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Determines the levels of acetylcholine (ACh), choline and dopamine (DA) in rat brain tissues after isatin administration to elucidate the role of isatin in regulating ACh levels. Description of isatin; ACh and DA concentrations in various brain regions before isatin administration to Wistar Kyoto rats; Effects of isatin microdialysis on ACh release from rat striatum.

We aimed to clarify the effect of nafamostat mesilate (nafamostat) on intestinal mucositis as well as the potentiation of intestinal 5-hydroxytryptamine (5-HT) dynamics induced by methotrexate, an anti-cancer drug, in rats. Rats received intraperitoneal methotrexate at 12.5 mg/kg/day for 4 days. In addition, 1, 3, or 10 mg/kg/day of nafamostat was given subcutaneously for 4 days. Ninety-six hours after the first administration of methotrexate, jejunal tissues were collected for analysis. The results showed that 1 mg/kg, but not 3 or 10 mg/kg, of nafamostat significantly ameliorated the methotrexate-induced body weight loss. Moreover, 1 mg/kg of nafamostat significantly improved methotrexate-induced mucositis, including villus atrophy. Nafamostat (1 mg/kg) significantly inhibited the methotrexate-induced mRNA expression of pro-inflammatory cytokines and cyclooxygenase-2, as well as methotrexate-induced 5-HT content and tryptophan hydroxylase (TPH) activity. In addition, it tended to inhibit the number of anti-TPH antibody-positive cells and significantly inhibited the number of anti-substance P antibody-positive cells. These findings suggest that low-dose nafamostat ameliorates tissue injury and 5-HT and substance P synthesis in methotrexate-induced mucositis. Nafamostat may be a novel therapeutic strategy for the prevention and treatment of mucositis as well as 5-HT- and/or substance P-related adverse effects in cancer chemotherapy., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B 2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B 2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B 2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B 2 was impaired, and intracellular levels of vitamin B 2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B 2 -dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B 2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The effects of cyclophosphamide on 5-hydroxytryptamine (5-HT) synthesis in the intestinal tissue of rats were investigated. Rats received 120 mg/kg cyclophosphamide intraperitoneally as a single administration, and kaolin and food intake was measured by an automatic monitoring apparatus. Ileal tissues were collected at either 24 or 72 h after administration. Cyclophosphamide caused a significant increase in kaolin intake at the acute and the delayed phases and was associated with a decrease in food intake, and body weight. Cyclophosphamide had no significant effect on intestinal mucosal morphology, or inducible nitric oxide synthase and cyclooxygenase-2 expression in the intestine. Cyclophosphamide significantly increased tryptophan hydroxylase 1 (TPH1) mRNA expression, number of anti-TPH antibody-positive cells, and 5-HT content in the intestine. Cyclophosphamide also significantly increased the expression of Tac1 mRNA, encoding preprotachykinin-1, which is a preprotein of substance P, and the number of anti-substance P antibody-positive cells in the intestine. Cyclophosphamide significantly increased Lgr5, Bmi1, and Atoh1 mRNA levels, which are markers for the proliferation and differentiation of stem cells. This study demonstrated that cyclophosphamide induced pica in rats, and potentiated 5-HT synthesis associated with hyperplasia of substance P-containing enterochromaffin cells without causing severe intestinal injury., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a common neurodegenerative disease that causes progressive cognitive decline. Deposition of amyloid-β (Aβ) peptides is the most important pathophysiological hallmark of AD. Oxidative stress induced by the generation of reactive oxygen species (ROS) is a prominent phenomenon in AD and is known to occur early in its course. Several reports have suggested a relationship between changes in redox status and AD pathology, including progressive Aβ deposition, glial cell activation, and inflammation. In the present study, we employed a newly designed three-dimensional continuous-wave digital electron paramagnetic resonance (EPR) imager with a blood-brain barrier (BBB)-permeable redox-sensitive piperidine nitroxide probe, 4-oxo-2,2,6,6-tetramethyl-piperidine-d 16 -1-oxyl, for early detection of changed brain redox status. Using this system, we noninvasively compared age-matched 7-month-old AD model mice with normal littermates (WT mice). The obtained brain redox images of AD and WT mice clearly showed impaired brain redox status of AD mice compared to WT, suggesting that oxidative damage had already increased in 7-month-old AD mice compared with age-matched WT mice. The pathological changes in 7-month-old mice in this study were detected earlier than in previous studies in which only AD mice older than 9 months of age could be imaged. Since EPR images suggested that oxidative damage was already increased in 7-month-old AD mice compared to age-matched WT mice, we also evaluated antioxidant levels and the activity of superoxide dismutase (SOD) in brain tissue homogenates of 7-month-old AD and WT mice. Compared to WT mice, decreased levels of glutathione and mitochondrial SOD activity were found in AD mice, which supports the EPR imaging results indicating impaired brain redox status. These results indicate that the EPR imaging method developed in this study is useful for early noninvasive detection of altered brain redox status due to oxidative disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Administration of cisplatin and methotrexate significantly increased 5-hydroxytryptamine (5-HT) release from intestinal tissues isolated at 72 h after administration in rats. Daily administration with nafamostat mesilate, a potent serine protease inhibitor, significantly inhibited the release of 5-HT induced by methotrexate, but not by cisplatin, in a dose-dependent manner. When applied to isolated ileal tissues in vitro, nafamostat mesilate also significantly inhibited the release of 5-HT induced by methotrexate, but not by cisplatin, in a concentration-dependent manner. These results suggest that serine proteases are involved in the mechanism of the methotrexate-induced release of 5-HT from the rat small intestine.

