Neuron-specific recombination by Cre recombinase inserted into the murine tau locus

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 ‘floxed’ mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.     

Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex

Interactions of TBP-interacting protein (TIP26), TBP, and TFB from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with TATA-DNA were examined by electrophoretic mobility shift assay. Tk-TFB formed a ternary complex with Tk-TBP and TATA-DNA. Tk-TIP26 did not inhibit the formation of this ternary complex, but interacted with it to form a TIP26/TFB/TBP/DNA quaternary complex. This interaction is rather weak, and a large excess of Tk-TIP26 over Tk-TBP is required to fully convert the TFB/TBP/DNA ternary complex to the quaternary complex. However, determination of the concentration of Tk-TIP26 and Tk-TBP in KOD1 cells by Western blotting analysis indicated that the concentration of Tk-TIP26 is approximately ten times that of Tk-TBP, suggesting that the quaternary complex might also form in vivo.     

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly⁻⁸² to Gly³¹⁶), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca²⁺ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.     

Effect of inhaled indomethacin on distilled water-induced airway epithelial cell swelling.

We evaluated the mechanism of the anti-asthmatic effect of inhaled indomethacin (Indo) by using an animal model (guinea pigs) of airway inflammation. After being exposed to either ozone or room air at identical flow rates (5 l/min) for 2 h, guinea pigs were anesthetized, tracheostomized, and lung resistance (RL) was subsequently measured. Guinea pigs inhaled either saline or Indo (1.5 mg/ml) for 1 min before undergoing an ultrasonically nebulized distilled water (UNDW) inhalation test. RL increased significantly after 10 min of UNDW inhalation in the room air and ozone groups but more so in the ozone group. This increase in RL was significantly suppressed by pretreatment with Indo. In the morphometric assessment of airway mucosa, a significant swelling of the epithelial cells after UNDW inhalation was observed in both the room air and ozone groups but especially so in the ozone group. This increase was also suppressed with Indo pretreatment. These results suggest that the increase in RL and the swelling of airway epithelial cells induced by inhaled UNDW in ozone-exposed guinea pigs was suppressed by pretreatment of inhaled Indo and that this suppression may be one of the reasons for the anti-asthmatic effect of inhaled Indo.     

Phytoestrogens from the roots of Polygonum cuspidatum (Polygonaceae): structure-requirement of hydroxyanthraquinones for estrogenic activity.

The methanolic extract from the roots of Polygonum (P.) cuspidatum was found to enhance cell proliferation at 30 or 100 microg/mL in MCF-7, an estrogen-sensitive cell line. By bioassay-guided separation from P. cuspidatum with the most potent activity, emodin and emodin 8-O-beta-D-glucopyranoside were isolated as active principles. The methanolic extracts from Polygonum, Cassia, Aloe, and Rheum species, which were known to contain anthraquinones, also showed the MCF-7 proliferation. As a result of the evaluation of various anthraquinones from plant sources and synthetic anthraquinones, aloe-emodin, chrysophanol, chrysophanol 8-O-beta-D-glucopyranoside, and 1,8-dihydroxyanthraquinone showed weak activity. On the other hand, alizalin and 2,6-dihydroxyanthraquinone as well as emodin having the 2- and/or 6-hydroxyl groups showed potent activity. These results show that the unchelated hydroxyl group is essential for strong activity. Emodin and 2,6-dihydroxyanthraquinone also inhibited 17beta-estradiol binding to human estrogen receptors (ERs) with K(i) values of 0.77 and 0.31microM for ERalpha and 1.5 and 0.69 microM for ERbeta. These findings indicate that hydroxyanthraquinones such as emodin are phytoestrogens with an affinity to human estrogen receptors.     

