Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV).

This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.

Analysis of Methamphetamine in Urine by HPLC with Solid-Phase Dispersive Extraction and Solid-Phase Fluorescence Derivatization.

We have developed a fluorescence detection-liquid chromatography (HPLC-FL) method that involves sample pretreatment by solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization for the simple and rapid analysis of methamphetamine (MA) in urine. This method uses a reversed-phase polymeric solid-phase gel to clean up analytes in SPDE, followed by fluorescence derivatization with 9-fluorenylmethyl chloroformate (FMOC) in the solid-phase. The optimal conditions for SPDE and solid-phase fluorescence derivatization were obtained when J-SPEC PEP was used as the solid-phase gel and 0.5 mmol/L FMOC in 50 mmol/L borate buffer solution (pH 10) was used as the fluorescence derivatization reagent. The recovery experiment of MA in urine yielded a clean chromatogram with no interfering peaks, demonstrating the validity of our method; the recoveries were 83.6% when spiked at a low concentration level (100 ng/mL) and 80.7% when spiked at a high concentration level (1000 ng/mL). Compared with the conventional liquid-phase method, the reaction product (FMOC-MA) of solid-phase fluorescence derivatization had higher stability. Reaction rate constants were calculated by changing the temperature conditions, and physicochemical parameters, including activation energy and activation entropy involved in the degradation reaction, were obtained from the Arrhenius plot and analyzed thermodynamically. Taken together, our results suggest that the HPLC-FL method with SPDE and solid-phase fluorescence derivatization for sample pretreatment provides a simple and rapid means of analyzing MA in urine samples.

Elucidation of Degradation Behavior of Hydroxy Group-Containing Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (III).

The degradation behavior of three benzodiazepines (BZPs)-lormetazepam (LMZ), lorazepam, and oxazepam-with hydroxy groups on the diazepine ring in artificial gastric juice and the effect of storage pH conditions on drug degradability were monitored using an LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. Although the three BZPs degraded in artificial gastric juice, none could be restored, despite increasing the storage pH, implying that the degradation reaction was irreversible. As for LMZ, we discussed the physicochemical parameters, such as the activation energy and activation entropy involved in the degradation reaction as well as the reaction kinetics; one of the degradation products was isolated and purified for structural analysis. In the LMZ degradation experiment, peaks corresponding to degradation products, (A) and (B), were detected through the LC/PDA measurements. Regarding the degradation behavior, we hypothesized that LMZ was degraded into (B) via (A), where (A) was an intermediate and (B) was the final product. Although the isolation of degradation product (A) was challenging, degradation product (B) could be isolated and was confirmed to be "methanone, [5-chloro-2-(methylamino)phenyl](2-chlorophenyl)-" based on structure determination using various instrumental analyses. The compound exhibited axis asymmetry as determined using single-crystal X-ray structure analysis. Because the formation of degradation product (B) was irreversible, it would be prudent to target the final degradation product (B) and LMZ for identification when detecting LMZ in human stomach contents, such as during forensic dissection.

Management of refractory chylothorax in the neonatal intensive care unit: A 22-year experience.

BACKGROUND: The aim was to assess the therapeutic strategy of patients with chylothorax in a neonatal intensive care unit. METHODS: Twenty-eight infants with chylothorax were included in this study. Their clinical characteristics and outcomes were reviewed retrospectively. RESULTS: The male-to-female ratio was 1:1. The mean gestational age and birthweight were 35.1 ± 3.5 weeks and 2,692 ± 791 g, respectively. Eighteen patients were diagnosed with congenital chylothorax; chylothorax occurred postoperatively in 10 patients. Chromosomal anomalies were diagnosed in 8 patients. Six patients received surgical therapy, such as pleurodesis, thoracic duct ligation, or lymphaticovenous anastomosis. Two patients required surgery due to resistance to pleurodesis. In surgically managed patients, the daily maximum amount of pleural effusion (mL)/bodyweight (kg) ratio was significantly larger than in non-surgically managed patients: 229.0 ± 180.5 versus 59.7 ± 49.2 mL/kg. In the receiver operating characteristic analysis of the daily maximum amount of pleural effusion/bodyweight ratio, the area under the curve was 0.889 when the cut-off value was 101 mL/kg, and the sensitivity was 0.8333 and the specificity was 0.8095 (P = 0.0059). CONCLUSIONS: Pleurodesis using OK432 could become a surgical first-line therapy for chylothorax even for neonates. It was important to initiate pleurodesis for refractory chylothorax at an earlier stage. A daily chylous effusion/bodyweight ratio of >101 mL/kg was a good predictor and seemed to be a useful parameter for prompt surgical intervention.

