransporter 2 inhibitor tofogliflozin for up to 24 weeks. Sex differences, baseline haemoglobin A1c (HbA1c) and serum uric acid (UA) levels, and log10‐transformed urinary N‐acetyl‐β‐D‐glucosaminidase ratio were significantly correlated with the reduction in serum UA levels at both 4 and 24 weeks in multivariate analysis (respectively, P 8.6%) and lowest in the group with the lowest baseline HbA1c level (quartile 1; HbA1c ≤ 7.4%). The decrease in serum UA levels was greatest in the quartile 1 group and lowest in the quartile 4 group. In most groups, the maximum decrease in serum UA levels was seen in the first 4 weeks, while the maximum decrease in HbA1c was seen at week 24. Thus, serum UA levels were significantly decreased in patients with moderate HbA1c levels.

Aims Thymic carcinoma is a rare type of cancer without an established standard pharmaceutical treatment. This study investigated the antitumor effect of dimethyl itaconate (DI), a cell-permeable derivative of itaconate, on human thymic carcinoma cell line. Main methods Human thymic carcinoma cell line Ty82 was used to evaluate the effect of DI on cell viability. Western blotting and immunohistochemistry were performed to determine the molecular mechanism of antitumor effects of DI on Ty82. Key findings DI suppressed cell growth and promoted apoptosis of Ty82. The suppressive effect of DI on Ty82 was mediated by the downregulation of lactate dehydrogenase A (LDHA), and the subsequent decrease in the activity of mechanistic target of rapamycin (mTOR). DI exhibited synergistic antitumor effects with a specific inhibitor of large neutral amino acid transporter 1 (LAT1), an amino acid transporter currently being investigated as a novel target for cancer therapy. Significance Our findings demonstrate that DI is a novel potential strategy for thymic carcinoma treatment.

Background The activation of Th2 cells that play a pivotal role in the development of allergic eosinophilic inflammation is regulated by an L-type amino acid transporter (LAT) 1. However, the contribution of LAT1 to the pathogenesis of Th2 cell-mediated airway inflammation has not been investigated. Objective In this study, we investigated the effect of a LAT1 inhibitor, JPH203, on Th2 cell-mediated airway eosinophilic inflammation. Methods BALB/c mice were transferred with ovalbumin (OVA)-specific Th2 cell and challenged by corresponding allergen with or without administration of JPH203. Then, the infiltration of inflammatory cells including eosinophils and allergen-specific Th2 cells in the lungs and bronchial hyperresponsiveness (BHR) was assessed. Results Inflammatory responses in the lungs with massive accumulation of eosinophils and BHR were induced in Th2 cell-transferred mice upon challenge with OVA. The treatment with JPH203 significantly suppressed the allergen-induced BHR but not eosinophil infiltration. The infused Th2 cells were also accumulated in the lungs upon allergen challenge, though the response was not affected by JPH203 treatment. Conclusion JPH203 suppressed Th2 cell-mediated BHR through the mechanisms independently of the lung accumulation of eosinophils and Th2 cells.

L-type amino acid transporter 1 (LAT1), a transporter of large neutral amino acids, is rarely expressed in resting T cells, whereas its expression is remarkably upregulated upon activation. We have proven the essential role of T cells in the development of allergic inflammation in various target organs, though the contribution of LAT1 to T cell function in allergic diseases has not been clarified. Here, we investigated the effect of a LAT1 inhibitor, JPH203, on antigen-induced inflammatory responses in the skin, lungs, and nasal mucosa of immunized mice or mice transferred with in vitro-differentiated antigen-specific Th2 cells. Consistent with the augmented expression of LAT1 in skin-accumulated CD4+ T cells of atopic dermatitis patients, antigen-induced skin inflammation with eosinophil infiltration and Th2 cytokine production developed in immunized and Th2 cell-transferred mice was suppressed by JPH203 treatment. Essentially the same efficacy of JPH203 was seen in antigen-induced nasal eosinophilic inflammation accompanied by nasal hyperresponsiveness evoked in both mice. Remarkably, antigen-induced bronchial hyperresponsiveness was suppressed, whereas lung accumulation of eosinophils or antigen-specific T cells was not affected, by JPH203 in Th2 cell-transferred mice. The blockade of LAT1-mediated glycolysis, oxidative phosphorylation, and activating transcription factor 4-regulated amino acid starving responses in cytokine production and proliferation of Th2 was suggested to be involved in the efficacy of JPH203. LAT1 is essential for the pathogenesis of activated Th2 cell-mediated allergic inflammation and would be a promising target for the development of new means to treat allergic diseases.