The role of nitric oxide (NO) in the changes in enterochromaffin cells and ileal 5-hydroxytryptamine (5-HT) content induced by a single i.p. administration of methotrexate was investigated in rats. Methotrexate significantly increased inducible NO synthase (iNOS) mRNA and protein expressions in the intestinal tissue at 96 h. Methotrexate also significantly caused hyperplasia of the enterochromaffin cells at 96 h; this was associated with a significant increase in 5-HT content. The methotrexate-induced hyperplasia of enterochromaffin cells and increase in 5-HT content were, however, completely suppressed by daily treatment with dexamethasone, and with N G -nitro-l-arginine methyl ester (l-NAME); this was not observed when meloxicam was administered. Histological examination showed slight but not pronounced mucosal injury, at 96 h after methotrexate administration. The methotrexate-induced decrease in body weight did not fully recover to the control level up to 96 h; however, the methotrexate-induced decrease in food/water intake slightly returned to the control level up to 96 h. l-NAME had no significant effect on methotrexate-induced body weight loss and anorexia. To conclude, the present study suggests that NO derived from methotrexate-induced iNOS plays a critical role in the mechanism of hyperplasia of enterochromaffin cells containing 5-HT in the intestinal tissue of rats., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

We previously reported that cisplatin potentiated ileal 5-hydroxytryptamine (5-HT) metabolism and caused pathological changes with an inflammatory response in the delayed phase (72 h) after administration to rats. In the present study, we further investigated the time-dependent effect of cisplatin on ileal 5-HT metabolism and the effects of combining cisplatin and anti-inflammatory drugs on ileal tryptophan hydroxylase expression and pica (the consumption of non-nutritive materials such as kaolin). Cyclooxygenase-2 (COX-2) expression was significantly increased at 24 h after cisplatin (5 mg/kg, intraperitoneal) administration. Cisplatin significantly increased ileal 5-HT content at 48 h after administration and the number of L-tryptophan hydroxylase-expressing cells (i.e., enterochromaffin cells) in the ileal mucosa within 24 h after administration. It also caused a significant increase in the number of substance P-expressing cells. Immunohistochemical double staining revealed that most of the enterochromaffin cells contained substance P. Neither daily treatment with dexamethasone (1 mg/kg, subcutaneous) nor meloxicam (3 mg/kg, subcutaneous), a selective COX-2 inhibitor, affected the cisplatin-induced increase in the number of enterochromaffin cells. Meloxicam had no effect on cisplatin-induced pica, although dexamethasone almost completely inhibited it. This study demonstrated that cisplatin administration induced COX-2 expression and increased the number of enterochromaffin cells in the acute phase (i.e., within 24 h). However, COX-2 expression in the ileum seems to have little direct effect on the mechanism of the induction of enterochromaffin cells and pica.

