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Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E2 (PGE2) plays an important role in nephrotoxicity. The aim of this study was to examine whether albumin-bound fatty acids induce PGE2 production in human renal proximal tubular epithelial cell line HK-2. Fatty acid-bearing human serum albumin increased PGE2 release in the culture medium in concentration-dependent and time-dependent manners, but fatty acid-depleted albumin had no effect on PGE2 production. Next, we investigated the effect of arachidonic acid, a precursor of eicosanoids, on PGE2 production. Arachidonic acid with fatty acid-free albumin significantly enhanced the release of PGE2 into the medium in a concentration-dependent manner. Furthermore, we examined the effect of arachidonic acid on mRNA expression of hypoxia inducible factor-1α (HIF-1α). Arachidonic acid increased HIF-1α mRNA expression in a concentration-dependent manner. These findings suggest that fatty acids, at least in part arachidonic acid, bound to albumin increase PGE2 production and expression of HIF-1α mRNA and protein, possibly resulting in various cell responses induced by albumin overload.

Background and Objectives: Immune-checitors have been established as a novel standard treatment for non-small cell lung cancer (NSCLC). The aim of this study was to identify factors associated with efficacy and nivolumab-related interstitial pneumonia in NSCLC by evaluating clinical data at the initiation of and during treatment. Methods: We retrospectively reviewed the medical records of patients who underwent treatment with nivolumab between October 2015 and December 2017. Using pretreatment patient data, we investigated factors associated with overall survival (OS) and the onset of nivolumab-related pneumonitis. We investigated serum biochemistry during treatment to identify the determinants associated with progressive disease (PD) and the onset of nivolumab-related pneumonitis. Results: A total of 94 patients were included. Eleven patients continued treatment, and 54 patients were diagnosed with progressive disease. Nivolumab-related pneumonitis occurred in 15 patients. A pretreatment Eastern Cooperative Oncology Group Performance Status (ECOG PS) = 0 was linked to significantly longer OS than ECOG PS = 1 (median: 20.1 vs. 6.5 months, respectively; p < 0.001). There was a higher incidence of nivolumab-related pneumonitis in patients with a history of interstitial pneumonia than in those without it ( p = 0.008). During treatment, the level of albumin gradually decreased prior to PD and onset of nivolumab-related pneumonitis. Conclusion: These results suggest that the pretreatment ECOG PS is the determining factor that is associated with OS, whereas history of interstitial pneumonia is the factor associated with nivolumab-related pneumonitis. A decrease in albumin during treatment may be associated with both PD and nivolumab-related pneumonitis.

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E

Controllable transgene expression systems are indispensable tools for the production of animal models of disease to investigate protein functions at defined periods. However, in nonhuman primates that share genetic, physiological, and morphological similarities with humans, genetic modification techniques have not been well established; therefore, the establishment of novel transgenic models with controllable transgene expression systems will be valuable tools to understand pathological mechanism of human disease. In the present study, we successfully generated transgenic marmosets using a tetracyclin-inducible transgene expression (tet-on) system as a neurodegenerative disease model. The mutant human ataxin 3 gene controlled by the tet-on system was introduced into marmoset embryos via lentiviral transduction, and 34 transgene-introduced embryos were transferred into the uteri of surrogate mothers. Seven live offspring (TET1-7) were obtained, of which four were transgenic. Fibroblasts from TET1 and 3 revealed that inducible transgene expression had occurred after treatment with 10 μg/mL of doxycycline, while treatment with doxycycline via drinking water resulted in 1.7- to 1.8-fold inducible transgene expression compared with before treatment. One transgenic second-generation offspring (TET3-3) was obtained from TET3, and doxycycline-inducible transgene expression in its fibroblasts showed that TET3-3 maintained a high transgene expression level that matched its parent. In conclusion, we established a novel transgenic marmoset line carrying the mutant human ataxin 3 gene controlled by the tet-on system. The development of nonhuman primate models with controllable transgene expression systems will be useful for the identification of disease biomarkers and evaluation of the efficacy and metabolic profiles of therapeutic candidates.

The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 μM), higher sildenafil concentrations (25 and 250 μM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 μM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

The ovary of neonatal nonhuman primates contains the highest number of immature oocytes, but its cryopreservation has not yet been sufficiently investigated in all life stages. In the current study, we investigated cryodamage after vitrification/warming of neonatal ovaries from a nonhuman primate, the common marmoset (Callithrix jacchus). A Cryotop was used for cryopreservation of whole ovaries. The morphology of the vitrified/warmed ovaries was found to be equivalent to that of fresh ovaries. No significant difference in the number of oocytes retaining normal morphology per unit area in histological sections was found between the two groups. In an analysis of dispersed cells from the ovaries, however, the cell viability of the vitrified/warmed group tended to be decreased. The results of a comet assay showed no significant differences in DNA damage. These results show that cryopreservation of neonatal marmoset ovaries using vitrification may be useful as a storage system for whole ovaries.

