Sequential hydrolysis of FAD by ecto-5' nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B2 into cells.

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 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 B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 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 B2 was impaired, and intracellular levels of vitamin B2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B2-dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

Low-dose nafamostat mesilate ameliorates tissue injury and inhibits 5-hydroxytryptamine synthesis in the rat intestine after methotrexate administration.

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.

Administration of cyclophosphamide to rats induces pica and potentiates 5-hydroxytryptamine synthesis in the intestine without causing severe intestinal injury.

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.

Role of Nitric Oxide in the Change of 5-Hydroxytryptamine Synthesis in the Intestine by a Consecutive Administration of Methotrexate to Rats.

This study aimed to investigate whether the consecutive administration of methotrexate affects 5-hydroxytryptamine (5-HT) synthesis in the rat small intestine. Rats received methotrexate at a dose of 12.5 mg/kg intraperitoneally on 4 consecutive days. NG-nitro-L-arginine methyl ester (L-NAME) was given subcutaneously to inhibit nitric oxide (NO) synthase. Methotrexate moderately altered 5-HT synthesis, whereas the combined administration of methotrexate and L-NAME significantly changed 5-HT synthesis in the rat ileal tissue. These results suggest that endogenous NO has an antagonistic role in the induction of 5-HT synthesis in rats following the consecutive administration of methotrexate.

Nitric oxide plays a critical role in methotrexate-induced hyperplasia of enterochromaffin cells containing 5-hydroxytryptamine in 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 NG-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.

Screening of recombinant Escherichia coli using activation of green fluorescent protein as an indicator.

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.

Early detection of redox imbalance in the APPswe/PS1dE9 mouse model of Alzheimer's disease by in vivo electron paramagnetic resonance imaging.

Alzheimer's disease (AD) is a common neurodegenerative disease that causes progressive cognitive decline. Deposition of amyloid-ß (Aß) 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 Aß 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-d16-1-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.

Application of "homogeneous assay for fluorescence concentrated on membrane" to the analysis of the substrate specificity of protease.

The utility of the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) in the analysis of the substrate specificity of protease was investigated using tobacco etch virus (TEV) protease. The V(max) of TEV protease against variants of a substrate was obtained by a simple procedure. It was considered that HAFCOM was more accurate than other endpoint measurements of protease assay.

Effects of nafamostat mesilate on 5-hydroxytryptamine release from isolated ileal tissues induced by anti-cancer drugs in rats.

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.

Beneficial effects of sarpogrelate hydrochloride, a 5-HT2A receptor antagonist, supplemented with pioglitazone on diabetic model mice.

OBJECTIVE: Feeding behavior control and dietetics with consequent weight reduction can be the most efficacious and fundamental methods to normalize fasting blood glucose. However, pioglitazone treatment has been found to incrementally increase body weight. In this study, we investigated whether the combined application of a 5-HT(2A) receptor antagonist, sarpogrelate, with pioglitazone can provide a clinical benefit. METHODS: Diabetic male KK-A(y) mice were randomly assigned to four groups: those receiving 10 mg/kg/day pioglitazone treatment for 30 days (pioglitazone group, n = 7), those receiving 30 mg/kg/day sarpogrelate treatment for 30 days (sarpogrelate group, n = 7), those receiving both agents for 30 days (pioglitazone + sarpogrelate group, n = 7) and those receiving no treatment (control group, n = 7). RESULTS: Feed intake was lower in the pioglitazone + sarpogrelate group than in the pioglitazone group. Water intake was also significantly lower in the pioglitazone, sarpogrelate and pioglitazone + sarpogrelate groups than in the control group. Combined application (pioglitazone + sarpogrelate) resulted in a 176% increase in leptin concentration compared with vehicle control. Body weight was significantly higher in the pioglitazone group, and there was a trend toward a smaller increment in body weight in the pioglitazone + sarpogrelate group. Mean values, calculated by multiplying insulin concentration and nonfasting glucose concentration, were significantly lower in the pioglitazone + sarpogrelate group than in the control group. CONCLUSIONS: These results suggest that the combined application of sarpogrelate with pioglitazone provides therapeutic benefits not only in preventing adverse effects but also in the treatment of diabetes.

Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk.

Green fluorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated "Cleave-Checker" on which GFP-SpA(B) (domain B in staphylococcal protein A) is immobilized. The SpA(B) region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpA(B) region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. The Cleave-Checker used for these methods was stable in a dry state, and long-term preservation for at least several months was possible.

Anti-inflammatory drugs ameliorate opposite enzymatic changes in ileal 5-hydroxytryptamine metabolism in the delayed phase after cisplatin administration to rats.

The effects of anti-inflammatory drugs on ileal 5-hydroxytryptamine (5-HT) metabolic dynamics at 72 h after a single administration of cisplatin were investigated in rats. Cisplatin 5 mg/kg i.p. caused pathological changes, with an inflammatory response occurring 72 h after its administration. The inflammatory response was associated with the induction of cyclooxygenase-2, but not cyclooxygenase-1, in the ileal mucosa at 72 h after the cisplatin administration. Daily treatment with meloxicam 3 mg/kg s.c. ameliorated the cisplatin-induced mucosal damage, whereas dexamethasone 1 mg/kg s.c. did not. Cisplatin administration also caused a significant increase in cyclooxygenase-2 mRNA expression at 72 h after administration, which was blunted by dexamethasone, but not by meloxicam. Cisplatin increased the content of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), but had no effect on 5-HT turnover (5-HIAA/5-HT ratio). Meloxicam and dexamethasone did not significantly decrease 5-HT and 5-HIAA content. Cisplatin significantly decreased monoamine oxidase activity but increased tryptophan hydroxylase (TPH) activity and TPH(1) mRNA expression in ileal tissue. Meloxicam and dexamethasone significantly restored the decreased monoamine oxidase activity and inhibited the cisplatin-induced increase in tryptophan hydroxylase activity toward the control levels. These drugs also decreased the cisplatin-induced increase in TPH(1) mRNA expression. Neither cisplatin nor the anti-inflammatory drugs had significant effect on mRNA expression of the serotonin re-uptake transporter. These results suggest that the inflammatory response associated with cyclooxygenase-2 induction is involved in the opposite change in ileal tryptophan hydroxylase and monoamine oxidase activities in the delayed phase after single administration of cisplatin to rats.

