In vivo distribution and localization of chorein

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. In this study, we produced an antibody against chorein and examined its protein-level expression and localization in mouse. Immunoblot analysis revealed that chorein was expressed in a gene dose-dependent manner in the VPS13A deletion-mice that we recently developed, which confirms the sensitivity of the antibody. Chorein was highly expressed in testis, kidney, spleen, and brain, and was expressed ubiquitously in various brain regions. Subcellular analysis of the brain showed high levels of chorein in microsomal and synaptosomal fractions. Immunohistochemically, chorein-like immunoreactivity was ubiquitously observed in the brain in the neuronal perinuclear region, cytoplasm and fibers. In testis and kidney, clear cell-specific patterns of chorein-like immunoreactivity were detected. Our findings provide basic information on chorein in vivo and may contribute to taking the first step toward understanding molecular pathogenesis of ChAc.     

1,5-Benzodioxepin derivatives as a novel class of muscarinic M3 receptor antagonists

The structure-activity relationships of novel 1,5-benzodioxepin derivatives as muscarinic M(1)-M(3) receptor antagonists are reported. Some of these compounds were found to possess high binding affinity for the muscarinic M(3) receptor and potent effect on rhythmic increase in bladder pressure in unanesthetized rats following oral administration. These compounds displayed selectivity for the bladder over the salivary gland.     

Heat-induced antigen retrieval: mechanisms and application to histochemistry

Since the introduction of the fluorescence-labeled antibody method by Coons et al. [Immunological properties of antibody containing a fluorescent group. Proc Soc Exp Biol Med 47, 200-2002], many immunohistochemical methods have been refined to obtain high sensitivity with low background staining at both light and electron microscopic levels. Heat-induced antigen retrieval (HIAR) reported by Shi et al. in the early 1990s has greatly contributed to immunohistochemical analysis for formalin-fixed and paraffin-embedded (FFPE) materials, particularly in the field of pathology. Although antigen retrieval techniques including enzyme digestion, treatment with protein denaturants and heating have been considered tricky and mysterious techniques, the mechanisms of HIAR have been rapidly elucidated. Heating cleaves crosslinks (methylene bridges) and add methylol groups in formaldehyde-fixed proteins and nucleic acids and extends polypeptides to unmask epitopes hidden in the inner portion of antigens or covered by adjacent macromolecules. In buffers having an appropriate pH and ion concentration, epitopes are exposed without entangling the extended polypeptides during cooling process, since polypeptides may strike a balance between hydrophobic attraction force and electrostatic repulsion force. Recent studies have demonstrated that HIAR is applicable for immunohistochemistry with various kinds of specimens, i.e., FFPE materials, frozen sections, plastic-embedded specimens, and physically fixed tissues at both the light- and electron-microscopic levels, and have suggested that the mechanism of HIAR is common to aldehyde-fixed and aldehyde-unfixed materials. Furthermore, heating has been shown to be effective for flow cytometry, nucleic acid histochemistry (fluorescein in situ hybridization (FISH), in situ hybridization (ISH), and terminal deoxynucleotidyl transferase-mediated nick labeling (TUNEL)), and extraction and analysis of macromolecules in both FFPE archive materials and specimens processed by other procedures. In this article, we review mechanism of HIAR and application of heating in both immunohistochemistry and other histochemical reactions.     

Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in the adult zebrafish

Among vertebrates, fish and mammals show intriguing differences in their growth control properties with age. The potential for unlimited or indeterminate growth in a variety of fish species has prompted many questions regarding the senescent phenomena that appear during the aging process in these animals. Using zebrafish as our model system, we have attempted in our current study to examine the growth phenomena in fish in relation to the onset of senescence-associated symptoms, and to evaluate the effects of genotoxic stress on these processes. We observed in the course of these analyses that the zebrafish undergoes continuous growth, irrespective of age, past the point of sexual maturation with gradually decreasing growth rates at later stages. Animal population density, current body size and chronological age also play predominant roles in regulating zebrafish growth and all inversely influence the growth rate. Interestingly, the induction of genotoxic stress by exposure to ionizing radiation (IR) did not adversely affect this body growth ability in zebrafish. However, IR was found to chronically debilitate the regeneration of amputated caudal fins and thereby induce high levels of abnormal fin regeneration in the adult zebrafish. In addition, by resembling and mimicking the natural course of aging, IR treatments likewise enhanced several other symptoms of senescence, such as a decline in reproductive abilities, increased senescence-associated beta-galactosidase activity and a reduction in melatonin secretion. Our current data thus suggest that during the lifespan of zebrafish, the onset of senescence-associated symptoms occurs in parallel with continuous growth throughout mid-adulthood. Moreover, our present findings indicate that genotoxic DNA damage may play a role as a rate-limiting factor during the induction of senescence, but not in the inhibition of continuous, density-dependent growth in adult zebrafish.     

A Hereditary Enteropathy Caused by Mutations in the SLCO2A1 Gene,Encoding a Prostaglandin Transporter

Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn’s disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of “chronic enteropathy associated with SLCO2A1 gene” (CEAS).     

Population structure and succession in temperate forests of southwestern Japan

In the temperate forests of southwestern Japan, the population density of woody plants in the community increases in the early stage of secondary succession, reaches a peak in the old oak-chestnut forest, and decreases towards the climax beech forest. The species richness and diversity of woody plants also show a trend similar to that of the population density. The canopy-tree population decreases in the course of the succession while the basal area increases, showing a self-thinning process. The species richness, diversity and population density of herbaceous plants are much influenced by the dominance of the bamboo, Sasa palmata. The life-history traits of trees, lower trees and shrubs are discussed in relation to their shoot system, reproductive pattern and successional processes.