ステッピングモーターは、定速回転をおこなうモーターとは異なり、デジタル制御により断続的に回転と停止を繰り返すことを目的として開発された特殊モーターである。研究の結果、モーターの種類、制御プログラムの開発、回転体部分（作品）の軽量化を試みることで、直径150mmの円盤に、25 個の合計100g のものを載せても駆動することが可能となった。このメカニズムにより、これまでの古典的なストロボを使用した立体アニメーション技術にあらたな表現の可能性が加わることとなった。もはや暗い部屋は不要であり、明るい環境で目の前で物体が動き出すのである。 研究の成果は神戸市と連携して、メディアイベント「KOBE078」、「MAKERS BAZAAR」に出展し、発表をおこなった。またオランダ、デザインアカデミー・アイントホーフェンと連携して共同研究をおこない、バート・ヘンゼル氏の来日に合わせてその成果を展示、関連したワークショップを開催した。研究のコアとなるモーターの制御プログラムはWEB および印刷物で公開している。 また、2018 年5 月には国際写真展「KYOTOGRAPHIEKG+」に出展し、作品としての発表をおこなった。, Unlike a motor that performs constant speed rotation, the stepping motor is a special motor developed for the purpose of repeatedly rotating and stopping intermittently by digital control. As a result of the research, we tried to reduce the weight of the rotating body part by developing the type of motor, the control program, and driving weight even if loading 25 pieces totaling 100 g on a disk of 150 mm in diameter. It has become possible. With this mechanism, the possibility of new expression is added to the three-dimensional animation technology using classical strobe. The dark room is no longer needed, and the object moves in front of you in a bright environment. The results of the research collaborated with Kobe City and exhibited at the media event "KOBE 078", "MAKERS BAZAAR", and made an presentation. In collaboration with the Design Academy Eindhoven in the Netherlands, he collaborated with the workshop to display the results of Mr. Bart Haensel as he came to Japan and held related workshops. The control program of the motor, which is the core of research, is released on the web and printed matter. In addition, in May 2018 I participated in the international photo exhibition "KYOTOGRAPHIE KG +" and made a presentation as a work.

More than 250,000 reports of adverse drug events were included in the database of the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan. However, these data have not been utilized sufficiently for analysis. While valacyclovir is the antiviral agent used worldwide, it is reported to induce nephrotoxicity. The aim of this study was to clarify the profiles of valacyclovir-induced adverse events using the PMDA database.Case reports were screened in the PMDA adverse event database from 2004 to 2011. The profiles of patients with acute kidney injury (AKI) were analyzed by sex, age, diseases, concomitant suspected drugs, and outcomes.A total of 514 kidney-related adverse events were detected, and 344 were cases that included AKI. Of the AKI cases, 246 patients (71.5%) were female. There were 145 patients who were 70 to 79 years of age, which was the most affected of all age groups. Of the 344 patients, 183 patients had hypertension, and 65 had diabetes. Valacyclovir was the only drug used among 257 patients (74.1%).There were many reports of AKI involving valacyclovir and females, particularly in the 70- to 79-year age group in Japan. The results suggest that these patients were most likely to develop AKI after valacyclovir treatment.

The purpose of this study was to build regression models for the prediction of apparent oral clearance (CL/F) for small-molecule inhibitors in the pediatric population using data obtained from adults. Two approaches were taken; a simple allometric regression model which considers no interdrug or interindividual variability and an allometric regression model with mixed-effects modeling where some variability parameters are included in the model. Average CL/F values were obtained for 15 drugs at various dosages from 31 literatures (a total of 139 data sets) conducted in adults and for 15 drugs from 26 literatures (62 data sets) conducted in children. Data were randomly separated into the "modeling" or "validation" data set, and the 2 allometric regression models were applied to the modeling data set. The predictive ability of the models was examined by comparing the observed and model-predicted CL/F in children using the validation data set. The percentage root mean square error was 17.2% and 26.3% in the simple allometric regression model and the allometric regression model with mixed-effects modeling, respectively. The predictive ability of the 2 models seems acceptable, suggesting that they could be useful for predicting the CL/F of new small-molecule inhibitors and for determining adequate doses in clinical pharmacotherapy for children.

