Protein S-nitrosylation is involved in valproic acid-promoted neuronal differentiation of adipose tissue-derived stem cells.

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is greatly promoted by valproic acid (VPA) with cAMP elevating agents thorough NO signaling pathways, but its mechanism is not fully understood. In the present study, we investigate the involvement of protein S-nitrosylation in the VPA-promoted neuronal differentiation of ASCs. The whole amount of S-nitrosylated protein was increased by the treatment with VPA alone for three days in ASCs. An inhibitor of thioredoxin reductase (TrxR), auranofin, further increased the amount of S-nitrosylated protein and enhances the VPA-promoted neuronal differentiation in ASCs. On the contrary, another inhibitor of TrxR, dinitrochlorobenzene, inhibited the VPA-promoted neuronal differentiation in ASCs even with cAMP elevating agents, which was accompanied by unexpectedly decreased S-nitrosylated protein. It was considered from these results that increased protein S-nitrosylation is involved in VPA-promoted neuronal differentiation of ASCs. By the proteomic analysis of S-nitrosylated protein in VPA-treated ASCs, no identified proteins could be specifically related to VPA-promoted neuronal differentiation. The identified proteins, however, included those involved in the metabolism of substances regulating neuronal differentiation, such as aspartate and glutamate.

Differential expression of desmin in the uterine myometrium and cervix as a possible mechanism for successful parturition in rats.

Expression of desmin, an intermediate filament, in the myometrium and cervix were investigated in peripartum rats (full term day 22 of pregnancy (DP22)). Des mRNA was expressed in lesser amounts in the cervix at peripartum (DP17 and 21, and day of birth 1 (DB1)), compared to those in the cervixes of ovariectomized rats. Immunohistochemical analysis revealed that desmin protein was diffusely present in the myometrium, and locally in the epithelium of the cervix. Western blot analysis showed that desmin protein levels in the myometrium increased 4- to 6-fold at DP17, 21 and DB1, and decreased rapidly at DB2 to the basal level observed in ovariectomized or non-pregnant rats. In contrast, cervical desmin protein levels increased approximately 10-fold at DP21 compared to those in ovariectomized rats, but decreased rapidly at DB1, indicating its decrease at parturition and an inconsistency between mRNA and protein expression. The administration of 17ß-estradiol to ovariectomized rats increased desmin protein levels in the myometrium and cervix after 24 h. S-nitrosylated desmin protein was detected in the myometrium and cervix at DP21. The mRNA expression of inducible nitric oxide synthase was consistent with the expression of desmin protein. Thus, desmin, which is regulated by estradiol, is differentially expressed in the myometrium and cervix at peripartum possibly for successful pregnancy and parturition. In the cervix, desmin protein expression seems to be regulated by estradiol at the translational level. S-nitrosylation of desmin may have a potential role in the peripartum uterus.

Complementary mRNA expression of enzymes producing nitric oxide and prostaglandin in ductus arteriosus with respect to their role in maintaining patency.

mRNA expression of molecules related to the activity of nitric oxide or prostaglandin E2, the critical regulators maintaining the ductus arteriosus patency, was examined in rat ductus arteriosus at preterm (days 18.5 and 19.5 of pregnancy) and near term (days 20.5 and 21.5). The endothelial nitric oxide synthase mRNA level increased transiently at preterm and then decreased at near term. The cyclooxygenase 2 mRNA increased gradually from near-term to the term complementary to the reduced endothelial nitric oxide synthase mRNA. These results suggest that the role shift between nitric oxide and prostaglandin E2 in maintaining ductus arteriosus patency at preterm and near term may be due to complementary expression changes of endothelial nitric oxide synthase and cyclooxygenase 2 at the transcriptional level.

Combination of T cell-redirecting bispecific antibody ERY974 and chemotherapy reciprocally enhances efficacy against non-inflamed tumours.

Identifying a strategy with strong efficacy against non-inflamed tumours is vital in cancer immune therapy. ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that recognizes glypican-3 and CD3. Here we examine the combination effect of ERY974 and chemotherapy (paclitaxel, cisplatin, and capecitabine) in the treatment of non-inflamed tumours in a xenograft model. ERY974 monotherapy shows a minor antitumour effect on non-inflamed NCI-H446 xenografted tumours, as infiltration of ERY974-redirected T cells is limited to the tumour-stromal boundary. However, combination therapy improves efficacy by promoting T cell infiltration into the tumour centre, and increasing ERY974 distribution in the tumour. ERY974 increases capecitabine-induced cytotoxicity by promoting capecitabine conversion to its active form by inducing thymidine phosphorylase expression in non-inflamed MKN45 tumour through ERY974-induced IFNγ and TNFα in T cells. We show that ERY974 with chemotherapy synergistically and reciprocally increases antitumour efficacy, eradicating non-inflamed tumours.

Reasons for medical students selecting a rural prefecture in Japan for initial clinical training: a single-center-based cross-sectional study.

