Salt-inducible bionylon polymer from Bacillus megaterium

Poly-gamma-glutamate (PGA) is a chiral polyamide material that possesses a nylon-like backbone, a bionylon polymer. We examined the PGA productivity of Bacillus megaterium and found NaCl-responsive PGA production in the bacterium. In the system of B. megaterium, salt would be significant in controlling the yield, molecular size, and stereochemistry of bionylon.     

pH-dependent formation of membranous cytoplasmic body-like structure of ganglioside G(M1)/bis(monoacylglycero)phosphate mixed membranes

Membrane structures of the mixtures of ganglioside G(M1) and endosome specific lipid, bis (monoacylglycero) phosphate (BMP, also known as lysobisphosphatidic acid) were examined at various pH conditions by freeze-fracture electron microscopy and small-angle x-ray scattering. At pH 8.5-6.5, a G(M1)/BMP (1:1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditions (pH 5.5, 4.6) with the lamellar repeat distance of 8.06 nm. Since BMP alone exhibits a diffuse lamellar structure at a broad range of pH values and G(M1) forms a micelle, the results indicate that both G(M1) and BMP are required to produce closely stacked multilamellar vesicles. These vesicles resemble membranous cytoplasmic bodies in cells derived from patients suffering from G(M1) gangliosidosis. Similar to G(M1) gangliosidosis, cholesterol was trapped in BMP vesicles in G(M1)- and in a low pH-dependent manner. Studies employing different gangliosides and a G(M1) analog suggest the importance of sugar chains and a sialic acid of G(M1) in the pH-dependent structural change of G(M1)/BMP membranes.     

The variation in Fas localization and the changes in Fas expression level upon stimulation with growth factors

Although Fas (APO-1/CD95) is well known as a death receptor, its stimulation occasionally fails to induce apoptosis in malignant cells. On the contrary, Fas is reported to advance the cell cycle in cancer cells. Therefore, we investigated roles of Fas in cell growth and apoptosis using human lung cancer cell lines. Fas was localized in the cytoplasm in exponentially growing cells, whereas only confluent cells expressed Fas on the cell membrane. A stimulation of confluent cells by either of EGF, IGF-I or VEGF induced once a decrease in Fas expression level and its sequential recovery. Fas expression levels in confluent cells were negatively correlated with cell doubling times (r=0.757, p=0.0088). Fas remained on the cell membrane of IgM-treated cells even after the growth factor stimulation, leading to apoptosis with abnormal mitosis, whereas the same stimulation induced Fas internalization in IgG(1)-treated cells. From these results, we suggest that Fas remaining on the cell membrane amplifies to induce apoptosis. Conversely, Fas internalization may enable cancer cells to escape from apoptosis. Our results suggest that growth factor may contribute to the resistance of cancer cells to Fas-mediated apoptosis in an autocrine or paracrine fashion.     

Selenocysteine beta-lyase and methylselenol demethylase in the metabolism of Se-methylated selenocompounds into selenide

The lyase activity toward Se-methylated selenoamino acids and the demethylase activity toward methylselenol in the metabolism of selenium were characterized in vitro. The beta- and gamma-lyase activities toward selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), respectively, were compared under exactly identical conditions by incubating 77Se-SeMet and 76Se-MeSeCys simultaneously in a liver supernatant, and then estimated by the decreases in the labeled starting selenoamino acids (MeSeCys and SeMet), and also by the increases in the labeled enzyme products (methylselenol and selenide) after oxidation to methylseleninic acid (MSA(IV)) and selenite, respectively, by HPLC-inductively coupled plasma-mass spectrometry (ICP-MS). Only 76Se-MeSeCys was decreased and only 76Se-selenite was produced, suggesting that conversion of MeSeCys to methylselenol by beta-lyase followed by that of methylselenol to selenide by demethylase actively occurred in the liver supernatant. The demethylase activity was characterized by incubating 77Se-methylselenol produced in situ from 77Se-MSA(IV) and glutathione in a partially purified enzyme preparation. It was found that demethylation takes place directly through an attack by a hydroxide anion on the methyl group of methylselenol producing selenide and methanol, selenide being detected on HPLC-ICP-MS after oxidation to selenite, and methanol on GC-MS. It was concluded that beta- but not gamma-lyase activity could be detected in a liver supernatant, and that the resulting methylselenol product is demethylated through hydrolysis, with methanol and selenide being produced (MeSeCys-->CH3SeH-->HSeH + CH3OH).     