For in vitro growth and maturation of mouse oocytes (IVG-IVM), serum is added to media up to and including the stage of oocyte maturation; this subsequently supports oocytes through fertilization and early embryo development. However, problems may occur with sera, such as batch differences and issues of biosafety. The purpose of the present study was to determine the capacity for fertilization and pre- and post-implantation development of oocytes that underwent IVG-IVM with a serum substitute. Oocyte-granulosa cell complexes from preantral follicles were cultured in medium with either fetal bovine serum (FBS), Serum Substitute Supplement™ (SSS), or Knockout™ Serum Replacement (KSR) for 10days, and were then allowed to mature for 17 h. Subsequently, more than 90% of oocytes underwent germinal vesicle breakdown (GVBD) and more than 70% reached metaphase II, with no significant difference between the groups. A lower fertilization rate, presumably due to zona hardening, was found in the serum substitute groups. Nevertheless, more than 50% of the inseminated oocytes were fertilized and 35%-45% of them underwent first cleavage and developed to the blastocyst stage. Following embryo transfer, one and four live offspring were produced from the SSS and KSR groups, respectively. The present study demonstrated that murine IVG-IVM oocytes cultured in media with a serum substitute, achieved fertilization in vitro, pre- and post-implantation development, and the delivery of live pups, although the efficiency of the process is reduced compared to FBS supplementation.

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo-microdrilling of a 6-μm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo-microdrilling a 20-μm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo-scraping an 80-μm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80-μm opening, rather than 6-μm or 20-μm openings, is suitable to complete the hatching process in the marmoset embryo.

Organic cation transporter OCTs and multidrug and toxin extrusion (MATE) are involved in pharmacokinetics of various drugs. In the renal proximal epithelia, OCT2 mediates uptake of the drugs such as metformin or cimetidine at basolateral membrane, and MATE1 and MATE2-K mediated the secretion of cationic and zwitterionic drugs at brush-border membrane. In the liver, OCT1 are expressed on the sinusoidal membrane and MATE1 are expressed on the canalicular membrane. These transporters mediate the biliary excretion of drugs. The change of these transporter activities, caused by genetic alteration or drug–drug interaction, affected the pharmacokinetics of substrates. Inhibitors of the transporters reduce the biliary or urinary secretion of substrate drugs. In addition, OCTs and MATEs are involved in adverse drug reactions. For example, it was considered that renal toxicities of platinum agents cisplatine or oxaliplatin were affected by substrate specificities of renal OCT2 and MATEs. OCTs and MATEs play important roles for drug efficacies and toxicities especially in the liver and the kidney.

Yokukansan is a Japanese traditional medicine that has been used for behavioral and psychological symptoms of dementia. Although the efficacy of Yokukansan have been reported, few studies have focused on its adverse effects. In this study, the adverse effects in hospitalized patients treated with Yokukansan were evaluated retrospectively and compared with those listed in the Japanese Adverse Drug Event Report database from the Pharmaceutical and Medical Device Agency. A total of 21 patients who were prescribed Yokukansan at Rakuwakai Otowa Hospital from April 2013 to September 2013 were registered as subjects for this study. Patient profiles, such as age, gender, serum potassium levels, AST and ALT, were evaluated. Serum potassium levels decreased significantly from 4.3 ± 0.6 mEq/L to 3.6 ± 0.4 mEq/L after treatment with Yokukansan, and 61.9% of the patients demonstrated hypokalemia. In addition, the pre-treatment serum potassium levels were associated with the induction of hypokalemia by Yokukansan. The onset date of hypokalemia was varied from 2 to 1,154 days in Otowa data and from 2 to 1,533 days in JADER data. In terms of the number of days to the onset of hypokalemia, there was no significant difference between the Rakuwakai Otowa Hospital and results of Japanese Adverse Drug Event Report database. It is necessary to pay attention to patients during the treatment with Yokukansan, even if treatment period was long term.

In the renal proximal tubular cells, various transporters play important roles in the secretion and reabsorption of drugs. When metabolic acidosis is induced, a number of adaptive changes occur in the kidney. The purpose of this study was to clarify the changes of drug transporters under the acidosis and the effects of these changes on urinary drug excretion.Wistar/ST rats were given 1.5% NH₄Cl in tap water for 48 h to induce the acidosis. Pharmacokinetics of PSP or metformin was evaluated. In addition, expression levels of drug transporters were examined by Western Blotting.The renal clearance of PSP was markedly decreased, whereas the creatinine clearance and renal clearance of metformin were unchanged. Furthermore, Western blots indicated that the protein expression level of organic anion transporter (OAT) 3 was decreased. In contrast to OAT3 levels, OAT1 and organic cation transporter (OCT) 2 levels were unaffected. An immunohistochemical analysis showed that the OAT3 protein in the proximal tubules was localized in the basolateral membrane both of the normal and the acidosis rats.The decrease of renal excretion of anionic drugs during metabolic acidosis might be partly due to a reduction in the level of OAT3 protein.