Objective: Securing a sufficient number of medical residents to work in rural areas is an urgent issue. This study sought to clarify the factors that cause medical students at a rural university in Japan to select a particular place for their initial clinical training. Materials and Methods: A questionnaire was administered to all medical students at Saga University between February and March 2021. Participants were divided into two groups based on their training location choice: those who chose Saga Prefecture (Saga group) and those who selected other prefectures (non-Saga group). Then, logistic regression analysis was performed. Results: The questionnaire was answered by 300 students (46.3% response rate), of whom 291 agreed to participate in the study; 122 (41.9%) and 169 (58.1%) students were allocated to the Saga and non-Saga groups. Within the Saga group, the following factors were statistically significant: being admitted to Saga University's medical school through the system of special allotment of admission to applicants pledging to work in Saga Prefecture following graduation (or regional quota programs for admission) (odds ratio [OR], 19.18; 95% confidence interval [CI], 6.99-52.60); and being from Saga Prefecture (OR, 6.05; 95% CI, 2.24-16.35). With the non-Saga group, the desire to work in an urban area (OR, 0.03; 95% CI, 0.00-0.37) was statistically significant. Conclusion: To encourage medical residents to choose this prefecture for their initial clinical training, the focus should be on medical students who are from Saga Prefecture or admitted through the regional quota program.

Determination of starting dose of the T cell-redirecting bispecific antibody ERY974 targeting glypican-3 in first-in-human clinical trial.

Currently, ERY974, a humanized IgG4 bispecific T cell-redirecting antibody recognizing glypican-3 and CD3, is in phase I clinical trials. After a first-in-human clinical trial of an anti-CD28 agonist monoclonal antibody resulting in severe life-threatening adverse events, the minimal anticipated biological effect level approach has been considered for determining the first-in-human dose of high-risk drugs. Accordingly, we aimed to determine the first-in-human dose of ERY974 using both the minimal anticipated biological effect level and no observed adverse effect level approaches. In the former, we used the 10% effective concentration value from a cytotoxicity assay using the huH-1 cell line with the highest sensitivity to ERY974 to calculate the first-in-human dose of 4.9 ng/kg, at which maximum drug concentration after 4 h of intravenous ERY974 infusion was equal to the 10% effective concentration value. To determine the no observed adverse effect level, we conducted a single-dose study in cynomolgus monkeys that were intravenously infused with ERY974 (0.1, 1, and 10 µg/kg). The lowest dose of 0.1 µg/kg was determined as the no observed adverse effect level, and the first-in-human dose of 3.2 ng/kg was calculated, considering body surface area and species difference. For the phase I clinical trial, we selected 3.0 ng/kg as a starting dose, which was lower than the first-in-human dose calculated from both the no observed adverse effect level and minimal anticipated biological effect level. Combining these two methods to determine the first-in-human dose of strong immune modulators such as T cell-redirecting antibodies would be a suitable approach from safety and efficacy perspectives.Clinical trial registration: JapicCTI-194805/NCT05022927.

Hydrogen sulfide potently promotes neuronal differentiation of adipose tissue-derived stem cells involving nitric oxide-mediated signaling cascade with the aid of cAMP-elevating agents.

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is potently promoted by valproic acid (VPA) through a gaseous signaling molecule, nitric oxide (NO). Here, we investigated the involvement of hydrogen sulfide (H2S), another gaseous signaling molecule, in neuronal differentiation of ASCs. VPA-promoted neuronal differentiation of ASCs was accompanied by increased intracellular H2S and sulfane sulfur with increased mRNA expression of enzymes synthesizing sulfane sulfur including cystathionine ß-synthase (CBS), of which inhibition reduced the differentiation efficiency. H2S donors, GYY4137 (GYY) or NaHS, potently promoted neuronal differentiation of ASCs when cAMP-elevating agents, dibutyryl cyclic adenosine monophosphate and isobutyl methyl-xanthine, were added as neuronal induction medium (NIM). Neuronal differentiation of ASCs promoted by NaHS or GYY was accompanied by Ca2+ entry and increased mRNA expression of voltage-gated Ca2+ channels. NaHS or GYY also increased mRNA expression of enzymes of the NO-citrulline cycle including inducible NO synthase (iNOS). It was concluded from these results that H2S potently promoted differentiation of ASCs into neuronal cells expressing functional voltage-gated Ca2+ channels with the aid of cAMP-elevating agents, involving NO-mediated signaling cascade. These effects of H2S were also considered as a partial mechanism for the VPA-promoted neuronal differentiation of ASCs.

Valproic acid promotes differentiation of adipose tissue-derived stem cells to neuronal cells selectively expressing functional N-type voltage-gated Ca2+ channels.

The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca2+ entry. VPA (2 mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca2+ and the mRNAs of voltage-gated Ca2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca2+ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca2+ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity.

L-NAME, a nitric oxide synthase inhibitor, increases the protein expression of both executioner and inhibitor of apoptosis in the placental bed of mid-to-late pregnant rats.

