Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme

The gene encoding acetylcholinesterase (AChE) was cloned from common carp muscle tissue. The full-length cDNA was 2368 bp that contains a coding region of 1902 bp, corresponding to a protein of 634 amino acids. The deduced amino acid sequence showed a significant homology with those of ichthyic AChEs and several common features among them, including T peptide encoded by exon T in the C-terminus. Three yeast expression vectors were constructed and introduced into the yeast Pichia pastoris. The transformant harboring carp AChE gene lacking exon T most effectively produced AChE activity extracellularly. The replacement of the native signal sequence with the yeast α-factor prepro signal sequence rather decreased the production. A decrease in cultivation temperature from 30 to 15 °C increased the activity production 32.8-fold. The purified recombinant AChE lacking T peptide, eluted as a single peak with a molecular mass of about 230 kDa on the gel filtration chromatography, exhibited the specific activity of 4970 U/mg. On the SDS–PAGE, three proteins with molecular masses of 73, 54, and 22 kDa were observed. These proteins were N-glycosylated, and their N-terminal sequence showed that the latter two were produced from the former probably by proteolytic cleavage at the C-terminal region. Thus, the recombinant AChE is homotrimer of three identical subunits with 73 kDa. The optimal temperature and pH of the recombinant were comparable to those of the native enzyme purified previously, but the values of kinetic parameters and the sensitivities to substrate inhibition and inhibitors were considerably different between them.     

Elderly People With Low Body Weight May Have Subtle Low‐grade Inflammation

Low‐grade inflammation, which plays important roles in the development of fatal diseases, is commonly observed in obese people. However, this has not been evaluated in lean people, who have relatively increased mortality risk compared with people of normal weight. Here, we elucidate the association between systemic low‐grade inflammation and low body weight, with particular emphasis on aging. We examined the relationship between circulating C‐reactive protein (CRP) and BMI in a cross‐sectional study of 2,675 apparently healthy adults who had undergone a medical check‐up. Overall, subjects with low BMI (<21.0 kg/m², n = 585) showed a favorable cardiovascular profile without being undernourished. In the elderly (≥55 years old), logarithmic CRP (LogCRP) showed a sigmoid curve against BMI with a base at BMI 21.0–22.9 kg/m², but not against waist circumference (WC), even in nonsmokers. In contrast, in middle‐aged people, LogCRP showed an almost linear relationship with both BMI and WC. LogCRP levels in elderly nonsmokers with low BMI, but not normal or high BMI, were significantly higher than those in middle‐aged with corresponding BMI (P < 0.05). After adjustment for age, sex, smoking status, and weight change over the past 2 years, the adjusted means of LogCRP still had a similar sigmoid curve against BMI in the elderly. These results suggest that elderly people with low body weight may have subtle low‐grade inflammation irrespective of a favorable cardiovascular risk, which remains to be confirmed in further studies.     

Floral scents of hawkmoth-pollinated flowers in Japan

Similarity among the floral scents of hawkmoth-pollinated plants was investigated with headspace samplings. Six of seven plant species belonging to different families were found to be rich in isoprenoids, among which linalool was the most common compound. Linalool showed rhythmicity with a nocturnal increase inLonicera japonica. These findings suggest that linalool is a common attractant for nocturnal hawkmoths. However, the composition of other isoprenoids, benzenoids and fatty acid derivatives varied markedly among the plant species examined. There was a significant correlation between species composition of flower-visiting hawkmoths and specific floral scents, suggesting that attractiveness to each hawkmoth species is dependent upon floral scent.     

Study on RNA structure by pyrene-labeled 2'-O-methyloligoribonucleotides.

Properties of 2'-O-methyloligoribonucleotides containing 2'-O-(1-pyrenylmethyl)uridine were investigated as the fluorescent probe to search the single strand regions on RNA secondary and tertiary structure. The pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) showed remarkable increase of fluorescence intensity to 333-fold at 375 nm when hybridized with the complementary oligoribonucleotide. When OMUpy, complementary to loop or stem regions, was applied to E. coli 5S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The difference of the fluorescence intensities corresponds to the higher-order structure of 5S-rRNA, suggesting that pyrene-labled 2'-O-methyloligoribonucleotide can be applicable to search single strand regions of RNA.     