A decline in fertility occurs with age. Because the extracellular matrix has a vital role in maintaining the integrity and determining the tissue function of numerous organs, the reasons for this fertility reduction may be associated with changes occurring in the ovarian matrix. To demonstrate this hypothesis, by using Picrosirius Red (PSR), a connective tissue dye specific for fibres of collagen I and III, we examined the ovarian tunica albuginea (TA), a part of the cortex composed by dense connective tissue that protects the ovaries and whose rupture is critical for the occurrence of ovulation events. According to an approval from the Ethics Committee of Okayama University (approval number 1818), ovaries were obtained from patients with gender identity disorder at the Okayama University Hospital. Sliced ovaries were fixed in 4% paraformaldehyde and included in paraffin. Sections of 5 μm thickness were stained with PSR. For each individual, 2 non-overlapping brightfield images were taken of each section with a fluorescence microscope and the attached viewer software (Keyence BZ-X710, Keyence Corp., Osaka, Japan). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify the distance of the dense connective tissue by selecting 5 random points manually per image and, to quantify the density of the tissue; we detected PSR staining above a determined threshold kept constant for all the images analysed. Statistical analyses of data from 3 replicates per individual were performed by Pearson’s correlation, and a P-value

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 microm in diameter and had competence to resume meiosis in vitro. When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences (P < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.

We investigated the pharmacokinetics and pharmacodynamics of paclitaxel with carboplatin or gemcitabine in patients with urogenital cancer to clarify the significance of monitoring of the serum concentration of paclitaxel.Paclitaxel was administered at 175 mg/m(2) or 150 mg/m(2) to patients with hormone-refractory prostate cancer (n = 10) or advanced transitional cell carcinoma (n = 6) along with carboplatin or gemcitabine, respectively. The relationships between pharmacokinetic parameters and hematological adverse effects, as well as pharmacological effects, were examined. The effects of patient characteristics, including single-nucleotide polymorphisms of MDR1(ABCB1), CYP2C8, CYP3A4, and CYP3A5, on the total body clearance of paclitaxel were evaluated.Total body clearance and volume of distribution at a steady-state after the intravenous infusion of paclitaxel were not significantly different between patients with carboplatin or gemcitabine. The percent decreases in neutrophils and platelets for the regimen with gemcitabine were significantly greater than those with carboplatin, and showed a significant positive relationship with the observed concentration at the end of infusion or time above 0.1-microM concentration of paclitaxel. Post-therapy decreases in prostate-specific antigen were not positively correlated with the extent of paclitaxel exposure in the prostate cancer patients. Neither the polymorphisms at exon 26 (C3435T) and at exon 21 (G2677A/T) in MDR1 nor the CYP3A5*1 allele significantly affected the total body clearance of paclitaxel.The hematological side effects of paclitaxel were intensified by gemcitabine, and were correlated with paclitaxel pharmacokinetics. Monitoring of the serum concentration of paclitaxel will facilitate the therapy, with less myelosuppression and without any loss of therapeutic efficacy.

The immunosuppressant mycophenolate mofetil (MMF) is frequently administered with calcineurin inhibitors and corticosteroids to recipients of organ transplantations. However, the renal handling of the active metabolite mycophenolic acid (MPA) and 7-O-MPA-glucuronide (MPAG) has been unclear. The purpose of the present study was to assess the interaction of MPA and MPAG with the human renal organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8), by conducting uptake experiments using HEK293 cells stably expressing these transporters. MPA and MPAG inhibited the time-dependent uptake of p-[(14)C]aminohippurate by hOAT1 and that of [(3)H]estrone sulfate by hOAT3. The apparent 50% inhibitory concentration (IC(50)) of MPA for hOAT1 and hOAT3 was estimated at 10.7 and 1.5 microM, respectively. In the case of MPAG, the IC(50) values were calculated at 512.3 microM for hOAT1 and 69.1 microM for hOAT3. Eadie-Hofstee plot analyses showed that they inhibited hOAT1 noncompetitively and hOAT3 competitively. No inhibitory effects of tacrolimus, cyclosporin A and azathioprine on transport of p-[(14)C]aminohippurate by hOAT1 and of [(3)H]estrone sulfate by hOAT3 were observed. No transport of MPA by these transporters was observed. On the other hand, the uptake of MPAG into cells was stimulated by the expression of hOAT3, but not hOAT1. These findings propose the possibility that the administration of MMF decreases the renal clearance of drugs which are substrates of hOAT1 and hOAT3. Present data suggest that hOAT3 contributes to the renal tubular secretion of MPAG.