The involvement of nitric oxide (NO) signaling in apoptosis was examined in the placental bed of mid-to-late pregnant rats. Pregnant rats were treated with l-NAME, a nitric oxide synthase inhibitor, by subcutaneous infusion for 48 hours before the examination at day 13.5, 17.5, or 21.5. l-NAME induced apoptosis in the placental bed to a limited extent at days 13.5 and 17.5, but not at day 21.5. When the placental bed was examined at day 17.5, the protein expression of both executioner (C-Cas3) and inhibitor (XIAP) of apoptosis was increased by l-NAME, but they did not co-localized with apoptosis. It was presumed that placental bed apoptosis induced by l-NAME is regulated through the expression of both executioner and inhibitor, possibly involving protein S-nitrosylation.

Valproic acid up-regulates the whole NO-citrulline cycle for potent iNOS-NO signaling to promote neuronal differentiation of adipose tissue-derived stem cells.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.

Valproic acid promotes mature neuronal differentiation of adipose tissue-derived stem cells through iNOS-NO-sGC signaling pathway.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing ßIII-tubulin protein, a neuronal marker, by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2 h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400 W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as ßIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS-NO-sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification.

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140-170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.

An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors.

Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker ßIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.

Valproic acid, a histone deacetylase inhibitor, decreases proliferation of and induces specific neurogenic differentiation of canine adipose tissue-derived stem cells.

Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.

Aqueous extracts of Rhizopus oryzae induced nitric oxide production in rat hepatocyte cell line RLN-10.

Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22(phox) and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.

Nitric oxide induces vascular endothelial growth factor expression in the rat placenta in vivo and in vitro.

We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

Nitric oxide production and its contribution to hepatocyte proliferation in normal juvenile rats.

Nitric oxide (NO) has been reported as a key mediator in enhancing hepatocyte proliferation during liver regeneration. Juvenile hepatocytes have a strong ability to proliferate while still in their undifferentiated state but the mechanism of NO production and its contribution to hepatocyte proliferation are not yet fully understood. The present study was designed to investigate NO production in the normal liver and its contribution to hepatocyte proliferation in juvenile rats. Endogenous NO production was evaluated quantitatively using a spin trap followed by electron paramagnetic resonance spectroscopy with the Fe-N, N-diethyldithiocarbamate complex as an NO-trapping reagent in the rat liver. NO production in the liver significantly peaked at 3 weeks after birth, but NO synthase (NOS) 3 expression did not change between 2 to 5 weeks after birth, while NOS 1 and NOS 2 mRNA were not detected. Hepatocyte proliferation, measured by the incorporation of 5-bromo-2'-deoxyuridine into the DNA, was found to decline significantly when endogenous NO production was inhibited by the administration of the NOS inhibitor N(G)-nitro- (L)-arginine methyl ester. These findings indicate that endogenous NO production peaked at 3 weeks after birth and hepatocyte proliferation declined significantly when NO production was inhibited. Thus, this study provides a novel insight into the contribution of NO to hepatic growth and liver maturation in juveniles.

[Analysis of blood concentrations following oral administration of beclomethasone dipropionate for gut GVHD].

In this study, we investigated the level of gut absorption following oral beclomethasone dipropionate (BDP) administration by measuring the blood concentration of its metabolites measured by LC-MS/MS using the HPLC method. Five patients who were administered BDP orally for gut GVHD were included. The blood concentrations of beclomethasone-17-monopropionate (17BMP), which is one of the active metabolites of BDP, were 618 approximately 1, 749 pg/mL in 4 of the studied 5 patients, which was comparable to that after inhalation of BDP; however, it was relatively higher in one patient (2,439+/-161 pg/mL). As the blood concentration of 17BMP in this study patient was higher compared with healthy volunteers administered a single oral BDP 4 mg, GVHD patients might have a higher concentration than healthy volunteers. Given that a higher grade of gut GVHD was associated with a higher blood level of 17BMP, BDP absorption might be associated with gut mucosal injury. Thus, the systemic adverse effect following oral BDP administration might not be negligible especially in gut GVHD patients.

Expression of genes responsible for biomineralization of Pinctada fucata during development.

This study compares the expression levels of nacrein, N16, MSI60, Prismalin-14, aspein and MSI31 genes during the ontogeny of Pinctada fucata. Several novel findings were obtained: 1) The early calcitic prismatic layer was distinguished as a thin membrane-like structure. 2) Initial formation of the nacreous layer started from the mantle pallial region at the age of 31days. 3) 18S rRNA of P. fucata was determined to be more suitable as a real-time PCR reference gene compared with GAPDH and beta-actin genes. 4) A relationship was recognized between the expression levels of the above six organic matrix genes and biomineralization of the larval shell. The lack of calcite in the shells of the veliger and pediveliger stages, when MSI31 and Prismalin-14 genes were expressed, makes a role of polymorph control by these genes less likely. The hypothetical involvement of N16 and MSI60 proteins in aragonitic nacreous layer formation was corroborated by the expression levels of N16 and MSI60 genes during ontogeny. Our results are important with respect to the control of CaCO(3) crystal polymorphism and shell microstructures in P. fucata.