The effect of the addition of sequential C-terminal tryptophan residues on the fluorescence intensity of GFP was investigated. Tandem repeats of six tryptophan residues markedly decreased fluorescence intensity. This phenomenon is likely to occur because of the inhibition of GFP folding, resulting in insolubility. Exploiting this phenomenon, we constructed a cloning vector that facilitates the identification of recombinant colonies of Escherichia coli by the activation of GFP.

3-oxohexadecanoyl-CoA was synthesized for the study of D-bifunctional protein (EC 4. 2. 1. 107, EC 4. 2. 1. 119, EC 1. 1. 1. n12) and L-bifunctional protein (EC 4. 2. 1. 17, EC 5. 3. 3. 8, EC 1. 1. 1. 35). First, tetradecanal was subjected to the Reformatsky reaction with ethyl bromoacetate, and the product was then converted into ethyl 3-oxohexadecanoate. After acetalization of the 3-oxo ester with ethylene glycol, 3,3-ethlenedioxyhexadecanoic acid was obtained by alkaline hydrolysis. The acid was condensed with coenzyme A (CoA) by the mixed anhydride method, and the resulting CoA ester was deprotected with 4 M HCl to obtain 3-oxohexadecanoyl-CoA. In addition, the behavior of the CoA ester under several conditions of high-performance liquid chromatography (HPLC) was also investigated. We established separation detection of (R)-3-hydroxyhexadecanoyl-CoA, (S)-3-hydroxyhexadecaboyl-CoA, 3-oxohexadecanoyl-CoA, and trans-2-hexadecenoyl-CoA.

d-bifunctional protein (d-BP) deficiency is thought to lead to severe lipid metabolism disorders. To investigate the effect of naturally occurring missense mutations in the hydratase domain in d-BP, we constructed several d-BP hydratase variants and measured their activities. Missense mutations at sites whose conservation rates among 30 eukaryotes were < 70% did not affect hydratase activity. We predicted that missense mutations of highly conserved amino acids would markedly reduce activity. However, R562H and R562L, naturally occurring missense mutations of highly conserved amino acids, did not reduce activity. This result suggests that a missense mutation in a highly conserved amino acid does not always lead to severe lipid metabolism disorders. We also investigated the effect of G525V, which had been found in a mildly symptomatic patient with d-BP deficiency who was heterozygous for G525 and G658X. G525V markedly reduced hydratase activity. We had predicted that heterozygous G525V and G658X would lead to severely disordered lipid metabolism. However, the symptoms were inconsistent with this prediction. Characterizing mutations in the d-BP gene and the symptoms of d-BP deficiency may require pleiotropy, not only in vitro, studies.

The effects of methotrexate on 5-hydroxytryptamine (5-HT) metabolism in the intestinal tissue of rats were investigated during the delayed phase after a single administration. Rats were i.p. injected with methotrexate or with saline as a control, and kaolin and food intakes were measured by an automatic monitoring apparatus. At 96 h after administration, dissected-out ileal tissue was frozen rapidly in liquid nitrogen for further analysis or fixed for immunohistochemical staining. Methotrexate at a dose of 50 mg/kg caused a time-dependent increase in kaolin intake lasting up to 72 h after administration, which returned to the control level at 96 h after administration. This dose of methotrexate caused a gradual decrease in body weight, food intake, and water intake lasting up to 72 h, which approached the control level at 96 h. Methotrexate caused pathologic changes, including a moderate inflammatory response in the ileal tissue and an increase in the number of L-tryptophan hydroxylase (TPH)-expressing cells in the ileal mucosa. Methotrexate also caused a significant increase in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content and in TPH1 mRNA expression in the ileal tissues. It had no significant effects on mRNA expression of serotonin transporter, COX-1, or COX-2 or on myeloperoxidase activity. This study demonstrated, for the first time, that methotrexate caused a change in the ileal 5-HT metabolism associated with hyperplasia of mucosal enterochromaffin cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel cloning vector that can be used to identify recombinant Escherichia coli colonies by activation of the green fluorescent protein gene (GFP) was constructed. Screening using the vector does not require special reagents. The recombinant plasmid activates GFP, and the rate of false-positive results is low., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The nucleotide sequences of the proximal promoters of UDP-glucuronosyltransferase (UGT) 1A8 and 1A9 genes are very similar. However, UGT1A8 and 1A9 are mainly expressed in extra-hepatic and hepatic cells, respectively. Using mutants of UGT1A8 and 1A9 proximal promoters, we revealed their critical differences in terms of promoter activity and the role of the T-repeat region (T-region) conserved in both promoters. In extra-hepatic cells, Caco2, the activity of UGT1A9 proximal promoter increased to 73.4 ± 8.5% of that of the UGT1A8 proximal promoter with only 4 base changes: -160C, -152A, -62T, and -59G. The derivatives of the T-region showed that this region is not necessary for promoter activity, but the length of T repeats influences the activity somewhat. Therefore, the cause of the low activity of the UGT1A9 proximal promoter may be not only 4 base changes, but also the truncation of T repeats. From these results, the UGT1A9 proximal promoter was assumed to change into the non-active form from the original sequence, and this might be one of the reasons for the tissue-specific expression of UGT1A9.

