Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.     

Plasma organochlorine levels and subsequent risk of breast cancer among Japanese women: a nested case-control study

To our knowledge, no prospective study has examined the association between blood levels of organochlorines and breast cancer risk in Asian countries. Here, we tested the hypothesis that higher blood levels of organochlorines are associated with an increased risk of breast cancer in Japanese women. A total of 24,226 women subjects of the Japan Public Health Center-based Prospective Study aged 40 to 69 years who responded to the baseline questionnaire and provided blood in 1990-1995 were followed to December 2002. During 10.7 years follow-up, 144 cases of breast cancer were newly diagnosed. Two matched-controls for each case were selected from the cohort. Plasma levels of p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), and beta-hexachlorocyclohexane (beta-HCH) were measured. A conditional logistic regression was used to estimate the odds ratio (OR) of breast cancer according to cholesterol-adjusted organochlorine levels based on 139 matched pairs. We found no statistically significant positive association between plasma organochlorine level and breast cancer risk. Adjusted ORs for p,p'-DDT, HCB, and beta-HCH were less than 1. For p,p'-DDE, adjusted OR for the highest versus lowest quartile was 1.48 (95% confidence interval 0.70-3.13; p for trend=0.25). A stratified analysis by menopausal status showed positive associations for p,p'-DDT and p,p'-DDE in premenopausal but not postmenopausal women, although without statistical significance. Our data do not support the hypothesis that plasma levels of p,p'-DDT, p,p'-DDE, HCB, and beta-HCH are associated with an overall increased risk of breast cancer among Japanese women.     

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.     

Monoacylglycerols Activate Capsaicin Receptor, TRPV1

Transient receptor potential vanilloid subtype 1 (TRPV1) is known as capsaicin (CAP) receptor and activated by CAP. Activation of TRPV1 by CAP increases energy expenditure and thermogenesis in rodents or human. Therefore, TRPV1 may be target for energy expenditure enhancement and thermogenesis. To search for novel TRPV1 agonist, we screened 19 types of foods by using TRPV1-expressing HEK293 cells. TRPV1 was activated by hexane extract of wheat flour, and its functional compounds were 1-monoacylglycerols containing oleic, linoleic, and alpha-linolenic acids. Their potencies (EC50) were about 50 times larger than that of CAP and their efficacies (maximal response) were about half of that of CAP. TRPV1 was activated by 1-monoacylglycerols (MGs) having C18 and C20 unsaturated and C8-C12 saturated fatty acid (FA). Moreover, 2-MGs having C18 and C20 unsaturated FA acted on TRPV1 with the same potency. On the other hand, no activation of TRPV1 was induced by MGs having C16 and C18 saturated FA, di- or triacylglycerols of C18:1 FA. Pain-relating aversive responses were induced when TRPV1-activating 1-monoacylglycerols (50 mM) was administered subcutaneously into rat hind paw. These effects were inhibited by the co-injection of capsazepine (10 mM) which is a TRPV1 competitive antagonist. These results suggested that these 1-monoacylglycerols activate TRPV1 in vitro and in vivo.     

Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge

A culture-independent molecular technique using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes was applied to the characterization of bacterial communities of activated sludge taken from different municipal sewage treatment plants. 16S rDNA fragments from the bulk DNA of sludge were amplified by PCR with a Cy5-labeled forward primer corresponding to nucleotide positions 8 to 27 and a reverse primer complementary to positions 907 to 926 in the Escherichia coli numbering system. The 16S rDNAs thus obtained were digested with tetrameric restriction enzymes and analyzed using a Pharmacia automated DNA sequencer. A preliminary study on a model DNA mixture prepared from different bacterial species showed that the fluorescence intensity of terminal fragments (T-RFs) of 16S rDNAs amplified and detected was directly proportional to the 16S rRNA gene copy number rather than the amount of genomic DNA of each species present. 16S rDNA fragments amplified from the sludges and digested with HhaI usually generated at least 60 T-RFs, among which T-RFs of around 208 bp were the most abundant regardless of the time or area sampled. Southern blot hybridization with oligonucleotide probes specific to the 5' terminal regions of the 16S rDNA of different phylogenetic groups indicated that the T-RFs of around 208 bp were derived from members of the beta subclass of the class Proteobacteria. Hybridization with a probe specific to the class Actinobacteria failed to detect any appreciable signal. These results did not agree fully with those obtained by quinone profiling. The usefulness and limitations of the T-RFLP method for monitoring bacterial population dynamics in activated sludge were discussed.     

