Clinical characteristics and pathophysiological properties of newly discovered LRRK2 variants associated with Parkinson's disease.

Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein domains, including armadillo repeat, ankyrin repeat, leucine-rich repeat (LRR), Ras-of-complex (ROC), C-terminal of ROC (COR), kinase, and WD40 domains. In this study, we performed genetic screening of LRRK2 in our PD cohort, detecting sixteen LRRK2 rare variants. Among them, we selected seven variants that are likely to be familial and characterized them in terms of LRRK2 protein function, along with clinical information and one pathological analysis. The seven variants were S1120P and N1221K in the LRR domain; I1339M, S1403R, and V1447M in the ROC domain; and I1658F and D1873H in the COR domain. The kinase activity of the LRRK2 variants N1221K, S1403R, V1447M, and I1658F toward Rab10, a well-known phosphorylation substrate, was higher than that of wild-type LRRK2. LRRK2 D1873H showed enhanced self-association activity, whereas LRRK2 S1403R and D1873H showed reduced microtubule-binding activity. Pathological analysis of a patient with the LRRK2 V1447M variant was also performed, which revealed Lewy pathology in the brainstem. No functional alterations in terms of kinase activity, self-association activity, and microtubule-binding activity were detected in LRRK2 S1120P and I1339M variants. However, the patient with PD carrying LRRK2 S1120P variant also had a heterozygous Glucosylceramidase beta 1 (GBA1) L444P variant. In conclusion, we characterized seven LRRK2 variants potentially associated with PD. Five of the seven variants in different LRRK2 domains exhibited altered properties in kinase activity, self-association, and microtubule-binding activity, suggesting that each domain variant may contribute to disease progression in different ways.

α-Synuclein V15A Variant in Familial Parkinson's Disease Exhibits a Weaker Lipid-Binding Property.

BACKGROUND: The α-Synuclein (α-Syn) V15A variant has been found in two Caucasian families with Parkinson's disease (PD). However, the significance of this missense variant remained unclear. OBJECTIVE: We sought to elucidate whether V15A could increase aggregation or change phospholipid affinity. METHODS: A sequencing analysis for the SNCA encoding α-Syn from 875 patients with PD and 324 control subjects was performed. Comparing with known pathogenic missense variants of α-Syn, A30P, and A53T, we analyzed the effects of V15A on binding to phospholipid membrane, self-aggregation, and seed-dependent aggregation in cultured cells. RESULTS: Genetic screening identified SNCA c.44 T>C (p.V15A) from two Japanese PD families. The missense variant V15A was extremely rare in several public databases and predicted as pathogenic using in silico tools. The amplification activity of α-Syn V15A fibrils was stronger than that of wild-type α-Syn fibrils. CONCLUSIONS: The discovery of the V15A variant from Japanese families reinforces the possibility that the V15A variant may be a causative variant for developing PD. V15A had a reduced affinity for phospholipids and increased propagation activity compared with wild-type. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A Novel LRRK2 Variant p.G2294R in the WD40 Domain Identified in Familial Parkinson's Disease Affects LRRK2 Protein Levels.

Leucine-rich repeat kinase 2 (LRRK2) is a major causative gene of late-onset familial Parkinson's disease (PD). The suppression of kinase activity is believed to confer neuroprotection, as most pathogenic variants of LRRK2 associated with PD exhibit increased kinase activity. We herein report a novel LRRK2 variant-p.G2294R-located in the WD40 domain, detected through targeted gene-panel screening in a patient with familial PD. The proband showed late-onset Parkinsonism with dysautonomia and a good response to levodopa, without cognitive decline or psychosis. Cultured cell experiments revealed that p.G2294R is highly destabilized at the protein level. The LRRK2 p.G2294R protein expression was upregulated in the patient's peripheral blood lymphocytes. However, macrophages differentiated from the same peripheral blood showed decreased LRRK2 protein levels. Moreover, our experiment indicated reduced phagocytic activity in the pathogenic yeasts and α-synuclein fibrils. This PD case presents an example wherein the decrease in LRRK2 activity did not act in a neuroprotective manner. Further investigations are needed in order to elucidate the relationship between LRRK2 expression in the central nervous system and the pathogenesis caused by altered LRRK2 activity.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Impaired function of aorta and perivascular adipose tissue in IL-18-deficient mice.

