Thirteen novel deoxynivalenol-degrading bacteria are classified within two genera with distinct degradation mechanisms

The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by species of the plant pathogen Fusarium, causes serious problems in cereal crop production because of its toxicity towards humans and livestock. A biological approach for the degradation of DON using a DON-degrading bacterium (DDB) appears to be promising, although information about DDBs is limited. We isolated 13 aerobic DDBs from a variety of environmental samples, including field soils and wheat leaves. Of these 13 strains, nine belonged to the Gram-positive genus Nocardioides and other four to the Gram-negative genus Devosia. The degradation phenotypes of the two Gram types were clearly different; all washed cells of the 13 strains degraded 100 μg mL(-1) DON to below the detection limit (0.5 μg mL(-1)), but the conditions inducing the DON-degrading activities differed between the two Gram types. The HPLC profiles of the DON metabolites were also distinct between the two genera, although all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed DON assimilation in media containing DON as a carbon source, whereas the Gram-negatives did not. Our results suggest that aerobic DDBs are distributed within at least two phylogenetically restricted genera, suggesting independent evolution of the DON-degradation mechanisms.     

Development of an enzyme-linked immunosorbent assay system for the determination of asymmetric dimethylarginine using a specific monoclonal antibody

We produced a monoclonal antibody (mAb) against N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM-100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

The Identification of a Novel Gene,MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O⁶-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.     

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.     

The Arabidopsis Protein SHI Represses Gibberellin Responses in Arabidopsis and Barley

The current model of gibberellin (GA) signal transduction is based on a derepressible system and a number of candidate negative regulators have been identified in Arabidopsis. We previously have reported the identification of the Arabidopsis gene SHORT INTERNODES (SHI) that causes suppression of GA responses when constitutively activated. In this paper, we show by using reporter gene analysis that the SHI gene is expressed in young organs, e.g. shoot apices and root tips. The model predicts a suppressor of GA responses to be active in these tissues to prevent premature growth or development. To study the effect of SHI on GA signaling, we used a functional assay that measures effects of signaling components on a well-defined GA response; the up-regulation of alpha-amylase in barley (Hordeum vulgare) aleurones in response to GA treatment. We found that SHI was able to specifically block the activity of a high-isoelectric point alpha-amylase promoter following GA(3) treatment, which further supports that SHI is a suppressor of GA responses. We have identified two putative loss-of-function insertion alleles of SHI and lines homozygous for either of the new alleles show no phenotypic deviations from wild type. Because SHI belongs to a gene family consisting of nine members, we suggest that SHI and the SHI-related genes are functionally redundant. We also show that a functional ERECTA allele is able to partly suppress the dwarfing effect of the shi gain-of-function mutation, suggesting that the erecta mutation harbored by the Landsberg erecta ecotype is an enhancer of the shi dwarf phenotype.     

Antivitamin B-6 effect of 1-aminoproline on rats

By intraperitoneal injection of 1-aminoproline, death after severe convulsion was observed in rats (LD₅₀ of 1-amino-l-proline, 26 mg per kg of body weight for young male rats fed a normal diet). The vitamin B-6-deficient rats were more sensitive to this hydrazino acid than the normal rats. The toxic effect was completely prevented by the administration of pyridoxine. 1-Amino-d-proline was less toxic than the l-isomer. By the 1-aminoproline treatment, the most remarkable changes in the free amino acid levels were the striking increases in the concentrations of α-aminodipic acid, citrulline and cystathionine in all the tissues tested, except in brain. Some unidentified ninhydrin-positive substances appeared. These results indicate that 1-aminoproline greatly disturbed the amino acid pattern, i.e. the amino acid metabolism in rats.     

Ability of the endangered species Papaver fauriei to produce hybrids with a cultivated poppy (Papaver sp.)

