First Identification of 5,11-Dideoxytetrodotoxin in Marine Animals,and Characterization of Major Fragment Ions of Tetrodotoxin and Its Analogs by High Resolution ESI-MS/MS

Even though tetrodotoxin (TTX) is a widespread toxin in marine and terrestrial organisms, very little is known about the biosynthetic pathway used to produce it. By describing chemical structures of natural analogs of TTX, we can start to identify some of the precursors that might be important for TTX biosynthesis. In the present study, an analog of TTX, 5,11-dideoxyTTX, was identified for the first time in natural sources, the ovary of the pufferfish and the pharynx of a flatworm (planocerid sp. 1), by comparison with totally synthesized (−)-5,11-dideoxyTTX, using high resolution ESI-LC-MS. Based on the presence of 5,11-dideoxyTTX together with a series of known deoxy analogs, 5,6,11-trideoxyTTX, 6,11-dideoxyTTX, 11-deoxyTTX, and 5-deoxyTTX, in these animals, we predicted two routes of stepwise oxidation pathways in the late stages of biosynthesis of TTX. Furthermore, high resolution masses of the major fragment ions of TTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX were also measured, and their molecular formulas and structures were predicted to compare them with each other. Although both TTX and 5,6,11-trideoxyTTX give major fragment ions that are very close, m/z 162.0660 and 162.1020, respectively, they are distinguishable and predicted to be different molecular formulas. These data will be useful for identification of TTXs using high resolution LC-MS/MS.     

Response of soybean plants to two inoculation methods with arbuscular mycorrhizal fungus of Glomus sp. strain R-10 under field condition

ABSTRACT

Inoculation of arbuscular mycorrhizal (AM) fungi has a great potential to reduce input of phosphorus fertilizer. In this study, we tested the hypothesis that transplanting of pre-inoculated plants (pre-inoculation) with AM fungal inoculum Glomus sp. strain R-10 (R-10) is more effective for increasing AM fungal colonization and soybean yield than placing R-10 inoculum into field soil (direct inoculation). We cultivated pre-inoculated and direct inoculated plants with and without R-10 in the same field. On the contrary to the hypothesis, hyphal colonization was increased by direct inoculation, but decreased by pre-inoculation in an early growth stage. Shoot phosphorus concentration, shoot dry weight, and yield also showed the same trend as the hyphal colonization. These results indicated that pre-inoculation with R-10 would be less effective for increasing AM fungal colonization and yield than direct inoculation. It may be due to a colonization strategy of R-10 and short duration for establishment of seedling.     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

Contribution of the type II secretion system in systemic infectivity of <Emphasis Type="Italic">Ralstonia </Emphasis><Emphasis Type="Italic">solanacearum</Emphasis> through xylem vessels

Ralstonia solanacearum strain OE1-1 (OE1-1) systemically invades tobacco plants and causes bacterial wilt. A type II secretion system (T2SS)-deficient mutant of OE1-1, derived from EZ::TN<KAN-2>transposon-insertion, retained the ability of the parent strain to produce exopolysaccharide in vitro and grow in intercellular spaces immediately after invasion of host plants, but lost the ability to systemically infect the host. With transmission electron microscopy, the mutant was not observed in xylem vessels. These findings suggest that the T2SS contributes to systemic infection by enabling the bacteria to invade xylem vessels.     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.     

Necrotising encephalitis in the Yorkshire terrier: a case report and literature review

A four-year-old, spayed female Yorkshire terrier was presented with a two-month history of lameness in the left forelimb, circling and falling. A magnetic resonance imaging (MRI) examination 11 days after presentation revealed dilation of the right lateral ventricle. Following euthanasia, which was performed about 10 months after the onset of clinical signs, there was gross evidence of degeneration and cavitation of the cerebrum and dilation of the lateral ventricle on the right side. Microscopically, cavitation and necrosis were observed in the white and grey matter of the right cerebrum and there was abundant gemistocytic and fibrillary astrocytosis. Haemorrhage and marked perivascular cuffing with mononuclear cells were found in the mesencephalon. Inflammatory lesions consisting of lymphocytic infiltration and glial proliferation were also present in the dorsal funiculus of the cervical spinal cord. This case was diagnosed as necrotising encephalitis in the Yorkshire terrier (NEYT) with involvement of the spinal cord. NEYT is a chronic progressive neurological disorder, resulting from widespread, destructive non-suppurative inflammation of the central nervous system of unknown cause. In the past decade, 12 cases have been documented in adult to aged Yorkshire terriers. Computed tomography and MRI can detect the characteristic multifocal cavitations and ventriculomegaly, facilitating premortem diagnosis.     

Detection of a major QTL related to smut disease resistance inherited from a Japanese wild sugarcane using GRAS-Di technology

Smut disease of sugarcane causes considerable yield losses and the use of resistant varieties is the best control practice. Our group identified a Japanese wild sugarcane with highly smut disease resistance named ‘Iriomote8’. In this study, we conducted QTL analysis for smut disease resistance using a mapping population derived from a resistant variety ‘Yaenoushie’, in which resistance is inherited from ‘Iriomote8’. We identified 4813 non-redundant markers using GRAS-Di technology and developed a linkage map of mapping parents. We evaluated smut disease resistance of the mapping population by the inoculation test. Consequently, a large number of clones did not show the disease symptoms and the distribution of smut disease incidence tended to be “L shaped”. Composite interval mapping detected an identical QTL for indices of smut disease incidence with a markedly high LOD score (26.6~45.6) at the end of linkage group 8 of ‘Yaenoushie’. This QTL explained approximately 50% of the cases of smut disease incidence. In the mapping population, there were no correlations between the indices of smut disease incidence and other agronomic traits. In conclusion, this QTL could be used for marker-assisted selection to significantly improve smut disease resistance without negative effects on other agronomic traits.     

Characterization of Thiamin‐Binding Protein from Wheat Germ

A thiamin‐binding protein was isolated from wheat germ (Triticum aestivum). Its molecular mass was estimated as 120,000 Da consisting of two 56,000‐subunits. The protein contained a large amount of glutamine or glutamic acid (15.4 mol%), arginine (12.5 mol%), and glycine (12.0 mol%). The levels of tyrosine, methionine, tryptophan, and cysteine in the protein were low. Optimum pH for its thiamin‐binding activity was pH 8.0. It bound free thiamin specifically, not thiamin phosphates. The apparent dissociation and maximum bound values for the thiamin‐binding were 2.52 μM and 9.34 nmol/mg of protein, respectively. Properties of the thiamin‐binding protein from wheat germ were similar to those of the thiamin‐binding protein from rice seeds, but not from buckwheat, sesame, or sunflower seeds.     

Relationship between abundance and localization of maternal messenger RNA and egg quality in artificially matured Japanese eel Anguilla japonica

Fisheries Science - Artificial maturation and ovulation of Japanese eel do not always result in the production of good-quality eggs. The molecular basis for compromised egg quality in artificially...