Potential theory for gate adsorption on soft porous crystals

We demonstrate that an adsorption potential at the gate adsorption pressure of soft porous crystals (SPCs) based on the Polanyi's potential theory of adsorption shows a constancy to temperature. This was done using grand canonical Monte Carlo simulations and free energy analysis, which were carried out with a simplified stacked-layer SPC model. This finding implies that the characteristic curve obtained from an experimental gate adsorption isotherm on SPCs can be used to predict the temperature dependence of the gate-opening pressure, even though the potential theory of adsorption does not take into account the deformation of porous solids during the adsorption. We develop a modified potential theory for gate adsorption and show that the derived relation has a form that the Gibbs free energy change due to the host framework deformation per guest molecule, ? ΔG_host/N, and a correction term, C, are added to the expression of the original potential theory of adsorption. The term C is not an empirical correction factor but is the difference of intermolecular interaction potential and entropy between the bulk liquid phase at the saturated state and the adsorbed phase, originating from spatial constraint of adsorbed guest molecules in the host. By evaluating the modified expression for gate adsorption using the simulation results, we demonstrate that the constancy of the adsorption potential to temperature results from a compensation effect between three terms: guest–host interaction potential per guest molecule, ? ΔG_host/N and C, which have a temperature dependence.     

Use of high pressure liquid chromatography for analysis of deoxyribonucleotides in the epidermis

An analytical method for free deoxyribonucleotide (nucleoside monophosphate) in the epidermis is presented. Free nucleotides were extracted from tissue using a methylal ethanol mixture. The deoxyribonucleotides were separated using DEAE-cellulose and DBAE-cellulose. The analysis of free deoxyribonucleotides was carried out by high pressure liquid chromatography on a column of Lichrosorb-NH₂ with a single buffer of potassium phosphate. The elution order of nucleotides was CMP, AMP, UMP, IMP, GMP, and XMP. This procedure employing high pressure liquid chromatography for the detection of deoxyribonucleotides in the epidermis makes it possible to elucidate the biological characteristics and significance of DNA metabolism in normal epidermis and changes that occur in pathological conditions.     

Use of bilateral information to determine the walking direction during orientation to a pheromone source in the silkmoth Bombyx mori

Odor source localization is an important animal behavior. Male moths locate mates by tracking sex pheromone emitted by conspecific females. During this type of behavior, males exhibit a combination of upwind surge and zigzagging flight. Similarly, the male walking moth Bombyx mori responds to transient pheromone exposure with a surge in movement, followed by sustained zigzagging walking. The initial surge direction is known to be influenced by the pheromone input pattern. Here, we identified the sensory input patterns that determine the initial walking direction of males. We first quantified the stimulus by measuring electroantennogram values, which were used as a reference for subsequent tests. We used a brief stimulus pulse to examine the relationship between sensory stimulus patterns and the turning direction of initial surge. We found that the difference in input timing and intensity between left and right antennae affected the walking direction, indicating that B. mori integrate bilateral pheromone information during orientation behavior. When we tested pheromone stimulation for longer periods, turning behavior was suppressed, which was induced by stimulus cessation. This study contributes toward understanding efficient strategies for odor-source localization that is utilized by walking insects.     

Elucidation of gastrointestinal dysfunction in response to irradiation using metabolomics

Gastrointestinal toxicity is frequently observed secondary to accidental or therapeutic radiation exposure. However, the variation in the intestinal metabolites after abdominal radiation exposure remains ambiguous. In the present study, C57BL/6 mice were exposed to 0, 2, and 20 Gy irradiation dose. The Head and chest of each mouse were covered with a lead shield before x-ray irradiation. 24 h post-irradiation treatment, intestinal tissue of each mouse was excised and prepared for metabolites measurement using gas chromatography-mass spectrometry (GC-MS). Our comprehensive analysis of metabolites in the intestinal tissues detected 44 metabolites after irradiation, including amino acids, carbohydrates, organic acids, and sugars. Amino acid levels in the intestinal tissue gradually rose, dependent on the radiation dose, perhaps as an indication of oxidative stress. Our findings raise the possibility that amino acid metabolism may be a potential target for the development of treatments to alleviate or mitigate the harmful effects of oxidative stress-related gastrointestinal toxicity due to radiation exposure.