Elucidation of Degradation Behavior of Nitrazepam and Other Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (II).

The degradation behavior of eight benzodiazepines (BZPs): alprazolam, etizolam, diazepam, triazolam, nitrazepam (NZP), flunitrazepam (FNZ), bromazepam, and lorazepam, in artificial gastric juice was monitored by a LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. For drugs that were degradable, such physicochemical parameters as reaction rate constant were measured to evaluate the effect of storage conditions on drug degradability, such as whether the degradation proceeds faster by increasing storage temperature, or whether the degradation reaction is reversible by adjusting pH. As a result, it was confirmed that although the eight BZPs degraded in artificial gastric juice, most of them could be restored when pH was increased, and the restoration rates differed depending on the pH and the type of BZP. As for NZP, an Arrhenius plot was drawn to obtain the physicochemical parameters, such as activation energy and activation entropy involved in the degradation reaction, and the reaction kinetics was discussed. In addition, two substances were confirmed as the degradation products of NZP in artificial gastric juice: one was a reversible degradation product (A) (intermediate) and the other was an irreversible degradation product (B) (final degradation product). The intermediate was identified as 2-amino-N-(2-benzoyl-4-nitrophenyl)-acetamide, and the final degradation product was 2-amino-5-nitrobenzophenone. Therefore, when detecting NZP in human stomach contents, such as during judicial dissection, it would be prudent to target NZP as well as the intermediate (A) and the final degradation product (B).

A novel irreversible FLT3 inhibitor, FF-10101, shows excellent efficacy against AML cells with FLT3 mutations.

An activating mutation of Fms-like tyrosine kinase 3 (FLT3) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication (FLT3-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3-ITD or FLT3-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.

Identification of a Novel Bone Marrow Cell-Derived Accelerator of Fibrotic Liver Regeneration Through Mobilization of Hepatic Progenitor Cells in Mice.

Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.

Elucidation of Degradation Behavior of Tricyclic Antidepressant Amoxapine in Artificial Gastric Juice.

The degradation behavior of eight tricyclic antidepressants (TCAs; amitriptyline, amoxapine (AMX), imipramine, clomipramine, desipramine, doxepin, dothiepin, and nortriptyline) in artificial gastric juice was investigated to estimate their pharmacokinetics in the stomach. As a result, among the eight TCAs, only AMX was degraded in artificial gastric juice. The degradation was a pseudo first-order reaction; activation energy (Ea) was 88.70 kJ/mol and activation entropy (ΔS) was -80.73 J/K·mol. On the other hand, the recovery experiment revealed that the degradation product did not revert to AMX and accordingly, this reaction was considered to be irreversible. In the AMX degradation experiment, peaks considered to be degradation products A (I) and B (II) were detected at retention times of around 3 min and 30 min in LC/UV measurements, respectively. Structural analysis revealed that compound (I) was [2-(2-aminophenoxy)-5-chlorophenyl]-piperazin-1-yl-methanone, a new compound, and compound (II) was 2-chlorodibenzo[b,f][1,4]oxazepin-11(10H)-one. As for the degradation behavior, it was estimated that AMX was degraded into (II) via (I), i.e., (II) was the final product. The results are expected to be useful in clinical chemistry and forensic science, including the estimation of drugs to be used at the time of judicial dissection and suspected drug addiction.

Abnormal axon guidance signals and reduced interhemispheric connection via anterior commissure in neonates of marmoset ASD model.

In autism spectrum disorder (ASD), disrupted functional and structural connectivity in the social brain has been suggested as the core biological mechanism underlying the social recognition deficits of this neurodevelopmental disorder. In this study, we aimed to identify genetic and neurostructural abnormalities at birth in a non-human primate model of ASD, the common marmoset with maternal exposure to valproic acid (VPA), which has been reported to display social recognition deficit in adulthood. Using a comprehensive gene expression analysis, we found that 20 genes were significantly downregulated in VPA-exposed neonates. Of these, Frizzled3 (FZD3) and PIK3CA were identified in an axon guidance signaling pathway. FZD3 is essential for the normal development of the anterior commissure (AC) and corpus callosum (CC); hence, we performed diffusion tensor magnetic resonance imaging with a 7-Tesla scanner to measure the midsagittal sizes of these structures. We found that the AC size in VPA-exposed neonates was significantly smaller than that in age-matched controls, while the CC size did not differ. These results suggest that downregulation of the genes related to axon guidance and decreased AC size in neonatal primates may be linked to social brain dysfunctions that can happen later in life.

Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration.

Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.

