Metabolic Profiling of the Uncaria Hook Alkaloid Geissoschizine Methyl Ether in Rat and Human Liver Microsomes Using High-Performance Liquid Chromatography with Tandem Mass Spectrometry

Geissoschizine methyl ether (GM) is an indole alkaloid found in Uncaria hook, which is a galenical constituent of yokukansan, a traditional Japanese medicine. GM has been identified as the active component responsible for anti-aggressive effects. In this study, the metabolic profiling of GM in rat and human liver microsomes was investigated. Thirteen metabolites of GM were elucidated and identified using a high-performance liquid chromatography with tandem mass spectrometry method, and their molecular structures were proposed on the basis of the characteristics of their precursor ions, product ions, and chromatographic retention times. There were no differences in the metabolites between the rat and human liver microsomes. Among the 13 identified metabolites, there were two demethylation metabolites, one dehydrogenation metabolite, three methylation metabolites, three oxidation metabolites, two water-adduct metabolites, one di-demethylation metabolite, and one water-adduct metabolite followed by oxidation. The metabolic pathways of GM were proposed on the basis of this study. This study will be helpful in understanding the metabolic routes of GM and related Uncaria hook alkaloids, and provide useful information on the pharmacokinetics and pharmacodynamics. This is the first report that describes the separation and identification of GM metabolites in rat and human liver microsomes

High Frequency of a Single Nucleotide Substitution (c.-6-180T>G) of the Canine MDR1/ABCB1 Gene Associated with Phenobarbital-Resistant Idiopathic Epilepsy in Border Collie Dogs

A single nucleotide substitution (c.-6-180T>G) associated with resistance to phenobarbital therapy has been found in the canine MDR1/ABCB1 gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9%) in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies

Identification of the <i>PLA2G6</i> c.1579G>A Missense Mutation in Papillon Dog Neuroaxonal Dystrophy Using Whole Exome Sequencing Analysis

<div><p>Whole exome sequencing (WES) has become a common tool for identifying genetic causes of human inherited disorders, and it has also recently been applied to canine genome research. We conducted WES analysis of neuroaxonal dystrophy (NAD), a neurodegenerative disease that sporadically occurs worldwide in Papillon dogs. The disease is considered an autosomal recessive monogenic disease, which is histopathologically characterized by severe axonal swelling, known as “spheroids,” throughout the nervous system. By sequencing all eleven DNA samples from one NAD-affected Papillon dog and her parents, two unrelated NAD-affected Papillon dogs, and six unaffected control Papillon dogs, we identified 10 candidate mutations. Among them, three candidates were determined to be “deleterious” by <i>in silico</i> pathogenesis evaluation. By subsequent massive screening by TaqMan genotyping analysis, only the <i>PLA2G6</i> c.1579G>A mutation had an association with the presence or absence of the disease, suggesting that it may be a causal mutation of canine NAD. As a human homologue of this gene is a causative gene for infantile neuroaxonal dystrophy, this canine phenotype may serve as a good animal model for human disease. The results of this study also indicate that WES analysis is a powerful tool for exploring canine hereditary diseases, especially in rare monogenic hereditary diseases.</p></div

Pedigree analysis of Case 1.

<p>Some paternal and maternal individuals were repetitively used for breeding, resulting in an increase in genetic homogeneity.</p

Overview of all variants identified by exome sequencing and <i>in silico</i> pathogenicity analysis.

<p>Overview of all variants identified by exome sequencing and <i>in silico</i> pathogenicity analysis.</p

iPLA₂β immunohistochemistry of Case 1 (a, c) and a normal (b, d) canine brain.

<p>(a) Spheroids in the cerebellum showed faint granular expression (black arrowheads). Cytoplasmic granular expression was also observed in the Purkinje cells (white arrowheads). (b) The expression was very weak in the cerebellum of the control dog. In the brainstem (c, d), intense cytoplasmic and nuclear expression was detected in the neurons of both the NAD and control dogs. Axonal spheroids in the neuropil of the brainstem of Case 1 showed an intense granular expression (arrowheads). Bar = 50 μm.</p