Induced pluripotent stem cells (iPSCs) are opening up new possibilities for medicine. Understanding the regulation of iPSC biology is important when attempting to apply these cells to disease models or therapy. Changes of lipid metabolism in iPSCs were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS). Analysis revealed changes of the intensity and distribution of peaks at m/z 782.5 and 798.5 in iPSC colonies during spontaneous differentiation. Two phosphatidylcholines (PCs) were identified: C44H81NO8P, PC(36:4)[M+H]+ at m/z 782.5 and C42H82NO8P, PC(34:1)[M+K]+ at m/z 798.5. The intensity of PC(36:4) showed an inverse relation between undifferentiated and differentiated iPSC colonies. PC(34:1) displayed a diffuse distribution in undifferentiated iPSC colonies, while it showed a concentric distribution in differentiated iPSC colonies, and was localized at the border of the differentiated and undifferentiated areas or the border between undifferentiated iPSC and feeder cells. These findings suggested that the distribution of lipids changes during the growth and differentiation of iPSCs and that MALDI-TOF-IMS was useful for analyzing these changes. PC(36:4) might play a role in maintaining pluripotency, while PC(34:1) might play a role in the differentiation and spread of iPSCs.

降圧薬などいくつかの薬剤は本来の薬理作用とは別に尿酸降下作用を持つものがあり,水溶性ヨード系造影剤もその一つでiodipamideやdiatrizoateでの尿酸排泄亢進が報告されていた.長らく不明のままであった腎尿酸輸送機構の分子実体は2002年の腎尿細管尿酸トランスポーターURAT1(Urate Transporter 1)の分子同定によりその理解が飛躍的に進んだ.本研究ではURAT1と水溶性造影剤のiodipamideおよびdiatrizoateの相互作用を検討することで,その尿酸排泄促進作用の分子機序の解明を目的とする.URAT1の尿酸輸送活性の測定にはURAT1安定発現HEK293細胞(HEK-URAT1)細胞を用いた.IodipamideはHEK-URAT1細胞でのRI標識尿酸取込みを著明に阻害した(IC_:1.19±0.08?μM)のに対し,diatrizoateは1?mMまでの範囲では50%以上の阻害作用を示さなかった.1?mMまでのiodipamideはHEK-URAT1細胞の生存率に影響を与えなかった.IodipamideによるURAT1媒介尿酸輸送への阻害作用のキネティクス解析の結果,その阻害は競合阻害であり,阻害定数Ki値は11.03?μMであった.以上より,iodipamideは尿酸トランスポーターURAT1と相互作用をすることを初めて確認できた.このことからiodipamideは細胞外からURAT1の尿酸結合部位に結合し,競合して阻害を行うことで,腎尿細管の経上皮性尿酸再吸収を抑制し,ひいては血清尿酸値を低下させるものと考えられた., Drug-induced hypouricemia has been found in several drugs such as probenecid, benzbromarone and angiotensin II receptor blocker(ARB)losartan. Xray contrast agents such as iodipamide and diatrizoate, used for the intravenous cholangiography and excretory urography, were reported to have uricosuric effct beside their original action. After the molecular identification of renal apical urate transporter URAT1 as an entrance of urate into the epithelial cells of proximal tubules, this protein is thought to be major determinant for renal reabsorption of urate that affect the blood urate levels in human. The purpose of this study is to examine whether iodipamide and diatrizoate act on URAT 1 . In URAT 1 -stably expressing HEK293(HEK-URAT1)cells, iodipamide inhibited [^C] urate uptake dose-dependently(IC_ , 1.19±0.08 μM), while diatrizoate did not. Up to the concentration of 1 mM, iodipamide incubation for 24 hr did not affect the viability of HEK293-URAT1 cells. Lineweaver-Burk plot of the kinetic analysis by URAT1-mediated urate uptake with or without iodipamide indicated that its interaction occurs in a competitive manner(Ki:11.03 μM). These results suggest that uricosuric effect of iodipamide can be explained by the interaction of iodipamide with urate-binding site of URAT1, and the inhibition of urate reabsorption from extracellular side by iodipamide causes uricosuria leading to induce hypouricemia.