Docetaxel is frequently used in the treatment of a wide variety of solid tumors, including breast cancer. The aim of this study is to obtain the population pharmacokinetic parameters of docetaxel in Japanese female patients with breast cancer. Blood samples from 24 patients were collected sequentially before and after docetaxel infusion. Genomic DNA was isolated from the peripheral blood and genotyped for the selected polymorphisms in the candidate genes of drug transporters and metabolizing enzymes. The influence of patient characteristics on the pharmacokinetics of docetaxel was evaluated using the nonlinear-mixed-effect modeling program, NONMEM. As a basis for comparison, the pharmacokinetics of another taxane paclitaxel in 41 separate female patients with breast cancer was calculated. A two-compartment model adequately described the pharmacokinetic profiles of docetaxel. The population mean estimates of the total body clearance for patients aged 58 years or less and the central volume of distribution for docetaxel were 32.6 L/h and 5.77 L, respectively. In patients over 58 years, the clearance was 24 % higher than that in the younger patients. No influences of the genotypes examined were noted on the clearance of docetaxel. The clearance of paclitaxel was not affected by patient age. Patients over the age of 58 years showed significantly higher clearance of docetaxel than that in patients aged 58 years or less. Since the clearance of paclitaxel was not affected by the age, it is possible that the pharmacokinetic mechanisms of docetaxel might be specifically affected by age in females.

Mammalian oocytes undergo growth and development through prenatal and postnatal periods. This is an investigation of development in vitro of oocytes and ovarian tissues from fetal gonads of the 12.5 days post coitus. Cultured tissues survived and the oocytes entered the growth phase, subsequently achieving long-term (14-30 days) culture. Growing oocytes were observed in their general morphology and were enclosed within 1-2 layers of granulosa cells, which resembled primary or secondary follicles. However, no follicular structures were also found in the cultures despite the existence of growing oocytes. The oocytes enclosed in very flattened cells were detected as in histological structure on day 18 of culture. The oocyte diameter became approximately 50 μm under culture conditions supplemented with 15% fetal bovine serum or Ehrlich ascites tumor fluid. These results indicate that the maximum culture period for oocyte growth in this organ culture is approximately 3 weeks and that the oocytes can grow in vitro to at least a medium size even if normal follicular structure is not formed.

To investigate the developmental competence in vitro of female germ cells in fetal gonads, gonadal ridges of 12.5 days post coitus (dpc) were organ-cultured for 18 days, and then oocyte-granulosa cell complexes (OGCs) were isolated from the cultures. Collected OGCs were further cultured for 11 days. The oocytes grown in vitro reached 64.1 ± 0.4 μm. Transmission electron microscopy analysis showed that oocytes grown in vitro had maintained connection with surrounding somatic cells. Some oocytes resumed meiosis by 1 μM okadaic acid or 10 ng/ml epidermal growth factor (EGF). The oocytes that underwent germinal vesicle breakdown reached 71.3 ± 0.5 μm. This study showed growth and meiotic resumption in vitro of oocytes through the support of ovarian tissue from fetal gonads.

Our previous studies showed that the mRNA level of human organic anion transporter (hOAT) 3 in the kidney was correlated with the rate of elimination of an anionic antibiotic cefazolin. However, the correlation coefficient was not so high. In the present study, therefore, we enrolled more patients to examine whether additional factors were responsible for the correlation. hOAT mRNA levels in renal biopsy specimens were quantified using the real-time polymerase chain reaction method. The elimination rates for the free fraction of cefazolin were determined in patients with various renal diseases. In the present study, the coefficient of correlation between the hOAT3 mRNA level and the elimination rates for the free fraction of cefazolin was not so high in the patients overall as in our previous study (r = 0.536). However, following the classification of renal diseases, a better correlation was obtained in patients with mesangial proliferative glomerulonephritis (r = 0.723). In contrast, multiple regression analyses including gender, age, and liver function did not result in any improvements in the correlation coefficients. These results suggest that the hOAT3 mRNA level is a significant marker of pharmacokinetics with which to predict the rate of elimination of cefazolin in patients with mesangial proliferative glomerulonephritis.