Comparative analysis of microbial diversity and bacterial seedling disease‐suppressive activity in organic‐farmed and standardized commercial conventional soils for rice nursery cultivation

The outbreak of rice plant diseases can be effectively suppressed in organic farming systems. However, the mechanisms of disease suppression by organic farming systems are not well understood. When Burkholderia‐infected rice seeds were sown and cultivated on nine organic‐farmed soils which were supplied by nine independent organic rice farmers or standardized commercial conventional soils, the emergence of bacterial seedling diseases was suppressed to equivalent degrees in nine organic‐farmed soils, whereas the diseases occurred in two commercial conventional soils. In any organic or commercial conventional soil sown with healthy rice seeds as a control, the diseases did not appear. Upon physicochemical analysis of the nine organic‐farmed soils, component common to these organic‐farmed soils seemed to not be directly associated with disease‐suppressive activity. However, microbiome analyses indicated that the bacterial population in these nine organic‐farmed soils was more diverse than those in commercial conventional soils. Intriguingly, the diverse bacterial population structures of organic‐farmed soils were preserved after irrigating and sowing rice seeds, but that of commercial conventional soils was clearly changed by them. Thus, organic‐farmed soils seem to maintain robust bacterial populations despite the irrigation and seedling growth. Indeed, pathogenic Burkholderia in infected rice seeds also did not proliferate in the seedling grown on organic‐farmed soils. Taken together, the common feature of organic‐farmed soils might be the correlation between bacterial seedling disease‐suppressive activity and higher robustness of the diversified microbiome.     

Effects of co-inoculation of two different plant growth-promoting bacteria on duckweed

Aseptic Lemna minor was soaked for 4 h in pond water where wild L. minor was naturally flourishing. Seven of the eight surface-colonizing bacterial strains were found capable of promoting the growth of L. minor. This high appearance of plant growth-promoting bacteria (PGPB) suggests that association of environmental bacteria is generally beneficial rather than harmful for host plants. One of the PGPB, Pseudomonas sp. Ps6, enhanced the growth of L. minor by 2–2.5-fold in 10 days. This activity was higher than that previously reported for Acinetobacter calcoaceticus P23, which enhanced growth of L. minor by 1.5–2-fold. Ps6 mostly adhered to and colonized the root rather than the frond, a leaf-like structure of duckweed where P23 preferentially adheres. It was expected that these two strains can share niches, coexist, and enhance the growth of duckweed additively upon co-inoculation. However, contrary to expectation, the growth of L. minor was enhanced by only 2.3-fold by co-inoculation of these two bacteria. P23 lowered the initial adhesion of Ps6 cells by 98.2% on the fronds and by 79.5% on the roots. However, initial adhesion of P23 cells to the roots increased dramatically, by 47.2-fold, following co-inoculation with Ps6. However, the number of P23 cells decreased dramatically to 0.7% on the root and to 3.6% on the frond after 10 days, whereas Ps6 cells increased by 12.5-fold on the frond and kept 69% on the root, thereby eventually restoring the population on the plant surfaces. Because duckweed is the fastest growing vascular plant and it is easy to grow an aseptic and axenic plant, the duckweed/bacteria co-culture system will be a model platform for studying multiple interactions among host plants and the associated bacteria.     

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model

Francisella tularensis, which causes tularemia, is an intracellular gram‐negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 10⁶ CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD₅₀) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD₅₀ of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.     

Acclimatization and response of minipigs toward humans.

As a first step toward making an efficient acclimatization methodology for minipigs, the reaction of G?ttingen minipigs, 3-24 months of age, toward humans was investigated. All minipigs were kept in an individual cage, and the reaction toward humans (acclimatization index) was evaluated by simple observations. The acclimatization index was evaluated as the total number of points scored (0-30 points) based on the following criteria: (1) the position of the minipig when the cage door was opened; (2) the reaction of the minipig when the observer approached; and (31 the reaction of the minipig when the observer touched it. Subsequently, each animal was ranked by total points scored: 30 points = AA, 20 points < or = A < 30 points, 10 points < or = B < 20 points, 0 points < or = C < 10 points. Based on this evaluation, the reactions of minipigs under three conditions were investigated. The following findings were confirmed: first, minipig reaction to humans was influenced by monthly age; second, taming was possible under ordinary conditions of care, but we had to wait until 10 months of age on average for this to occur; third, if simple contact was made during care time, minipigs became tame within less than 4 weeks after the commencement of contact. We therefore consider it possible to artificially control the reaction of minipigs toward humans, and to make minipigs more available for experiments by adding control of the hereditary factors that influence this reaction.