Effect of sequential C-terminal tryptophans on green fluorescent protein fluorescence.

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.

Cisplatin increases the number of enterochromaffin cells containing substance P in rat intestine.

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.

Synthesis and Detection by HPLC of 3-Oxohexadecanoyl-CoA for the Study of Peroxisomal Bifunctional Proteins.

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.

Pharmacological aspects of anticancer drug-induced emesis with emphasis on serotonin release and vagal nerve activity.

Cytotoxic drug-induced nausea and vomiting are the side effects most feared by cancer patients. Emesis is an instinctive defense reaction caused by the somatoautonomic nerve reflex, which is integrated in the medulla oblongata. Emesis caused by cytotoxic drugs such as cisplatin is associated with an increase in the concentration of 5-hydroxytryptamine (5-HT) in the intestine and the brainstem. It is proposed that cytotoxic drugs evoke 5-HT release from the enterochromaffin (EC) cells in the intestinal mucosa and that the released 5-HT stimulates the 5-HT receptors on the adjacent vagal afferent nerves. The depolarization of the vagal afferent nerves stimulates the vomiting center in the brainstem and eventually induces a vomiting reflex. 5-HT released from EC cells seems to mediate the cisplatin-induced emesis sensitive to 5-HT(3) receptor antagonists. The release of 5-HT from the EC cells, however, is regulated by polymodal mechanisms on autoreceptors or heteroreceptors. The precise role of 5-HT on the occurrence of vomiting has not been fully elucidated. The present review aims to describe the role of 5-HT in anticancer drug-induced emesis from the viewpoint of 5-HT release and afferent vagus nerve activity. Various methods for predicting emesis are also evaluated.

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity.

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.

Nicotine compensates for the loss of cholinergic function to enhance long-term potentiation induction.

Long-term potentiation (LTP) in the hippocampal CA1 region is widely regarded to be the cellular substrate of learning and memory, and its induction critically depends on the activation of N-methyl-D-aspartate receptors (NMDARs). Nicotine reverses memory deficits caused by a lesion of the cholinergic system in animals. The mechanisms underlying this effect and the effect of nicotine on LTP after cholinergic degeneration are unknown. Here we show that cholinergic lesions impaired the induction of LTP, and nicotine reversed this effect and promoted the induction of LTP. The compensatory action of nicotine appears to be due to the enhancement of NMDAR responses mediated by nicotine-induced disinhibition of pyramidal cells. This may represent the cellular basis of nicotine-mediated cognitive enhancement observed in the presence of cholinergic deficits.

Isatin, an endogenous MAO inhibitor, improves bradykinesia and dopamine levels in a rat model of Parkinson's disease induced by Japanese encephalitis virus.

Isatin, an endogenous monoamine oxidase (MAO) inhibitor, has an important role in the control of neurotransmitter concentration. We previously reported that exogenously administered isatin significantly increased acetylcholine (ACh) and dopamine (DA) levels in the rat striatum. In order to test the possibility of treating Parkinson's disease by isatin, we evaluated DA levels in the striatum and bradykinesia using a rat model of Parkinson's disease induced by the Japanese encephalitis virus (JEV).We have already reported that in adult Fischer rats infected with JEV at day 13, there was a marked decrease of tyrosine hydroxylase-positive neurons in the bilateral substantia nigra after 12 weeks. Effects of isatin were investigated in JEV-induced post-encephalitic parkinsonism rats by a pole test and high performance liquid chromatograph (HPLC) with an electrochemical detector (ECD). Isatin (100 mg/kg per day for 1 week, intraperitoneal injection) improved the bradykinesia observed in the JEV-induced parkinsonism rats. Dopamine (DA) concentrations in the JEV-infected rats were profoundly reduced in the striatum as compared with controls. Isatin also increased DA in the striatum of parkinsonism rats. These results suggest that isatin could be a possible treatment for Parkinson's disease as well as for post-encephalitic parkinsonism.

Reduced expressions of inducible nitric oxide synthase and cyclooxygenase-2 in vascular smooth muscle cells of stroke-prone spontaneously hypertensive rats.

Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are expressed in vascular smooth muscle cells stimulated with interleukin-1beta (IL-1beta), resulting in the production of nitric oxide (NO) and prostaglandins (PGs) such as PGI2. The iNOS and COX-2 proteins and their mRNA expressions in cultured vascular smooth muscle cells isolated from 6-7 week-old stroke-prone spontaneously hypertensive rats (SHRSP) were compared with those in the cells isolated from age-matched normotensive Wistar Kyoto rats (WKY). The IL-1beta-induced NO production and iNOS expression in vascular smooth muscle cells of SHRSP were significantly lower than those in cells of WKY. Similarly, PGI2 production and COX-2 expression were significantly lower in vascular smooth muscle cells of SHRSP than WKY, whereas there was no difference in the COX-1 expression. There were no significant differences in iNOS and COX-2 mRNA expressions between the two strains, suggesting that these protein expression may be reduced at the post-transcriptional level in cells of SHRSP. These results further suggest that the reduction of iNOS and COX-2 expressions in vascular smooth muscle cells may have relevance to the pathophysiology in SHRSP.