Hyperuricemia has recently been recognized as one of the risk factors for cardiovascular diseases. Some calcium channel blockers(CCBs), commonly used in the treatment of hypertension, have been reported to decrease serum urate level. Here, we tried to elucidate the molecular mechanism of the urate-lowering effects of CCBs. We performed [^C]urate uptake in cells stably expressing human urate transporter 1, a major contributor of renal urate reabsorption and a major target of uricosuric drugs such as benzbromarone and losartan(HEK-URAT1), together with mock(HEK-mock)cells to analyze the uricosuric action of CCBs. We also measured the activity of human xanthine oxidase(XO)to determine whether CCBs have inhibitory effects on urate production. The CCBs tested were nifedipine, nilvadipine, nitrendipine, benidipine, nisoldipine, nicardipine, efonidipine, amlodipine, azelnidipine, verapamil and diltiazem. We found for the first time that at least seven CCBs in the dihydropyridine subgroup interacted with URAT1-mediated urate uptake in HEK-URAT1 cells. Among these CCBs, nifedipine, nilvadipine and nitrendipine strongly inhibited URAT1-mediated urate uptake. Their IC_s were 15.8, 0.018 and 0.40?μM, respectively. In contrast, urate production mediated by XO was weakly inhibited by nifedipine and nisoldipine. In summary, URAT1 interacted with various CCBs differently, whereas XO, a major enzyme for urate production in the liver, did not interact with most of CCBs. Although CCBs were not excreted from the urine basically, their urate-lowering effects may be associated with the inhibition of renal urate reabsorption mediated by renal urate transporters such as URAT1 with their metabolites, and the results for structure-activity information in this study will provide a clue for developing new uricosuric drugs targeting URAT1.

Aims Thymic carcinoma is a rare epithelial tumor, for which, optimal pharmacotherapeutic methods have not yet been established. To develop new drug treatments for thymic carcinoma, we investigated the effects of fluvastatin-mediated pharmacological inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) on thymic carcinoma. Main methods Thymic carcinoma tissue was surgically excised and HMGCR expression was assessed by immunohistochemistry. Ty82 human thymic carcinoma cells were treated with fluvastatin (1–10 μM) and their growth was monitored. Key findings HMGCR was expressed on carcinoma cells but not on normal epithelial cells in thymic tissue. Inhibition of HMGCR by fluvastatin suppressed cell proliferation and induced the death of Ty-82 human thymic carcinoma cells. Fluvastatin mediated its antitumor effects by blocking the production of geranylgeranyl-pyrophosphate (GGPP), an isoprenoid that is produced from mevalonate and binds to small GTPases, which promotes cell proliferation. Significance Fluvastatin showed marked antitumor effects on thymic carcinoma. The results suggest that the statin has clinical benefits in thymic carcinoma management.