IL-18 is ubiquitously produced by both hematopoietic and non-hematopoietic cells. The present study examined the thoracic aorta, including the surrounding perivascular adipose tissue (PVAT), of IL-18KO mice from functional and histological perspectives. IL-18KO mice exhibited raised blood pressure compared with wild-type mice. Echocardiographic examination showed a thickened vascular wall and narrowed vascular diameter of the aorta. Examination by the Magnus test demonstrated dysfunction of endothelial cells (ECs) in the IL-18KO thoracic aorta and impairment of the anticontractile function of IL-18KO PVAT. Histological examination showed no inflammatory lesions in the aorta but indicated progressive fibrosis in the vessel and conversion of PVAT from brown adipose tissue-like features to white adipose tissue-like features. Electron microscopic observation suggested several deformed mitochondria in the aorta and vacuole-like structures in ECs from IL-18KO mice. In addition, activity of complex IV was lower and production of reactive oxygen species was augmented in the mitochondria of IL-18KO aorta. Although expression of LC3 B was higher, rapamycin-induced autophagy flux was impaired in the IL-18KO PVAT. Moreover, Western blot analysis revealed that LAMP 1/2 was increased in IL-18KO PVAT, and measurement of cathepsin-D activity indicated decreased levels in IL-18KO PVAT. The IL-18KO thoracic aorta thus showed defects in physiological functions related to histological alterations, and the inflammasome/IL-18 system was suggested to play a protective role in cardiovascular cells, probably through quality control of mitochondria via promotion of autophagosome/autophagolysosome formation.NEW & NOTEWORTHY IL-18 deficiency caused aortic abnormalities in terms of morphology and functions in parallel with an accumulation of damaged mitochondria and anomalous turnover of protein complexes, such as PGC-1 and LAMP1 and -2 in PVAT. These findings suggested that IL-18 plays roles in maintaining the homeostasis of vessels and PVAT around the aorta, possibly by promoting autophagy.

TENOR: Database for Comprehensive mRNA-Seq Experiments in Rice.

Here we present TENOR (Transcriptome ENcyclopedia Of Rice, http://tenor.dna.affrc.go.jp), a database that encompasses large-scale mRNA sequencing (mRNA-Seq) data obtained from rice under a wide variety of conditions. Since the elucidation of the ability of plants to adapt to various growing conditions is a key issue in plant sciences, it is of great interest to understand the regulatory networks of genes responsible for environmental changes. We used mRNA-Seq and performed a time-course transcriptome analysis of rice, Oryza sativa L. (cv. Nipponbare), under 10 abiotic stress conditions (high salinity; high and low phosphate; high, low and extremely low cadmium; drought; osmotic; cold; and flood) and two plant hormone treatment conditions (ABA and jasmonic acid). A large number of genes that were responsive to abiotic stresses and plant hormones were detected by differential expression analysis. Furthermore, several responsive genes were found to encode transcription factors that could control the transcriptional network of stress responses, but the timing of the induction of these genes was not uniform across conditions. A significant number of cis-regulatory elements were enriched in the promoter regions of the responsive genes and were shared among conditions. These data suggest that some key components of gene regulation networks are shared between different stress signaling pathways. All the resources (novel genes identified from mRNA-Seq data, expression profiles, co-expressed genes and cis-regulatory elements) can be searched for and are available in TENOR.

Novel functional small RNAs are selectively loaded onto mammalian Ago1.

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1-4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein-Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1-4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

Genomic inversion caused by gamma irradiation contributes to downregulation of a WBC11 homolog in bloomless sorghum.

Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight-a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F2 bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.

Global transcriptome analysis reveals distinct expression among duplicated genes during sorghum-interaction.