In order to clarify the physiological significance of stereospecificities of peroxisomal multifunctional enzyme (MFE) type 1 (MFE1) and MFE2, we developed a chiral separation analysis for 3-hydroxyacyl-CoA using high performance liquid chromatography (HPLC) equipped with a chiral separation column. To demonstrate the utility of this technique, we cloned the hydratase domain from wild-type human MFE2 hydratase (MFE2Hwt) and expressed it as a GFP-tagged protein (GFP-MFE2Hwt) in Escherichia coli (E. coli). GFP-MFE2H was purified by diethylaminoethyl (DEAE) Sephacel from an E. coli sonication solution. As anticipated, we observed the formation of 3R-hydroxyhexadecanoyl-CoA (3R-OH-16-CoA) on the HPLC chromatogram after incubating trans-2-enoyl-CoA (16eno-CoA) with GFP-MFE2Hwt. GFP-MFE2Hwt was readily purifiable and could be assayed because of its traceability. We used site-directed mutagenesis to construct GFP-MFE2H variants corresponding to 17 reported MFE2H missense mutations and measured their hydratase activities using our HPLC method. Hydratase activity was completely lost or markedly decreased in the same variants corresponding to MFE2H mutations in patients with D-bifunctional protein (DBP) deficiency type II. On the other hand, the nonpathological variants did not markedly affect hydratase activity.

The intestinal absorption mechanism of domoic acid (DA) was investigated using Caco-2 cells. DA is a tricarboxylic amino acid that contains a glutamic acid moiety, and causes deficits in short-term memory by binding to glutamate receptors as an agonist of glutamic acid. Caco-2 cell monolayers cultured on permeable membranes were incubated with 100 μM DA on either the apical or basolateral side, and the transcellular transport of DA was measured. The transcellular transport of DA from the apical to basolateral side was about twofold that in the opposite direction. The transcellular transport of DA from the apical side was optimal at a neutral pH, and was temperature- and Cl(-)-dependent, but was Na(+)-independent. Coincubation of DA with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, significantly decreased the apical-to-basolateral transport of DA by 48%, and coincubation with probenecid (a non-specific anion transport inhibitor) significantly decreased the transport of DA by 31%. In contrast, coincubation with glutamic acid, succinic acid (a dicarboxylic acid), or citric acid (a tricarboxylic acid) did not decrease the transport of DA. These results suggest that the apical-to-basolateral transport of DA across the Caco-2 cell monolayers is mediated by DIDS-sensitive anion transporters., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of 2-enoyl-CoA to yield 3-hydroxyacyl-CoA in mitochondrial and peroxisomal β-oxidation. However, the stereospecificities of these hydratases differ from each other. To provide clear evidence of the stereospecificities of hydratases, the absolute configuration of 3-hydroxyhexadecanoyl-CoAs was determined, and they were subjected to a high-performance liquid chromatography (HPLC) using a chiral separation column. The retention time of 3(R)-hydroxyhexadecanoyl-CoA was shorter than that of 3(S)-hydroxyhexadecanoyl-CoA. The HPLC analysis carried out using a chiral separation column is considered to be useful for the study of enoyl-CoA hydratase.