Transgenic rice expressing amyloid β-peptide for oral immunization

Various vaccine therapies for Alzheimer's disease (AD) have been investigated. Here we report transgenic rice expressing amyloid β-peptide (Aβ). The Aβ42 gene fused with a green fluorescent protein gene was introduced into rice using the Agrobacterium method. When transgenic brown rice expressing Aβ was orally administered to mice, serum anti-Aβ antibody titers were elevated. The same results were observed when mice were fed boiled, transgenic brown rice. The results indicate that an edible vaccine against AD using rice may be feasible. A vaccine derived from rice would be far cheaper than existing medical vaccines.     

Neonatal lethality in knockout mice expressing the kinase-dead form of the gefitinib target GAK is caused by pulmonary dysfunction

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd(+/+) mice but not in GAK-kd(-/-) pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.     

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as “granule bodies”, which were found only in the pericentral cells of the thallus. This research was supported by Ministry of Education, Culture, Sports, Science and Technology to R.S. (13660206).     

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.     

Superoxide dismutase (SOD) as a potential inhibitory mediator of inflammation via neutrophil apoptosis

Superoxide dismutase (SOD) is supposed to be an effective agent for neutrophil-mediated inflammation in the area of critical medicine. We investigated the involvement of SOD in the regulation of neutrophil apoptosis. Exogenously added SOD effectively induced neutrophil apoptosis, and the fluorescence patterns determined using annexin-V and the 7-AAD were similar to those seen in Fas-mediated neutrophil apoptosis. Neutrophils are short-lived leukocytes that need to be removed safely by apoptosis. The clearance of apoptotic neutrophils from sites of inflammation is a crucial determinant of the resolution of inflammation. Catalase inhibited the neutrophil apoptosis and caspase-3 activation. Spontaneous apoptosis, hydrogen peroxide and anti-Fas antibody-induced apoptosis of neutrophils were accelerated in Down's syndrome patients, in whom the SOD gene is overexpressed. Hydrogen peroxide was thought to be a possible major mediator of ROS-induced neutrophil apoptosis in caspase-dependent manner. Neutrophil apoptosis represents a crucial step in the mechanism governing the resolution of inflammation and has been suggested as a possible target for the control of neutrophil-mediated tissue injury. SOD may be a potential inhibitory mediator of neutrophil-mediated inflammation.     

Carbon cycling and budget in a forested basin of southwestern Hokkaido,northern Japan

Quantification of annual carbon sequestration is very important in order to assess the function of forest ecosystems in combatting global climate change and the ecosystem responses to those changes. Annual cycling and budget of carbon in a forested basin was investigated to quantify the carbon sequestration of a cool-temperate deciduous forest ecosystem in the Horonai stream basin, Tomakomai Experimental Forest, northern Japan. Net ecosystem exchange, soil respiration, biomass increment, litterfall, soil-solution chemistry, and stream export were observed in the basin from 1999–2001 as a part of IGBP-TEMA project. We found that 258 g C m^–2 year^–1 was sequestered annually as net ecosystem exchange (NEE) in the forested basin. Discharge of carbon to the stream was 4 g C m^–2 year^–1 (about 2% of NEE) and consisted mainly of dissolved inorganic carbon (DIC). About 43% of net ecosystem productivity (NEP) was retained in the vegetation, while about 57% of NEP was sequestered in soil, suggesting that the movement of sequestered carbon from aboveground to belowground vegetation was an important process for net carbon accumulation in soil. The derived organic carbon from aboveground vegetation that moved to the soil mainly accumulated in the solid phase of the soil, with the result that the export of dissolved organic carbon to the stream was smaller than that of dissolved inorganic carbon. Our results indicated that the aboveground and belowground interaction of carbon fluxes was an important process for determining the rate and retention time of the carbon sequestration in a cool-temperate deciduous forest ecosystem in the southwestern part of Hokkaido, northern Japan.     

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

PRIME: automatically extracted PRotein Interactions and Molecular Information databasE

With the exponentially increasing amount of information in the biomedical field, the significance of advanced information retrieval and information extraction, as well as the role of databases, has been increasing. PRIME is an integrated gene/protein informatics database based on natural language processing. It provides automatically extracted protein/family/gene/compound interaction information including both physical and genetic interactions, gene ontology based functions, and graphic pathway viewers. Gene/protein/family names and functional terms are recognized based on dictionaries developed in our laboratory. The interaction and functional information are extracted by syntactic dependencies and various phrase patterns. We have included about 920,000 (non-redundant) protein interactions and 360,000 annotated gene-function relationships for major eukaryotes. By combining the sequence and text information, the pathway comparison between two organisms and simple pathway deduction based on other organism interaction data, and pathway filtering using tissue expression data, are also available. This database is accessible at http://prime.ontology.ims.u-tokyo.ac.jp:8081.     

A guideline for ecological risk management procedures

A practical guideline for community-level ecological risk management is proposed, with particular emphasis on the mutual interdependencies of the scientific analysis, public consensus building, and an adaptive management. The procedure we recommend spans the screening of potential ecological risks, the involvement of related stakeholders, the conceptual development from the undesired event over assessment endpoints to measures of effect and stress factors, the risk assessment for the no-action case, the planning phase from the public decision to become active and the setting of goals over a specification of monitoring and control methods to an assessment of feasibility and a public approval of the management plan and finally the adaptive management from initiation over continued monitoring to revisions of the plan, if required. The procedure contains several checkpoints, alternative routes, and possibilities to correct previous decisions.