Transposon-tagged mutagenesis in the rat

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.     

Characterization of ABC transporter genes,sll1180, sll1181, and slr1270, involved in acid stress tolerance of Synechocystis sp. PCC 6803

Photosynthesis Research - Over 50 ATP-binding cassette (ABC) transporter-related genes are detected in the Synechocystis sp. PCC 6803 genome by genome sequence analysis. Deletion mutants of other...     

Morphological and molecular characterization of the strawberry black leaf spot pathogen referred to as the strawberry pathotype of Alternaria alternata

The remaining unclarified taxon among the seven known pathotypes of host-selective toxin (HST)-producing Alternaria alternata, namely, the strawberry pathotype (the strawberry black leaf spot pathogen), is taxonomically revised and re-described herein. According to our morphological observations, reference isolates of strawberry and Japanese pear pathotypes, which are toxic to leaves of Japanese pear ‘Nijisseiki’, have conidia that are formed in chains of 3–13, usually without lateral branches, after 7?d incubation on potato-carrot agar. The mean size of the conidia is 27–31?×?11–13?μm. Morphological characteristics of the examined isolates are identical to those of A. gaisen rather than A. alternata. A phylogenetic tree obtained by analysis of a combined dataset of ITS, gapdh, rpb2, tef1, Alt a 1, and endoPG sequences also strongly supports both pathotypes as one species, A. gaisen. We re-describe the fungus as A. gaisen Nagano ex Bokura and propose two formae speciales of the species, A. gaisen f. sp. fragariae producing AF-toxin and f. sp. pyri producing AK-toxin. The epitype specimen and ex-epitype culture of A. gaisen are newly designated.     

Predator avoidance in phytophagous mites: response to present danger depends on alternative host quality

We studied whether volatiles released by putative host plants affect the antipredator response of an herbivorous mite, Tetranychus urticae, when the patch was invaded by Phytoseiulus persimilis. Tetranychus urticae laid a lower number of eggs on tomato leaves than on lima bean leaves, suggesting that lima bean is a preferred host food source for T. urticae. In addition, T. urticae preferred lima bean plant volatiles to tomato plant volatiles in a Y-tube olfactometer test. To investigate the antipredator response of T. urticae, we examined the migration of T. urticae from a lima bean leaf disc to a neighbouring leaf disc (either a tomato or lima bean leaf disc) when ten predators were introduced into the original lima bean disc. A Parafilm bridge allowed for migration between the leaf discs. No migrations occurred between leaf discs when there were no predators introduced to the original leaf disc. However, when predators were introduced migrations did occur. When the neighbouring leaf disc was upwind of the original disc, the migration rate of the mite from original lima bean leaf disc to a neighbouring tomato leaf disc was significantly lower than that to a neighbouring lima bean leaf disc. By contrast, when the neighbouring leaf disc was downwind of the original leaf disc, there was no difference in the migration rates between lima bean leaf discs and tomato leaf discs. The number of T. urticae killed by P. persimilis for each treatment was not different, and this clearly shows that the danger was the same in all treatments regardless of the decision made by T. urticae. From these results, we conclude that T. urticae change their antipredator response by evaluating the difference in host plant volatiles in the patch they inhabit.     

Amelioration of type II diabetes in db/db mice by continuous low-dose-rate gamma irradiation

Low-dose-rate radiation modulates various biological responses including carcinogenesis, immunological responses and diabetes. We found that continuous irradiation with low-dose-rate gamma rays ameliorated type II diabetes in db/db mice, diabetic mice that lack leptin receptors. Whole-body exposure of db/db mice to low dose-rate gamma radiation improved glucose clearance without affecting the response to insulin. Histological studies suggested that degeneration of pancreatic islets was significantly suppressed by the radiation. Insulin secretion in response to glucose loading was increased significantly in the irradiated mice. These results suggest that low-dose-rate gamma radiation ameliorates type II diabetes by maintaining insulin secretion, which gradually decreases during the progression of diabetes due to degeneration of pancreatic islets. We also inferred that protection from oxidative damage is involved in the anti-diabetic effect of low-dose-rate gamma rays because expression and activity of pancreatic superoxide dismutase were significantly elevated by low-dose-rate gamma radiation.     

Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice

Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and leads to liver injury and neurodegeneration, but the details of abnormalities related to impaired autophagy are largely unknown. Here we used mouse genetic analyses to define the roles of autophagy in the aforementioned events. We report that the ubiquitin- and LC3-binding protein "p62" regulates the formation of protein aggregates and is removed by autophagy. Thus, genetic ablation of p62 suppressed the appearance of ubiquitin-positive protein aggregates in hepatocytes and neurons, indicating that p62 plays an important role in inclusion body formation. Moreover, loss of p62 markedly attenuated liver injury caused by autophagy deficiency, whereas it had little effect on neuronal degeneration. Our findings highlight the unexpected role of homeostatic level of p62, which is regulated by autophagy, in controlling intracellular inclusion body formation, and indicate that the pathologic process associated with autophagic deficiency is cell-type specific.     

Release of thioredoxin from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with environmental stimuli: solubilization of thioredoxin with ethanol

Thioredoxin is crucial for the maintenance of the redox status of cells of all types. Mammalian thioredoxin is secreted from various types of cells, although the mechanism underlying has not yet been clarified. Previously, we demonstrated that thioredoxin was released from Saccharomyces cerevisiae after treatment with ethanol. In this paper, we show that as well as ethanol, low-pH shock and hypoosmotic shock release thioredoxin. Low-molecular-weight proteins in yeast cells were preferentially released by treatment with ethanol and low-pH shock. A cell wall integrity pathway seems partially involved in the hypoosmotic shock-induced release of thioredoxin. Considerable amounts of thioredoxin were present in the insoluble fractions of the cells, a portion of which was associated with lipid microdomains that are resistant to nonionic detergent at 4°C. The intracellular localization of thioredoxin may influence the efficiency of its release from yeast cells with ethanol.     

Development of a convenient in vitro fertilization method using interspecific hybrids between Oryzias latipes and Oryzias curvinotus

Medaka is a small Asian freshwater teleost and has been an excellent model for fertilization studies for more than 50 years. Therefore, experimental procedures for in vitro fertilization (IVF) and cryopreservation of sperm are well established. In contrast, since the eggs or early embryos can not be cryopreserved, many females are killed to obtain unfertilized eggs for IVF. Recent progress in genomics is establishing medaka as a new model animal in functional genomics, and numerous mutant and transgenic strains have been established and stored as frozen sperm. Accumulated preserved resources require a simple and reliable recovery method for IVF. In this paper, we describe a method for obtaining a large number of unfertilized eggs without killing females, using sterile interspecific hybrids between Oryzias latipes and O. curvinotus. However, there is no report about the normality of offspring that were obtained by IVF using unfertilized eggs spawned in mating with the sterile hybrid male. In this paper, we have confirmed the reliability of the method regarding the influences on the next generation and also assessed conditions for efficient collection of unfertilized eggs. The method would be useful not only for fertilization studies but also for keeping transgenics and mutants, including a mutant library for a reverse genetic approach.     

A novel embryotoxic estimation method of VPA using ES cells differentiation system

Valproic acid (VPA), which has a wide range of therapeutic applications, is known as a potent teratogen that induces neural tube defects in vertebrates. Here, we have characterized the tissue-specific, embryotoxic effects of VPA on developmental processes using a novel system with differentiating mouse ES cells. Under our cultivating condition, ES cells differentiated into cardiomyocytes, although various cell types can be differentiated. VPA affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The analysis of tissue-specific markers also revealed that VPA potently inhibited mesodermal and endodermal development but promoted neuronal differentiation in a lineage-specific manner. Taking the in vivo teratogenicity of VPA into account, this assay system could be useful in predicting the degree of embryotoxicity of VPA. We, thus, propose that the in vivo embryotoxic effects of various medicines can be estimated fast and accurately using this in vitro cell differentiation system.     

Domains 16 and 17 of tropoelastin in elastic fibre formation

AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39-43 residue long Abeta (amyloid-beta)-peptide. The most abundant species, Abeta(1-40) and Abeta(1-42), are both present within senile plaques, but Abeta(1-42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Abeta-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Abeta(1-40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Abeta(1-42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Abeta(1-40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two beta-strands connected with a bend. This protection pattern partly resembles the pattern found in Abeta(1-42) fibrils, but the Abeta(1-40) fibrils display a significantly increased protection for the N-terminal residues Phe4-His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37-Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.