DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.     

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.     

Development of a strain for efficient degradation of polychlorinated biphenyls by patchwork assembly of degradation pathways

Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.     

Discrimination of psychoactive fungi (commonly called "magic mushrooms") based on the DNA sequence of the internal transcribed spacer region

'Magic mushrooms' (MMs) are psychoactive fungi containing the hallucinogenic compounds, psilocin (1) and psilocybin (2). Since June 6, 2002, these fungi have been regulated by the Narcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the internal transcribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese markets to classify them by a genetic approach. Based on the size and nucleotide sequence of the ITS region amplified by PCR, tested MMs were classified into 6 groups. Furthermore, a comparison of the DNA sequences of the MMs with those of authentic samples or with those found in the databases (GenBank, EMBL and DDBJ) made it possible to identify the species of tested MMs. Analysis by LC revealed that psilocin (1) was contained at the highest level in Panaeolus cyanescens among the MMs, but was absent in the Amanita species.     

Establishment of a simple system to analyse the molecular interaction in the agglutination of Saccharomyces cerevisiae

Saccharomyces cerevisiae a-agglutinin, which is involved in mating and covalently anchoring to the cell wall, consists of two components, Aga1p and Aga2p, whose syntheses are individually regulated. To facilitate the analysis of the protein-protein interaction on agglutination between a- and alpha-agglutinins, the construction of a yeast strain (MATa) with the functional protein prepared by genetic fusion of Aga1p- and Aga2p-encoding genes and by the expression system using the UPR-ICL promoter derived from the n-alkane-assimilating yeast, Candida tropicalis, which is functional under the condition of lower glucose concentration was tried and the agglutination ability of the constructed strain was evaluated with a yeast strain (MATa) which expressed AGalpha1 encoding alpha-agglutinin under the control of the same promoter. The genes were integrated into the yeast chromosomes. Cell agglutination between both (MATa) strains was observed microscopically when these two strains were mix-cultured to a glucose-decreased concentration. The agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed Aga1p-Aga2p fusion protein was enoughly functional for the interaction with the Agalpha1 protein, and that this phenomenon occurred dependent on glucose concentration, but independent of the peptide pheromones secreted by the cells of the opposite mating types. Using this system, the role of two disulphide linkages between Aga1p and Aga2p on the binding activity between Aga2p and Aga1p was first evaluated. Under the treatment by the SH-compound (dithiothreitol), in which Agalpha2p is easily released into the medium from the intact cell surface, the Aga1p and Aga2p fusion protein was a good tool to make clear the role of the disulphide linkages. As a result, the linkages had a significant effect on not only the assembly but also the binding activity. The novel and simple system described here may further facilitate the study of molecular interaction in agglutination.     

Sonochemical decomposition of perfluorooctane sulfonate and perfluorooctanoic acid

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are shown to be globally distributed, environmentally persistent, and bioaccumulative. Although the toxicities of these compounds were reported, the cleanup procedure from the environment is not developed because of their inertness. In this report the sonochemical degradations of PFOS and PFOA to the products through the fission of the perfluorocarbon chains were observed and the half-life times of the PFOS and PFOA degradations under an argon atmosphere determined to be 43 and 22 min, respectively. The shortening of perfluorocarbon chain of PFOS and PFOA leads to the lowering of the toxicity in view of the decrease of the persistence, and the technique would contribute to the remediation of the environmental pollution by these compounds.     

DNA and chemical analyses of commercial fly agaric-related products

Since June 6, 2002, psilocin and psilocybin-containing fungi (commonly called "magic mushrooms") have been regulated by the Narcotics and Psychotropics Control Law in Japan. However, various fly agaric-related products are now entering the Japanese market via the internet. In this study, fly agaric-related products available in this way were investigated for raw materials by DNA analysis and for additives by chemical analysis. Nucleotide sequence analysis of the mitochondrial 12S rDNA region suggested that these fly agaric-related products originate from A. muscaria or A. muscaria var. persicina. Furthermore, they were classified into three strains based on the ITS2-LSU nucleotide sequence. Harmine derivatives and/or tryptamine derivatives were detected in some of these products by LC/MS analysis. In accordance with this, the matK gene of Peganum harmala was found in all of the harmine derivative-containing samples.     

Effects of plasticizers on the properties of edible films from skin gelatin of bigeye snapper and brownstripe red snapper

The effects of type and concentration of plasticizers on the mechanical properties (tensile strength, TS and elongation at break, EAB), water vapor permeability, light transmission, transparency and color of fish skin gelatin edible films from bigeye snapper (Priacanthus marcracanthus) and brownstripe red snapper (Lutjanus vitta) were investigated. At the same plasticizer concentration, fish skin gelatin films from both species plasticized with glycerol (Gly) showed the greatest EAB (P<0.05), whereas ethylene glycol (EG) plasticized films showed the highest TS (P<0.05). Films prepared from brownstripe red snapper skin gelatin exhibited slightly greater TS than those of bigeye snapper skin gelatin (P<0.05) when Gly and sorbitol (Sor) were used. EG, polyethylene glycol 200 (PEG 200) and polyethylene glycol 400 (PEG 400) affected the mechanical properties of both films differently. Films generally became more transparent and EAB, water vapor permeability (WVP), as well as light transmission of films increased, but TS and yellowness decreased with increasing plasticizer concentrations.     

Crystal growth and scintillation properties of Nd-doped Lu3Al5O12 single crystals with different Nd concentrations

Nd 0.1%, 0.5%, 1% and 3% doped Lu₃Al₅O₁₂ (Nd:LuAG) single crystals were grown in the nitrogen atmosphere by the micro-pulling down (μ-PD) method. The grown crystals had a single-phase confirmed by powder XRD analysis. In absorption spectra, some weak absorption lines due to Nd³⁺ 4f-4f transitions were observed and their intensity increased with the increase of Nd concentration. When excited by ²⁴¹Am α-ray, a broad emission peak due to defects in the host lattice at 320 nm and some sharp lines due to Nd³⁺ 4f-4f transitions at wavelength longer than 400 nm were observed. The decay time profiles of Nd:LuAG under γ-ray excitation were well approximated by two exponential function of 340-760 ns and 3-5 μs for each sample. By pulse height measurement using ¹³⁷Cs, Nd 0.5%:LuAG showed the highest light yield of 7600 ± 760 photons/MeV.