We examined the substrate specificity of human organic anion transporter (hOAT) 1 and hOAT3 for various cephalosporin antibiotics, cephaloridine, cefdinir, cefotiam, ceftibuten, cefaclor, ceftizoxime, cefoselis and cefazolin by using HEK293 cells stably transfected with hOAT1 or hOAT3 cDNA (HEK-hOAT1, HEK-hOAT3). Additionally, we examined the uptake of various compounds by these transfectants. The mRNA level of hOAT3 in HEK-hOAT3 was about three-fold that of hOAT1 in HEK-hOAT1. Functional expression of hOAT1 and hOAT3 was confirmed by the uptake of p-[14C]aminohippurate and [3H]estrone sulfate, respectively. p-[14C]Aminohippurate, [3H]estrone sulfate, [14C]captopril, [3H]methotrexate, [3H]ochratoxin A, [3H]leucovorin and [3H]cimetidine were shown to be substrates for hOAT1 and hOAT3, and [3H]dehydroepiandrosterone sulfate was shown to be a substrate for hOAT3. All cephalosporin anitibiotics tested were shown to inhibit the uptake of p-[14C]aminohippurate and [3H]estrone sulfate via hOAT1 and hOAT3, respectively, in a dose-dependent manner, and the IC50 values of these antibiotics, except for cefaclor, for the hOAT1-mediated uptake of p-[14C]aminohippurate were within four-fold of those for the hOAT3-mediated uptake of [3H]estrone sulfate. The uptake of cephaloridine, cefdinir and cefotiam by HEK-hOAT3 was 35-50-fold greater than that by control cells. Moreover, the accumulation of the other cephalolsporin antibiotics was significantly greater in HEK-hOAT3 than in control cells. In contrast, the uptake of these antibiotics by HEK-hOAT1 was within two-fold of that by control cells. In conclusion, hOAT3 plays a more important role than hOAT1 in the renal secretion of cephalosporin antibiotics.

OBJECTIVE To report a patient with a high tacrolimus blood concentration after lansoprazole administration and assess the potential interaction between tacrolimus and lansoprazole. CASE SUMMARY A 34-year-old Japanese man underwent a living-donor kidney transplantation having received tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression. Lansoprazole was administered from postoperative day 4 as prophylaxis of ulcers. The trough concentration of tacrolimus increased markedly after the introduction of lansoprazole, while results of liver function tests were within normal limits. Lansoprazole was stopped on day 15 and was replaced with famotidine on day 17. The trough concentration of tacrolimus returned to the therapeutic range after administration of lansoprazole ceased. Genetic analysis revealed a heterozygous mutation at exon 5 of the CYP2C19 gene (CYP2C19*1/*2) in this patient. DISCUSSION Lansoprazole is metabolized by 2 enzymes, CYP2C19 and CYP3A4. Since tacrolimus is also metabolized by CYP3A4, the blood concentration of tacrolimus in this patient who had a CYP2C19 gene mutation may have been elevated by decreased hepatic elimination of lansoprazole. An objective causality assessment revealed that this interaction was probable. CONCLUSIONS Trough concentrations of tacrolimus should be monitored closely for optimizing the dosage regimen in patients receiving concomitant lansoprazole.

We have evaluated the functional consequences of genetic variations in human organic cation transporter hOCT1 (SLC22A1). Three coding single nucleotide polymorphisms (cSNPs) resulted in the amino acid changes Pro283Leu, Arg287Gly and Pro341Leu were assessed. Uptake experiments with transient expression system using HEK293 cells revealed that the variants Pro283Leu and Arg287Gly had completely diminished transport activity. The other variant Pro341Leu had a significantly, but not completely, decreased transport activity. Western blot analysis showed that the expression levels of all three variant proteins in the crude membranes of HEK293 cells were comparable to those of wild type hOCT1. Moreover, the expression of variant proteins at the plasma membrane was confirmed by indirect immunofluorescence, indicating that these SNPs did not affect the membrane localization of hOCT1. Present results suggest that the amino acid residues Pro283 and Arg287 have a substantial role in substrate recognition of hOCT1.