Epidermal growth factor receptor/heme oxygenase-1 axis is involved in chemoresistance to cisplatin and pirarubicin in HepG2 cell lines and hepatoblastoma specimens.

PURPOSE: To investigate the possibility that the antioxidant stress protein Heme oxygenase-1 (HO-1) is involved in the acquisition of chemoresistance in cisplatin and pirarubicin (CITA) therapy. METHODS: Human hepatoblastoma-derived cell line (HepG2) was used to generate a knockdown cell line of HO-1 by small interfering RNA (siRNA). Expression of HO-1, epidermal growth factor receptor (EGFR), Akt, and extracellular signal-regulated kinase1/2 (ERK1/2) was examined by Western blot. The cytotoxic effect of cisplatin, pirarubicin, and EGFR inhibitor was examined by trypan blue staining. In human hepatoblastoma specimens (n = 5), changes of HO-1 expression were examined immunohistochemically before and after CITA therapy. RESULTS: HO-1 expression in HepG2 cells was increased by the treatment of cisplatin (CDDP) and pirarubicin (THP) dose-dependently. In HO-1 knockdown HepG2 cells, the HO-1 was not expressed and the percentage of trypan blue-positive cells (dead cells) was significantly increased after treatment of CDDP and THP. The EGFR inhibitor decreased the levels of HO-1, phospho-Akt and phospho-ERK1/2 in HepG2 cells. Combination treatment of EGFR inhibitor with CDDP and THP increased the cytotoxic effect in HepG2 cells. In human hepatoblastoma specimens, 4 of the 5 patients (80%) showed HO-1 expression changed much stronger in the viable tumor cells after CITA therapy. CONCLUSION: The cytotoxic effects of CDDP and THP were both enhanced under HO-1 knockdown conditions as well as under conditions that inhibit the activation pathway of HO-1 by EGFR inhibitors. EGFR/HO-1 axis may be involved in acquiring chemoresistance in HepG2 cell lines as well as in human hepatoblastoma.

[LC/Tribrid Orbitrap Analysis of Phosphodiesterase-5 Inhibitors and Their Analogs as Adulterants in Dietary Supplements].

LC/Tribrid Orbitrap was developed to determine phosphodiesterase-5 (PDE-5) inhibitors and their analogs as adulterants in dietary supplements. High-resolution MS/MS and MS3 spectra of PDE-5 inhibitors and their analogs were obtained by LC/Tribrid Orbitrap using both higher-energy collisional dissociation and collision-induced dissociation. We investigated dietary supplements that claim to enhance men's sexual performance, and detected PDE-5 inhibitors and their analogs. We also estimated the structures of the PDE-5 inhibitor analogs and the impurities of PDE-5 inhibitors and their analogs in the dietary supplements.

Evidence of exposure to chemicals and heavy metals during pregnancy in Japanese women.

Purpose: Prenatal exposure to environmental chemicals is a growing concern, because such exposures have been shown to be associated with various diseases. The levels of chemicals and heavy metals in maternal blood, cord blood, maternal urine and amniotic fluid in Japanese pregnant women were investigated. Methods: A total of 145 women, including 14 fetal growth restriction cases, were included in the present study. The levels of phthalates (di[2-ethylhexyl]phthalate and mono[2-ethylhexyl]phthalate), perfluorinated compounds (perfluorooctane sulfonate, perfluorohexanoic acid, perfluorooctanoic acid, and perfluorononanoic acid), pesticides (dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, 3-phenoxybenzoic acid, and octachlorodipropyl ether), bisphenol A, nicotine (nicotine, nornicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine), polybrominated diphenyl ethers, and heavy metals were measured. The relationship between fetal growth and the levels of chemicals and heavy metals were investigated. Results: Phthalates, perfluorinated compounds, pesticides, polybrominated diphenyl ethers, and heavy metals were detected in high frequency, whereas nicotine and bisphenol A were almost negative. Phthalates, perfluorinated compounds, and several heavy metals were transferred to the fetus. High perfluorononanoic acid levels in the maternal blood and cord blood, and low perfluorooctanoic acid level in the cord blood were significantly and negatively associated with fetal growth. Conclusions: The present study showed that pregnant women in Japan and their fetuses are exposed to a variety of chemicals and heavy metals.

An improved model of predicting hepatocarcinogenic potential in rats by using gene expression data.