Rheological and Morphological Comparison of Thermal and Hydrostatic Pressure-Induced Filamentous Myosin Gels

The structure and rheological properties of heat‐ and pressure‐induced myosin filament gels were investigated. The apparent elasticity of heat‐induced gel peaked at 55 °C (4.35 ± 0.57 kPa), whereas that of pressure‐induced gel increased with elevating pressure, and the gel formed at 500 MPa had a value of 4.79 ± 0.25 kPa. All pressure‐ and heat‐induced gels showed similar internal structure, namely, the gels were composed of a fine‐strand network. The detailed structures of the strands induced by pressure‐ and heat‐treatment of myosin filaments were observed using an atomic force microscope. The structural differences among the strands were not observed, whereas the elasticity of the strands measured by atomic force microscope revealed differences among the strands formed with varying heating temperature and pressure. The elasticities of the heat‐induced strands were 1.19 ± 0.09 MPa, 10.24 ± 1.16 MPa, and 3.09 ± 0.25 MPa at 40 °C, 55 °C, and 70 °C, respectively. On the other hand, the elasticity of the pressure‐induced strand increased with elevating pressure. The elasticity values were 1.24 ± 0.09 MPa, 2.32 ± 0.17 MPa, and 9.80 ± 0.84 MPa at pressures of 150, 300, and 500 MPa, respectively. Because the changes in the elasticity of the whole gel corresponded to those of the strand, it is concluded that the rheological properties of the constituting strands determine that of myosin filamentous gel.     

Dual-Hop Non-regenerative OFDM Relay Systems with Chunk-Based Power Allocation

In this paper, we propose dual-hop non-regenerative Orthogonal Frequency Division Multiplexing (OFDM) relay systems without signal buffering and processing delay due to Fast Fourier Transform (FFT) and Inverse FFT (IFFT) at the Relay Node (RN). One scheme uses per subcarrier Power Allocation (PA) at Source Node (SN) and per chunk PA at RN to improve the achievable rate. Another scheme employs per chunk PA at both SN and RN. Numerical results in mobile communication environments show that both schemes improve the achievable rates compared to conventional simple amplify-and-forward relay systems with per subcarrier PA at RN. In particular, the latter scheme can obtain almost the same or slightly higher achievable rates by selecting an appropriate chunk size in severe frequency selective channel and by setting any chunk size in highly frequency-correlated channels. Therefore, chunk-based PA at both SN and RN can be found a beneficial PA for dual-hop OFDM relay systems in terms of competitive achievable rates and relatively small amount of required channel information to calculate allocated power.     

Standard infrared absorption spectrum of betaine and optimal conditions for its measurement

The infrared absorption (IR) spectrum is often used as a standard reference in identification tests of food additives in Japan. In the case of betaine, many different IR spectra have been reported and, therefore, it is necessary to establish an IR spectrum that is reproducible and reliable enough to be used as a standard for identification. In the present study, suitable conditions to obtain a standard IR spectrum were examined from various viewpoints, including pretreatment, selection of method, and measuring technique. The KBr disk method, which has generally been used to identify betaine, was found to be humidity-dependent, and there was also an interaction between betaine and KBr. A reproducible IR spectrum suitable as a standard could be obtained by drying betaine at 105 degrees C for 3 hours over phosphorus pentoxide, and then measuring the IR spectrum by the liquid paraffin (Nujol) paste method.     

北京电视台

概念设计北京电视台是北京市的广播电视新枢纽,地点位于中央商务区,在天安门以东6km。由于市区空间紧凑,节目编辑、制作和管理部分围绕一个钢制"超级结构体"堆叠在一起,"超级结构体"的中心是一个大型中庭,通过这个中庭把各个部门联系在一起,促进了员工的交流。