The critical period for thyroid hormone responsiveness through thyroid hormone receptor isoform alpha in the postnatal small intestine

During second and third weeks after birth in rats, serum thyroid hormone level is elevated. In this study, we investigated the jejunal expression of thyroid hormone receptor (TR) alpha in developing rats. The TRalpha-1 mRNA level and TRalpha-1/TRalpha-2 mRNA ratio increased two-fold from 5 to 13 days after birth. This high level of TRalpha-1 mRNA was maintained until 20 days and then decreased to the basal level by the end of weaning period at 27 days; however, the level of TRalpha-2 mRNA remained unchanged throughout the developmental period. The increase in the TRalpha-1/TRalpha-2 mRNA ratio from 5 to 13 days was accompanied by an initial rise in the levels of mRNA for hexose transporters in the jejunum. Administration of T(3) during the suckling period (8-13 days) caused a 50% increase in the TRalpha-1/TRalpha-2 mRNA ratio, while administration of T(3) on days 12-17 and days 16-21, but not on days 22-27, caused a two to four-fold increase in the levels of mRNA for hexose transporters. These results suggest that a transient variation in the TRalpha-1/TRalpha-2 expression ratio is closely related to the critical period of thyroid hormone responsiveness for hexose transporters expression in the developing rat jejunum.     

Synthesis of a novel inhibitor against MRSA and VRE: preparation from zerumbone ring opening material showing histidine-kinase inhibition

Zerumbone ring-opening derivative 2 inhibited autophosphorylation of the essential histidine protein kinase (HPK), YycG, existing in Bacillus subtilis constituting a two-component system (TCS). However, it did not inhibit drug-resistant bacterium such as MRSA and VRE. Tryptophan derivative 34 also could be regulated by a TCS system like 2. In addition, 34 showed good inhibition against MRSA and VRE.     

Impaired Cerebellar Development in Mice Overexpressing VGF

VGF nerve growth factor inducible (VGF) is a neuropeptide precursor induced by brain-derived neurotrophic factor and nerve growth factor. VGF is increased in the prefrontal cortex and cerebrospinal fluid in schizophrenia patients. In our previous study, VGF-overexpressing mice exhibited schizophrenia-like behaviors and smaller brain weights. Brain developmental abnormality is one cause of mental illness. Research on brain development is important for discovery of pathogenesis of mental disorders. In the present study, we investigated the role of VGF on cerebellar development. We performed a histological analysis with cerebellar sections of adult and postnatal day 3 mice by Nissl staining. To investigate cerebellar development, we performed immunostaining with antibodies of immature and mature granule cell markers. To understand the mechanism underlying these histological changes, we examined MAPK, Wnt, and sonic hedgehog signaling by Western blot. Finally, we performed rotarod and footprint tests using adult mice to investigate motor function. VGF-overexpressing adult mice exhibited smaller cerebellar sagittal section area. In postnatal day 3 mice, a cerebellar sagittal section area reduction of the whole cerebellum and external granule layer and a decrease in the number of mature granule cells were found in VGF-overexpressing mice. Additionally, the number of proliferative granule cell precursors was lower in VGF-overexpressing mice. Phosphorylation of Trk and Erk1 were increased in the cerebellum of postnatal day 3 VGF-overexpressing mice. Adult VGF-overexpressing mice exhibited motor disability. All together, these findings implicate VGF in the development of cerebellar granule cells via promoting MAPK signaling and motor function in the adult stage.

     

Perturbation experiments and fluctuation enhancement in finite size of lattice ecosystems: Uncertainty in top-predator conservation

Study of perturbation experiments is crucial for conservation biology. We carry out Monte Carlo simulations on finite-size lattices composed of n species (n ≤ 4). The value of mortality rate m of top predator is altered to a higher or lower level, and a fluctuation enhancement (FE) is explored. Here FE means an uncertainty in population dynamics. It is found for n ≥ 2 that FE is observed, when m is decreased. Namely, when we protect the top predator, its population dynamics becomes very difficult to predict.     

MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice

Mutant populations are indispensable genetic resources for functional genomics in all organisms. However, suitable rice mutant populations, induced either by chemicals or irradiation still have been rarely developed to date. To produce mutant pools and to launch a search system for rice gene mutations, we developed mutant populations of Oryza sativa japonica cv. Taichung 65, by treating single zygotic cells with N-methyl-N-nitrosourea (MNU). Mutagenesis in single zygotes can create mutations at a high frequency and rarely forms chimeric plants. A modified TILLING system using non-labeled primers and fast capillary gel electrophoresis was applied for high-throughput detection of single nucleotide substitution mutations. The mutation rate of an M₂ mutant population was calculated as 7.4 × 10⁻⁶ per nucleotide representing one mutation in every 135 kb genome sequence. One can expect 7.4 single nucleotide substitution mutations in every 1 kb of gene region when using 1,000 M₂ mutant lines. The mutations were very evenly distributed over the regions examined. These results indicate that our rice mutant population generated by MNU-mutagenesis could be a promising resource for identifying mutations in any gene of rice. The modified TILLING method also proved very efficient and convenient in screening the mutant population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.