BACKGROUND: Sorghum (Sorghum bicolor L. Moench) is a rich source of natural phytochemicals. We performed massive parallel sequencing of mRNA to identify differentially expressed genes after sorghum BTx623 had been infected with Bipolaris sorghicola, a necrotrophic fungus causing a sorghum disease called target leaf spot. RESULT: Seventy-six-base-pair reads from mRNAs of mock- or pathogen-infected leaves were sequenced. Unannotated transcripts were predicted on the basis of the piling-up of mapped short reads. Differentially expressed genes were identified statistically; particular genes in tandemly duplicated putative paralogs were highly upregulated. Pathogen infection activated the glyoxylate shunt in the TCA cycle; this changes the role of the TCA cycle from energy production to synthesis of cell components. The secondary metabolic pathways of phytoalexin synthesis and of sulfur-dependent detoxification were activated by upregulation of the genes encoding amino acid metabolizing enzymes located at the branch point between primary and secondary metabolism. Coordinated gene expression could guide the metabolic pathway for accumulation of the sorghum-specific phytochemicals 3-deoxyanthocyanidin and dhurrin. Key enzymes for synthesizing these sorghum-specific phytochemicals were not found in the corresponding region of the rice genome. CONCLUSION: Pathogen infection dramatically changed the expression of particular paralogs that putatively encode enzymes involved in the sorghum-specific metabolic network.

Pathophysiological evaluation of the LRRK2 G2385R risk variant for Parkinson's disease.

Missense variants in leucine-rich repeat kinase 2 (LRRK2) lead to familial and sporadic Parkinson's disease (PD). The pathological features of PD patients with LRRK2 variants differ. Here, we report an autopsy case harboring the LRRK2 G2385R, a risk variant for PD occurring mainly in Asian populations. The patient exhibited levodopa-responsive parkinsonism at the early stage and visual hallucinations at the advanced stage. The pathological study revealed diffuse Lewy bodies with neurofibrillary tangles, amyloid plaques, and mild signs of neuroinflammation. Biochemically, detergent-insoluble phospho-α-synuclein was accumulated in the frontal, temporal, entorhinal cortexes, and putamen, consistent with the pathological observations. Elevated phosphorylation of Rab10, a substrate of LRRK2, was also prominent in various brain regions. In conclusion, G2385R appears to increase LRRK2 kinase activity in the human brain, inducing a deleterious brain environment that causes Lewy body pathology.

Plant biochemistry: anthocyanin biosynthesis in roses.

Anthocyanin is the principal pigment in flowers, conferring intense red-to-blue cyanic colours on petals and helping to attract pollinators. Its biosynthesis involves glycosylation steps that are important for the stability of the pigment and for its aqueous solubility in vacuoles. Here we describe anthocyanin biosynthesis in roses (Rosa hybrida), which is unlike the pathway used in other flowers in that it relies on a single enzyme to achieve glycosylation at two different positions on the precursor molecule. Phylogenetic analysis also indicates that this previously unknown glucosyltransferase enzyme may be unique to roses, with glycosylation having apparently evolved into a single stabilizing step in other plants.

α-Synuclein Seeding Assay Using Cultured Cells.

α-Synuclein, a presynaptic protein, is involved in synaptic vesicle dynamics in response to neuronal activity. Mutations of the α-synuclein gene and the neuronal deposition of α-synuclein, called Lewy bodies, are linked to the development of Parkinson's disease. α-Synuclein has a prion-like property that converts its physiological protein conformation to a pathogenic one, forming disease-causing fibrils. Aggregation of these fibrils and subsequent inclusion formation are suggested to interfere with vesicular trafficking and organelle function in neurons. Thus, detection of a prion-like property of α-synuclein and the evaluation of its modifying factors are required to understand the pathogenesis of Parkinson's disease and to develop new therapies. In this chapter, we describe a cell-based assay for detecting α-synuclein propagation.

Measurement of GCase Activity in Cultured Cells.