Age-associated neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3–4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases., Article, eNeuro.4(2):e0250(2017)

We have isolated a cDNA encoding a pH-sensing regulatory factor protein that modulates transport activity of the human H+/peptide cotransporter, hPEPT1, from the human duodenum cDNA library. The cDNA (1,724 bp) for the regulatory factor (hPEPT1-RF) had an open reading frame encoding a 208-amino acid. The 18-195 amino acid residues of hPEPT1-RF were completely consistent with the 8-185 amino acid residues of hPEPT1, whereas the 1-17 and 196-208 residues were unique sequences. Using a reticulocyte lysate, the in vitro synthesized hPEPT1-RF RNA translated a product of approximately 23 kDa. Northern blot analysis and reverse transcription-coupled PCR revealed that both hPEPT1 and hPEPT1-RF mRNA transcripts are expressed in Caco-2 cells. When expressed in Xenopus oocytes, hPEPT1-RF showed no transport activity of glycylsarcosine, but shifted pH profile of the dipeptide transport mediated by the coexpressed hPEPT1. The pH profile of glycylsarcosine uptake in oocytes coexpressing hPEPT1 and hPEPT1-RF was almost similar to that in the Caco-2 cells. This is the first demonstration of cDNA isolation of a regulatory factor which modulates hPEPT1 activity.

In recent years there have been over 30000 suicides annually in Japan. This is one of the most serious problems for Japanese society. Because mental disorder is closely associated with suicide, factors related to the increase in mental disorders and suicides should be clarified. In this study, various data regarding social factors were evaluated to assess the correlation of the number of patients with mental disorders and suicides among the 47 prefectures of Japan. Various data regarding social factors, such as income, savings, or rate of divorce, were obtained from the database of the Ministry of Health, Labour and Welfare of Japan. Among the factors, the annual income and the amount of savings were significantly correlated with the number of patients with mental disorder. On the other hand, while the annual income did not have a significant correlation with suicides, the amount of savings had a significant correlation with suicides. In conclusion, the annual income and amount of savings may both be one of the important factors involved in mental disorders, and the savings may also be a factor affecting suicides. These analyses are valuable in helping to clarify the causes of mental disease, and can hopefully contribute to the health and welfare of Japanese.

Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 μl or less) or a large volume (2-3 μl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 μl or less is essential for successful marmoset embryo transfer.

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.

In the kidney, human organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) are the major transporters for the secretion of cationic drugs into the urine. In the human kidney, OCT2 mediates the uptake of drugs from the blood at the basolateral membrane of tubular epithelial cells, and MATE1 and MATE2-K secrete drugs from cells into the lumen of proximal tubules. However, the expression of these transporters depends on the species of the animal. In the rodent kidney, OCT1 and OCT2 are expressed at the basolateral membrane, and MATE1 localizes at the brush-border membrane. Together, these transporters recognize various compounds and have overlapping, but somewhat different, substrate specificities. OCTs and MATEs can transport important drugs, such as metformin and cisplatin. Therefore, functional variation in OCTs and MATEs, including genetic polymorphisms or inter-individual variation, may seriously affect the pharmacokinetics and/or pharmacodynamics of cationic drugs. In this review, we summarize the recent findings and clinical importance of these transporters.

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.

The kidney plays an important role in the secretion of organic compounds including drugs, toxins and endogeneous metabolites. The renal elimination process of organic cations is mediated by two distinct transport systems expressed on the apical and basolateral membrane of proximal epithelial cells. In 2005, mammalian multidrug and toxin extrusion 1 (MATE1)/SLC47A1 was identified as an orthologue of bacterial NorM. MATE1 is the H(+)/organic cation antiporter at the apical membrane, which mediates the secretion of organic cations. Kidney-specific MATE2-K was isolated from human kidney and localized at the brush-border membrane of proximal tubules. Like MATE1, MATE2-K mediates the secretion of organic cations into urine. MATE1 and MATE2-K are involved in the excretion of important medications and the disruption of these transporters can cause severe pharmacological problems. Recent findings regarding the MATE/SLC47 family are summarized in this review.

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.

This study was undertaken to examine pre- and postimplantation developmental potency of cryopreserved embryos that had undergone in vitro growth (IVG), maturation (IVM) and fertilization (IVF) of oocytes from the preantral follicle stage. An oocyte culture system for IVG and IVM was used in oocyte-granulosa cell complexes (OGCs) derived from preantral follicles in 12-day-old mice. The rate of oocyte maturation was improved by the addition of gonadotropins (FSH/LH) and cytokines (IGF-I/SCF) to culture medium for IVG. During culture for IVG, estradiol-17β and progesterone concentrations increased progressively to the latter period of culture. This culture system enabled IVG, IVM, IVF and pre- and postimplantation development. From 90 cryopreserved 2-cell stage embryos transferred into recipients after warming, 10 live pups were produced. Cryopreservation of embryos by vitrification at the 2-cell stage showed no harmful effect on development to the blastocyst stage or on the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). This study demonstrated that embryos derived from oocytes grown in vitro have tolerance for vitrification and competence to develop to term after warming. This IVG-IVM-IVF technology combined with embryo cryopreservation might be useful for assisted reproduction in mice.