L-type amino acid transporter 1 (LAT1) functions to transport large neutral amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine. These amino acids are essential for cell growth and proliferation. Many studies have demonstrated LAT1 expression in various types of cancer, and its high expression level was associated with poor prognosis. However, the significance of LAT1 expression in thymic epithelial tumors is controversial. We conducted this retrospective study to investigate the LAT1 immunoreactivity in thymic epithelial tumors and its impact on prognosis. We analyzed 32 patients with thymoma and 14 patients with thymic carcinoma who underwent surgery at our institute. Immunohistochemical analysis was performed using formalin-fixed paraffin-embedded surgical tissues and an anti-LAT1 polyclonal antibody. We thus found that LAT1 immunoreactivity was undetectable in all of the thymoma specimens, regardless of the subtypes of thymoma. By contrast, LAT1 immunoreactivity was consistently detected in the cytosol of thymic carcinoma cells; namely, all 14 thymic carcinoma specimens demonstrated LAT1 immunoreactivity in the cytosol. Among these 14 thymic carcinoma specimens, four carcinoma specimens also showed LAT1 immunoreactivity in the cell membrane. Survival analysis indicated that the thymic carcinoma with the LAT1 membrane signal was associated with poor prognosis, compared with the specimens with the LAT1 cytosol signal. We therefore propose that LAT1 is expressed in the cytosol of thymic carcinoma cells, which could be a diagnostic marker of thymic carcinoma. Moreover, LAT1 expression in the cell membrane is a prognostic marker of thymic carcinoma.

Diabetes mellitus is a lifestyle-related disease that is characterized by inappropriate or diminished insulin secretion. Ex vivo pharmacological studies of hypoglycemic agents are often conducted using perfused pancreatic preparations. Pancreas preparations for organ bath experiments do not require cannulation and are therefore less complex than isolated perfused pancreas preparations. However, previous research has generated almost no data on insulin secretion from pancreas preparations using organ bath preparations. The purpose of this study was to investigate the applicability of isolated rat pancreas preparations using the organ bath technique in the quantitative analysis of insulin secretion from β-cells. We found that insulin secretion significantly declined during incubation in the organ bath, whereas it was maintained in the presence of 1 µM GLP-1. Conversely, amylase secretion exhibited a modest increase during incubation and was not altered in the presence of GLP-1. These results demonstrate that the pancreatic organ bath preparation is a sensitive and reproducible method for the ex vivo assessment of the pharmacological properties of hypoglycemic agents.

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na+-independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [14C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue–stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal’s body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

T cell response initiated by engagement of T cell receptor (TCR) is dependent on signal transduction events composed of protein kinases and adaptor proteins. However, the molecular mechanism for gene expression of these proteins is not entirely understood. Here we identified Heat Shock Protein 90 (HSP90) as an essential regulator for gene expression of Linker for activation of T cells (LAT) in primarily activated human T cells. Primarily activated T cells continuously synthesized LAT protein and treatment of cells with 17-AAG, a pharmacological inhibitor of HSP90, decreased LAT protein level following reduction of LAT mRNA. Furthermore, promoter activity of LAT gene was dramatically inhibited by 17-AAG. These results reveal a novel role of HSP90 as a positive regulator for expression of LAT gene in activated T cells.

In the present study, we investigated the transport of nephrotoxic mycotoxin ochratoxin A (OTxA) by a novel human organic anion transporter hNPT4 using the Xenopus oocyte expression system. hNPT4 mediated time- and concentration-dependent uptake of OTxA (Km: 802.8 μM) in a pH- and voltage-sensitive manner. Cis-inhibition experiments suggest that the substrate selectivity of hNPT4 is similar to that of hOAT4. The fact that the Km of OTxA for the efflux transporter hNPT4 was much higher than those for the uptake transporters hOAT1 and hOAT3 may favor the accumulation of OTxA in the tubular cell and lead to nephrotoxicity. Keywords:: ochratoxin A, organic anion transporter, nephrotoxicity

T-cell activation is an energy expenditure process and should be properly controlled in accordance with the availability of nutrients such as amino acids to eliminate wasteful energy consumption. However, the details of response to amino acids insufficiency in activated T cells remain largely unknown. Here we show that homeobox B9 (HOXB9), a member of the homeobox gene family that is known as a morphogenesis regulator, acts as a suppressor of activated human T cells to address amino acid starvation. The expression of HOXB9 was triggered by amino acid deprivation as well as functional inhibition of L-type amino acid transporter 1 (also known as SLC7A5) via activating transcription factor 4 in activated T cells. HOXB9 interfered the activities of NF-κB, nuclear factor of activated T-cells (NFAT) and AP-1 but not retinoic acid receptor-related orphan receptor, resulting in attenuation of the production of selective cytokines in activated T cells. Thus, the morphogenetic gene plays an unexpected role in the regulation of cellular metabolism with changes in the nutrition status in human T cells.