Glucocerebrosidase (GCase), which is encoded by the GBA1 gene, has lysosomal glycoside hydrolase activity that hydrolyzes glucosylceramide. Defects in GCase lead to the accumulation of glucosylceramide, which causes the development of the lysosomal storage disease known as Gaucher's disease. Loss-of-function mutations in the GBA1 gene are the most important genetic risk factor for synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies. Recent studies on PD genes associated with lysosomal function suggest that GCase activity is decreased in cell models of PD and in neurons derived from PD patients. In this chapter, we describe a protocol to measure GCase activity in cultured cells.

Derivatization-assisted enzyme-linked immunosorbent assay for identifying hallucinogenic mushrooms with enhanced sensitivity.

A sensitive immunochemical method for identifying hallucinogenic mushrooms (magic mushrooms) is required for regulating their illicit use. We have previously generated a monoclonal antibody (mAb) that targets psilocin (Psi), the major psychoactive compound in hallucinogenic mushrooms, and developed an enzyme-linked immunosorbent assay (ELISA). However, this ELISA failed to achieve the expected low-picomole-range sensitivity, as a result of insufficient affinity of the mAb to Psi. It is recognized that haptenic antigens with a larger molecular mass tend to induce antibodies with higher affinities. Thus, we herein report a "derivatization-assisted ELISA," in which the "real analyte" Psi was determined as a "surrogate analyte," the tert-butyldimethylsilyl ether analog thereof (TBS/Psi) having a 1.6-fold greater molecular mass (Mr 318.53) than Psi. A novel mAb against TBS/Psi, prepared by immunizing mice with a TBS/Psi-albumin conjugate showed a 69-fold higher affinity to TBS/Psi residues (Ka = 3.6 × 107 M-1 as IgG) than that of our previous mAb against Psi. This mAb consequently enabled a competitive ELISA for measuring TBS/Psi with the desired sensitivity: the dose-response curve midpoint (12.1 pmol per assay) was >100-fold lower than that of the previous ELISA for determining Psi. Extracts of dried mushroom powders were mixed with TBS triflate for 30 min at room temperature, converting Psi into TBS/Psi in approximately 50% yield. The reaction mixture was then subjected to an ELISA using the anti-TBS/Psi mAb to determine TBS/Psi. Psilocybe cubensis, a species of hallucinogenic mushrooms, gave rise to positive signals, indicating the presence of Psi therein in the expected quantity, while no detectable response was observed for four kinds of edible mushrooms available in the markets.

BMP and Hh signaling affects primordial germ cell division in Drosophila.

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up- and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.

[Discrimination of Kratom Products by an Improved PCR-RFLP Method].

In Japan, mitragynine, 7-hydroxymitragynine and Mitragyna speciosa Korth. (M. speciosa, "Kratom") were controlled as Designated Substances under the Pharmaceutical and Medical Device Act from March 2016. In this study, the origins of 16 Kratom products obtained from the illegal drug market in Japan were investigated by DNA analyses and LC-MS analyses. When the PCR-restriction fragment length polymorphism (RFLP) was performed using the restriction enzyme XmaI (as reported by Sukrong et al. to be able to distinguish M. speciosa), the same DNA fragment patterns were obtained from all 16 products. On the other hand, as a result of the identification of the plant species of each product by nucleotide sequence analyses, the sequences of M. speciosa were detected in only 14 products. Despite the facts that mitragynine and 7-hydroxymitragynine were detected also in the other two products by the LC-MS analyses, M. speciosa DNAs were not amplified from these products by the PCR. Moreover, the DNA amplicons of the other psychotropic plant (Mesembryanthemum sp., e.g. "Kanna") were detected. This plant PCR amplicon has the restriction site for the XmaI at the same position of the M. speciosa PCR amplicon and it is difficult to distinguish "Kratom" and "Kanna" by the conventional PCR-RFLP. When the restriction enzyme XhoI was used simultaneously with the Xmal, the specific DNA fragment was only observed from the M. speciosa amplicon and it was possible to distinguish both species using this improved PCR-RFLP method. This method is useful to identify the origin of Kratom products distributed in the illegal drug market.

Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms.

Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosphorylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20-20 or 0.040-2.0 µg/assay (limit of detection was 0.14 or 0.029 µg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8 % (or <0.5 %), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.

Heart and vessel pathology underlying brain infarction in 142 stroke patients.

OBJECTIVE: This study was designed to determine the histopathological characteristics of cardiac and vascular lesions responsible for various subtypes of ischemic stroke. METHODS: Postmortem pathological examination was performed on 142 patients who died within 30 days of the onset of ischemic stroke in the National Cardiovascular Center, Osaka, Japan. RESULTS: The numbers of cases with autopsy-proven diagnoses of atherothrombotic, cardioembolic, and lacunar strokes, ischemic stroke of other determined causes, and ischemic stroke of undetermined cause were 17, 107, 2, 12, and 4, respectively. Thrombi that developed at the culprit plaques of the cerebral arteries were responsible for atherothrombotic stroke. In 70% of the cases with cardioembolic stroke, the presence of thrombi as potential embolic sources were confirmed in the heart or, in some cases, in the venous circulation of patients with patent foramen ovale and tetralogy of Fallot. INTERPRETATION: In most atherothrombotic strokes, fibrin- and platelet-rich thrombi of various thicknesses develop at the culprit plaques of the cerebral arteries, which are finally occluded with fibrin- and red-cell-rich thrombi (red thrombi). In most cardioembolic strokes, red thrombi generated in the heart or peripheral veins are dislodged to embolize the cerebral arteries.

[Detection method for the ability of hemp (Cannabis sativa L.) seed germination by the use of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)].

Cannabis plants show a high Delta9-tetrahydrocannabinol content and are used as a psychoactive drug. Therefore the cultivation of hemp and its possession are prohibited by law in Japan. Meanwhile, Cannabis seeds have been used as a component of shichimi-togarashi (a Japanese spice), bird feed, or a crude drug (mashinin). To exclude the possibility of germination, it is officially noticed that hemp seeds must be killed. However, the number of violators has increased in recent years. To judge the ability of seed germination, a germination test is performed. However, the test requires several days and thus has not been used for on-site inspection. In this study, we developed a rapid detection method to determine the ability of Cannabis seeds to germinate using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC). The principle of the assay is as follows. The endogenous respiratory enzymes in hemp seeds convert added colorless TTC into red 1,3,5-triphenylformazan. Consequently, a living embryo is stained red, while red does not appear in the dead seeds. The reaction was active over a pH range of 8.0-9.0, and the optimum activity was found from 40 to 50 degrees C. Under the optimum conditions, we were able to determine the ability of seeds to germinate based on the presence of color within 20 min. Since this method is rapid and simple, it is applicable to on-site inspections. In addition, it could be used as an alternative technique to the germination test, because erroneous decisions is cannot occur under the assay principle.

[Analytical data of designated substances (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan, part I: GC-MS and LC-MS].

In the last 10 years, many analogs of narcotic substances have been widely distributed in Japan as easily available psychotropic substances and this has become a serious problem. They have been sold as video cleaners, incense and reagents via the Internet or in video shops. They are not controlled under the Narcotics and Psychotropics Control Law because their pharmacological effects have not yet been proved scientifically. As a countermeasure to prevent the abuse of these substances, the Ministry of Health, Labor and Welfare amended the Pharmaceutical Affairs Law in 2006 so that 31 non-controlled psychotropic substances (11 tryptamines, 11 phenethylamines, 6 alkyl nitrites, 2 piperazines and salvinorin A) and 1 plant (Salvia divinorum) are now controlled as "Designated Substances (Shitei-Yakubutsu)" as of April 2007. Five other compounds (4 phenethylamines and 1 piperazine) were also added to this category in January 2008. In this study, we developed simultaneous analytical methods for these designated substances using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) and present retention times, UV spectra, electron ionization (EI), GC-MS, and electrospray ionization (ESI) LC-MS data.