Organic cation transporters (OCTs) and organic anion transporters (OATs) (SLC22A family) play crucial roles in the renal secretion of various drugs. Messengar ribonucleic acid (mRNA) expression of transporters can be a key factor regulating interindividual differences in drug pharmacokinetics. However, the source of variations in mRNA levels of transporters is unclear. In this study, we focused on single nucleotide polymorphisms (SNP) in the promoter region [regulatory SNPs (rSNPs)] as candidates for the factor regulating mRNA levels of SLC22A. We sequenced the promoter regions of OCT2 and OAT1–4 in 63 patients and investigated the effects of the identified rSNPs on transcriptional activities and mRNA expression. In the OCT2 promoter region, one deletion polymorphism (−578_−576delAAG) was identified; −578_−576delAAG significantly reduced OCT2 promoter activity (p < 0.05), and carriers of −578_−576delAAG tend to have lower OCT2 mRNA levels, but the difference is not significant. There was no rSNP in the OAT1 and OAT2 genes. The five rSNPs of OAT3 and one rSNP of OAT4 were unlikely to influence mRNA expression and promoter activity. This is the first study to investigate the influences of rSNPs on mRNA expression of SLC22A in the kidney and to identify a regulatory polymorphism affecting OCT2 promoter activity.

Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of Cl−on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[14C]aminohippurate by HEK-hOAT1 and [3H]estrone sulfate by HEK-hOAT3 was greater in the presence of Cl−than in the presence of SO42−or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the Cl−-containing medium than the gluconate-containing medium, suggesting that the influences of Cl−are not dependent on substrate and that Cl−directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with Cl−did not change the Kmvalue for the uptake of p-[14C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate (Vmax) value. On the other hand, replacement of gluconate with Cl−decreased the Kmvalue for the uptake of [3H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the Vmaxvalue. In summary, Cl−upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that Cl−participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3.

Human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) are responsible for renal tubular secretion of an antifolic acid methotrexate, and are considered to be involved in drug interaction of methotrexate with nonsteroidal anti-inflammatory drugs (NSAIDs). In our hospital, a delay of methotrexate elimination was experienced in a patient with Hodgkin's disease, who took loxoprofen, a commonly used NSAID in Japan, which suggested a cause. In this study, we examined the drug interaction via hOAT1 and hOAT3, using Xenopus laevis oocytes. hOAT1 and hOAT3 mediated the methotrexate transport with low affinity (K(m) of 724.0 muM) and high affinity (K(m) of 17.2 muM), respectively. Loxoprofen and its trans-OH metabolite, an active major metabolite, markedly inhibited the methotrexate transport by both transporters. Their inhibition concentrations (IC(50)) were in the range of the therapeutic levels. These findings suggest that loxoprofen retards the elimination of methotrexate, at least in part, by inhibiting hOAT1 and hOAT3.

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.

Histamine H 2 receptor antagonist famotidine and cimetidine are commonly used for treatment of gastrointestinal ulcer diseases. Inasmuch as these drugs are mainly secreted by renal tubules, dosages have been adjusted according to renal function. Although many studies have been performed on the molecular mechanisms of renal handling of cimetidine, little is known about that of famotidine. In this study, to examine the recognition and transport of famotidine by human organic anion transporters (OATs; hOAT1, hOAT3) and human organic cation transporter (OCT; hOCT2), the uptake studies using Xenopus laevis oocytes were performed in comparison with cimetidine. The half-maximal inhibitory concentrations of famotidine for [ 3 H]estrone sulfate transport by hOAT3 and [ 14 C]tetraethylammonium transport by hOCT2 (300 μM and 1.8 mM, respectively) were higher than those of cimetidine (53 and 67 μM, respectively). While cimetidine inhibited p -[ 14 C]aminohippurate transport by hOAT1 in a concentration dependent manner, famotidine did not affect it at 5 mM. In addition, hOAT3 mediated famotidine uptake, but hOAT1 and hOCT2 did not show famotidine transport. These results indicate that there are marked differences between famotidine and cimetidine in the recognition and transport by organic ion transporters and that hOAT3 contributes to the renal tubular secretion of famotidine. Present findings should be useful information to understand the renal handling of famotidine and cimetidine.