Intake of nutrients from the environment is fundamental to cellular activity. The requirement of nutrients depends on the situation in which cells are placed. Activation of T cells changes the structure and scale of cellular metabolism, which requires a large amount of nutrients. Hydrophilic nutrients such as glucose and amino acids cannot diffuse beyond the cellular membrane; thus transporters are required to assist the incorporation of these nutrients into cells. Based on this observation, metabolic changes accompanying activation of T cells must occur simultaneously with the reorganization of transporters that are capable of providing sufficient nutrients to the cell. This review describes the functional advantages of using special nutrient transporters in activated T cells and discusses the mechanisms of responses to nutrient starvation in T cells.

Induction of T cell responses following engagement of the Ag-specific TCR depends on TCR-initiated rearrangements of the cellular actin cytoskeleton and highly coordinated and tightly regulated interactions and of diverse intracellular signaling proteins. In this study, we show that filamin A (FLNa), an actin-binding and signal mediator scaffolding protein, is required for T cell activation. Following Ag stimulation, FLNa was recruited to the T cell-APC contact area, where it colocalized with protein kinase C-θ (PKCθ). Depletion of FLNa by RNA interference did not affect TCR-induced early tyrosine phosphorylation or actin polymerization but, nevertheless, resulted in impaired IL-2 expression by human primary T cells and reduced activation of NF-κB, AP-1, and NFAT reporter genes in transfected T cells. TCR stimulation induced stable physical association of FLNa with PKCθ. Furthermore, the TCR/CD28-induced membrane translocation of PKCθ was inhibited in FLNa-depleted T cells. These results reveal novel role for FLNa in the TCR/CD28 signaling pathway leading to transcription factor activation and IL-2 production, and suggest that this role is mediated, in part, through the inducible interaction of FLNa with PKCθ.

The molecular basis of T cell anergy is not completely understood. We show that in antigen-primed anergic murine CD4(+) T cells the linker for activation of T cells (LAT) is hypophosphorylated upon CD3/CD28 restimulation. Signaling events downstream of LAT (PLCgamma1 phosphorylation and p85 [PI3-K] association) were impaired, whereas upstream events (CD3zeta and ZAP-70 phosphorylation) remained intact. LAT recruitment to the immunological synapse and its localization in detergent-resistant membrane (DRM) fractions were defective in anergic T cells. These defects resulted from impaired palmitoylation of LAT and were selective since the DRM localization and palmitoylation of Fyn were intact. This LAT defect was independent of Cbl-b and did not reflect enhanced LAT degradation. These results identify LAT as the most upstream target of anergy induction; moreover, they suggest that regulation of the amount of LAT in the immunological synapse and DRM by posttranslational palmitoylation contributes to the induction of T cell anergy.

The death or survival of double positive (DP) thymocytes is determined by the strength of their TCR signaling. Of the three Runx family proteins, the DP cells only express the Runx1 transcription factor. We introduced and expressed in murine thymocytes the Runt domain of Runx1, which antagonizes the activity of endogenous Runx1. The Runt transgenic DP thymocytes expressed higher levels of the proapoptotic molecules Fas and Bim compared with the wild-type cells. Furthermore, the Runt transgenic cells were more susceptible to apoptosis induced by the artificial cross-linking of the TCR by the anti-CD3 Ab. This susceptibility was partially abrogated by the lpr/lpr background. In addition, Runx1:HY-TCR-double transgenic DP thymocytes were resistant to the apoptosis induced by the endogenously presented HY Ag. We propose that Runx1 functions to suppress the apoptotic sensitivity of DP thymocytes in the context of TCR signaling.