Carcinogenicity studies using animals are expensive and time consuming. Therefore, the development of a highly accurate carcinogenicity prediction system to interpret short-term test results would be beneficial. The Ames test is popular for mutagens; however, it cannot detect non-genotoxic carcinogens. Previously, we reported a prediction system using gene expression data obtained from a short-term (28-day) study that screened candidate compounds for testing in long-term carcinogenicity studies. In this study, our system was improved by adding more gene expression data. To establish our new system, we used the data of 93 test compounds (41 hepatocarcinogens and 52 non-hepatocarcinogens). Analysis of liver gene expression data by dividing compounds into 'for training' and 'for test' categories (20 cases assigned randomly) using Support Vector Machine (SVM) identified a set of marker probe sets that could be used to predict hepatocarcinogenicity. The assigned 42 probe sets have included the cancer- or c-Myc-related genes such as Hsp90, Pink1, Hspc111, Fbx29, Hepsin, Syndecan2 and Synbindin. Compared with the older version, the improved system had a higher concordance rate with the training data and a good performance with the external test data.

Clinical and structural remission rates increased annually and radiographic progression was continuously inhibited during a 3-year administration of tocilizumab in patients with rheumatoid arthritis: A multi-center, prospective cohort study by the Michinoku Tocilizumab Study Group.

OBJECTIVE: To evaluate the clinical and structural efficacy of tocilizumab (TCZ) during its long-term administration in patients with rheumatoid arthritis (RA). METHODS: In total, 693 patients with RA who started TCZ therapy were followed for 3 years. Clinical efficacy was evaluated by DAS28-ESR and Boolean remission rates in 544 patients. Joint damage was assessed by calculating the modified total Sharp score (mTSS) in 50 patients. RESULTS: When the reason for discontinuation was limited to inadequate response or adverse events, the 1-, 2-, and 3-year continuation rates were 84.0%, 76.8%, and 72.2%, respectively. The mean DAS28-ESR was initially 5.1 and decreased to 2.5 at 6 months and to 2.2 at 36 months. The Boolean remission rate was initially 0.9% and increased to 21.7% at 6 months and to 32.2% at 36 months. The structural remission rates (ΔmTSS/year ≤ 0.5) were 68.8%, 78.6%, and 88.9% within the first, second, and third years, respectively. The structural remission rate at 3 years (ΔmTSS ≤ 1.5) was 66.0%, and earlier achievement of swollen joint count (SJC) of 1 or less resulted in better outcomes. CONCLUSIONS: TCZ was highly efficacious, and bone destruction was strongly prevented. SJC was an easy-to-use indicator of joint destruction.

Expression of the clustered NeuAcα2-3Galß O-glycan determines the cell differentiation state of the cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galß O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Developmental genetic profiles of glutamate receptor system, neuromodulator system, protector of normal tissue and mitochondria, and reelin in marmoset cortex: potential molecular mechanisms of pruning phase of spines in primate synaptic formation process during the end of infancy and prepuberty (II).

This is the second report of a series paper, which reports molecular mechanisms underlying the occurrence of pruning spine phase after rapid spinogenesis phase in neonates and young infant in the primate brain. We performed microarray analysis between the peak of spine numbers [postnatal 3 months (M)] and spine pruning (postnatal 6M) in prefrontal, inferior temporal, and primary visual cortices of the common marmoset (Callithrix jacchus). The pruning phase is not clearly defined in rodents but is in primates including the marmoset. The differentially expressed genes between 3M and 6M in all three cortical areas were selected by two-way analysis of variance. The list of selected genes was analyzed by canonical pathway analysis using "Ingenuity Pathway Analysis of complex omics data" (IPA; Ingenuity Systems, Qiagen, Hilden, Germany). In this report, we discuss these lists of genes for the glutamate receptor system, G-protein-coupled neuromodulator system, protector of normal tissue and mitochondria, and reelin. (1) Glutamate is a common neurotransmitter. Its receptors AMPA1, GRIK1, and their scaffold protein DLG4 decreased as spine numbers decreased. Instead, GRIN3 (NMDA receptor) increased, suggesting that strong NMDA excitatory currents may be required for a single neuron to receive sufficient net synaptic activity in order to compensate for the decrease in synapse. (2) Most of the G protein-coupled receptor genes (e.g., ADRA1D, HTR2A, HTR4, and DRD1) in the selected list were upregulated at 6M. The downstream gene ROCK2 in these receptor systems plays a role of decreasing synapses, and ROCK2 decreased at 6M. (3) Synaptic phagosytosis by microglia with complement and other cytokines could cause damage to normal tissue and mitochondria. SOD1, XIAP, CD46, and CD55, which play protective roles in normal tissue and mitochondria, showed higher expression at 6M than at 3M, suggesting that normal brain tissue is more protected at 6M. (4) Reelin has an important role in cortical layer formation. In addition, RELN and three different pathways of reelin were expressed at 6M, suggesting that new synapse formation decreased at that age. Moreover, if new synapses were formed, their positions were free and probably dependent on activity.

Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I).

The synapse number and the related dendritic spine number in the cerebral cortex of primates shows a rapid increase after birth. Depending on the brain region and species, the number of synapses reaches a peak before adulthood, and pruning takes place after this peak (overshoot-type synaptic formation). Human mental disorders, such as autism and schizophrenia, are hypothesized to be a result of either too weak or excessive pruning after the peak is reached. Thus, it is important to study the molecular mechanisms underlying overshoot-type synaptic formation, particularly the pruning phase. To examine the molecular mechanisms, we used common marmosets (Callithrix jacchus). Microarray analysis of the marmoset cortex was performed in the ventrolateral prefrontal, inferior temporal, and primary visual cortices, where changes in the number of dendritic spines have been observed. The spine number of all the brain regions above showed a peak at 3 months (3 M) after birth and gradually decreased (e.g., at 6 M and in adults). In this study, we focused on genes that showed differential expression between ages of 3 M and 6 M and on the differences whose fold change (FC) was greater than 1.2. The selected genes were subjected to canonical pathway analysis, and in this study, we describe axon guidance signaling, which had high plausibility. The results showed a large number of genes belonging to subsystems within the axon guidance signaling pathway, macrophages/immune system, glutamate system, and others. We divided the data and discussion of these results into 2 papers, and this is the first paper, which deals with the axon guidance signaling and macrophage/immune system. Other systems will be described in the next paper. Many components of subsystems within the axon guidance signaling underwent changes in gene expression from 3 M to 6 M so that the synapse/dendritic spine number would decrease at 6 M. Thus, axon guidance signaling probably contributes to the decrease in synapse/dendritic spine number at 6 M, the phenomenon that fits the overshoot-type synaptic formation in primates. Microglial activity (evaluated by quantifying AIF1 expression) and gene expression of molecules that modulate microglia, decreased at 6 M, just like the synapse/dendritic spine number. Thus, although microglial activity is believed to be related to phagocytosis of synapses/dendritic spines, microglial activity alone cannot explain how pruning was accelerated in the pruning phase. On the other hand, expression of molecules that tag synapses/dendritic spines as a target of phagocytosis by microglia (e.g., complement components) increased at 6 M, suggesting that these tagging proteins may be involved in the acceleration of pruning during the pruning phase.

Identification of indicator components for the discrimination of Cassia plants in health teas and development of analytical method for the components.

Components that could be used as indicators for the discrimination of senna (Cassia angustifolia) from other cassia plants contained in health teas were identified, and an analytical method for the components was developed. Our results revealed two components in senna that were not found in other Cassia spp. widely used in health teas, such as C. alata, C. corymbosa, C. obtusifolia, and C. occidentalis. Structural elucidation of the two components showed that they were isorhamnetin-3-O-gentiobioside and tinnevellin glucoside. We analyzed commercial health teas using the HPLC method developed in this study. The two indicator components were detected at 366 nm using an RP C18 column and gradient elution with a mixture of water and acetonitrile (with formic acid), as the mobile phase. Our analytical method by HPLC enabled the differentiation of senna from other Cassia plants present in health teas in which sennosides A and B were detected. Moreover, this method allowed us to predict the parts of senna in health teas from the amounts of isorhamnetin-3-O-gentiobioside and tinnevellin glucoside contained in the teas.

An improved Simon reaction method to discriminate between methamphetamine and false-positive substances.

The conventional methamphetamine (MA) detection method using the Simon reaction can be affected by false positives owing to compounds similar to aliphatic secondary amines. In this study, we examined the new Simon reaction to improve the qualitative accuracy of MA detection to discriminate substances that give false positives in a conventional Simon reaction. After the conventional Simon reaction for MA and false positives (N-isopropylbenzylamine (NIP-BA), N-methylbenzylamine (NMe-BA), L-proline (Pro), and L-hydroxyproline (HYP)), which are colored blue, di-tert-butyl dicarbonate (t-Boc) reagent was added, and color tone changes were observed. When t-Boc was added to the false positives (NIP-BA, NMe-BA, Pro, and HYP), the colors of MA, Pro, and HYP changed to purple; NIP-BA changed to blue; and NMe-BA changed to light pink after 3 min. These results suggested that MA can be differentiated from NIP-BA and NMe-BA. Furthermore, the solid-phase chromogenic method was examined, and it was confirmed that MA could be differentiated from Pro and HYP. The method developed in this study should increase the accuracy of MA appraisal at crime scenes and contribute to the reduction of misclassifications arising from false-positive substances.