Papaver fauriei is an endemic and endangered species that grows only on the gravelly alpine slopes of Mt. Rishiri, Japan. Cultivated poppy (Papaver sp.), the species name of which is unknown, has been introduced to the natural habitats of P. fauriei through human activities. Because the appearance and internal transcribed spacer (ITS) sequences of these two poppies are highly similar, it is of concern that they could produce hybrids in their natural habitats. Thus, first, the ability of these two poppies to produce hybrids was analyzed by artificial fertilization in this study. A large number of seeds were produced by reciprocal crosses between P. fauriei and the cultivated poppy, comparable with the number of seeds obtained by self‐ or cross‐fertilization of P. fauriei or the cultivated poppy. In addition, high germination was observed for seeds obtained from crosses between the two poppies, and deleterious phenotypes, such as albinism and dwarfism, were not detected in the F₁ generation. These results indicate that after pollination, there is no reproductive isolation between the two poppies. Second, we sequenced the internal transcribed spacer (ITS) region of 240 poppy individuals collected from the gravelly alpine slopes of Mt. Rishiri, and 66 showed the sequence of P. fauriei, whereas 174 showed the sequence of the cultivated poppy. However, the ITS sequence that confirms hybridism between the two poppies was not detected in these individuals, indicating that hybridization of P. fauriei and the cultivated poppy rarely occurs under natural conditions. Unknown mechanism(s) appear to prevent cross‐pollination between the two poppies.     

Cadmium-induced abnormality in strains of Euglena gracilis: morphological alteration and its prevention by zinc and cyanocobalamin

Euglena gracilis is susceptible to cadmium (Cd) at high concentrations. There are no comparative data on cytotoxicity or abnormality of CdCl₂ to E. gracilis Z and its achlorophyllous mutant SMZ. The present study examined the cytotoxicity of CdCl₂ under continual exposure at levels ranging from sub-ppm to ppm, and assessed the effects of zinc (Zn) or cyanocobalamin (VB₁₂) supplementation on the suppression of Cd-induced abnormal cell proliferation and hypertrophy. With Zn levels restricted to 1 ppm [as Zn⁺⁺], cell growth of both E. gracilis strains was reduced in proportion to Cd concentration. More abnormal cells (hypertrophied, V-shape and starfish-shape) were observed in both strains at sub-ppm levels of Cd. ZnSO₄ supplementation from 2 to 63 ppm significantly suppressed the incidence of Cd-induced abnormality. However, a significant increase in abnormal cells was observed following Zn supplementation at levels of 125 and 250 ppm, which produced remarkable differences in cell morphology. The incidence of abnormal cells varied with supplemented VB₁₂ levels ranging from 4 to 250 ppb in both E. gracilis strains.     

Neurocalcin-like immunoreactivity in the rat esophageal nervous system

Neurocalcin is a newly identified neuronal calcium-binding protein. We tried here to investigate the immunohistochemical distribution of neurocalcin in the rat esophagus. Nerve cell bodies having neurocalcin immunoreactivity were found throughout the myenteric plexus. In the myenteric ganglia, two types of nerve terminals showed neurocalcin immunoreactivity. One was varicose terminals containing numerous small clear vesicles and forming a synapse with nerve cells. The other terminals were characterized by laminar or pleomorphic structure and many mitochondria. These laminar terminals were supposed to be sensory receptors of the esophageal wall. In the motor endplates of the striated muscles, nerve terminals containing many small clear vesicles and mitochondria also had neurocalcin immunoreactivity. After left vagus nerve cutting under the nodose ganglia, the number of immunopositive thick nerve fibers, laminar endings and nerve terminals on the striated muscles decreased markedly. Retrograde tracing experiments using Fast Blue showed extrinsic innervation of esophagus from ambiguus nucleus, dorsal motor nucleus of vagus, superior cervical ganglia, celiac ganglia, nodose ganglia and dorsal root ganglia. In the celiac ganglia, nodose ganglia and dorsal root ganglia, retrogradely labeled nerve cells were neurocalcin-immunoreactive. Neurons in the celiac ganglia may project varicose terminals, while nodose and dorsal root neurons project laminar terminals. Although cell bodies of motoneurons in the ambiguus nucleus lacked neurocalcin immunoreactivity, these neurons may contain neurocalcin only in the nerve terminals in the motor endplates. Neurocalcin immunoreactivity is distributed in many extrinsic and intrinsic neurons in the esophagus and this protein may play important roles in regulating calcium signaling in the neurons.     

Hypomorphic phenotype of <Emphasis Type="Italic">Foxn1</Emphasis> gene-modified rats by CRISPR/Cas9 system

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.