Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.     

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.     

Interindividual differences in thermal comfort and the responses to skin cooling in young women

The present study aims to understand the effects of interindividual differences in thermal comfort on the relationship between the preferred temperature and the thermoregulatory responses to ambient cooling. Thirteen young women subjects chose the preferred ambient temperature (preferred T_a) in a climate chamber and were categorized into the H group (preferring ≥29 °C; n=6) and the M group (preferring <29 °C; n=7). The H group preferred warmer sensations than the M group (P<0.05) and the average of preferred T_a was 27.6 °C and 30.2 °C in the M group and H group, respectively. Then all subjects were exposed to temperature variations in the climate chamber. During T_a variations from 33 °C to 25 °C, the H group felt colder than the M group, although no difference was noted in the T_sk (mean skin temperature) and T_s-hand between the 2 groups. From the view of the relationship between the T_sk and thermal sensation, although the thermal sensitivity to the T_sk was almost similar in the H and M groups, the H group might have lower threshold to decreasing T_a than the M group.     

Amino acid changes in hemagglutinin contribute to the replication of oseltamivir-resistant H1N1 influenza viruses

Oseltamivir-resistant H1N1 influenza viruses emerged in 2007 to 2008 and have subsequently circulated widely. However, prior to 2007 to 2008, viruses possessing the neuraminidase (NA) H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. NA mutations that compensate for the deleterious effect of the NA H274Y mutation have since been identified. Given the importance of the functional balance between hemagglutinin (HA) and NA, we focused on amino acid changes in HA. Reverse genetic analysis showed that a mutation at residue 82, 141, or 189 of the HA protein promotes virus replication in the presence of the NA H274Y mutation. Our findings thus identify HA mutations that contributed to the replacement of the oseltamivir-sensitive viruses of 2007 to 2008.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

A study of seed dispersal by flood flow in an artificially restored floodplain

Riverine floodplains play many important roles in river ecosystems. However, many floodplains have suffered degradation or loss of ecological function due to excessive river improvements or through changes in agricultural systems. As a result, many floodplain restoration projects are being conducted worldwide. One of the many methods being implemented to restore floodplain vegetation is flood water seed dispersal. In this technique, precisely estimating the effect of seed dispersal by flood water is important in order to achieve successful floodplain revegetation. Here, we focus our attention on sediment transport by flood water into the Azamenose Swamp, a restored floodplain. We attempt to estimate the function of seed deposition in the restored floodplain and explain how the seeds are deposited in the floodplain by flood water. The result suggests that the restored floodplain functions as a more appropriate deposition site for seeds than the riverbanks of the main river. It was also found that the distance from the inflow site and the weight of the sediment were related to seed deposition.     

Alkannin,HSP70 Inducer,Protects against UVB-Induced Apoptosis in Human Keratinocytes

Alkannin is an active constituent from the root extract of Alkanna tinctoria of the Boraginaceae family and it may have utility as a heat shock protein 70 (HSP70) inducer in living organisms. Here, the effects of alkannin-induced HSP70 on ultraviolet (UV) B (40 mJ/cm²)-induced apoptosis were investigated in human keratinocyte HaCaT cells. Pretreatment of cells with alkannin (1 µM) caused significant inhibition of UVB-induced apoptosis and caspase-3 cleavage. On the other hand, the addition of KNK437 (HSP70 inhibitor) reversed the action of alkannin increasing UVB-induced apoptosis in a dose-dependent manner. In addition, differences in gene expression associated with the suppression of UVB-induced apoptosis in the presence of alkannin were investigated using Gene Chip assay. Our results indicate that alkannin suppresses UVB-induced apoptosis through the induction of HSP70 in human keratinocytes, and therefore, we suggest the usefulness of using alkannin as an antiaging agent.     

Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: Improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC₅₀: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI₅₀: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).     

Method for inferring and extracting reliable genetic interactions from time-series profile of gene expression

Recent advances in technologies such as DNA microarrays have provided an abundance of gene expression data on the genomic scale. One of the most important projects in the post-genome-era is the systemic identification of gene expression networks. However, inferring internal gene expression structure from experimentally observed time-series data are an inverse problem. We have therefore developed a system for inferring network candidates based on experimental observations. Moreover, we have proposed an analytical method for extracting common core binomial genetic interactions from various network candidates. Common core binomial genetic interactions are reliable interactions with a higher possibility of existence, and are important for understanding the dynamic behavior of gene expression networks. Here, we discuss an efficient method for inferring genetic interactions that combines a Step-by-step strategy (Y. Maki, Y. Takahashi, Y. Arikawa, S. Watanabe, K. Aoshima, Y. Eguchi, T. Ueda, S. Aburatani, S. Kuhara, M. Okamoto, An integrated comprehensive workbench for inferring genetic networks: Voyagene, Journal of Bioinformatics and Computational Biology 2(3) (2004) 533.) with an analysis method for extracting common core binomial genetic interactions.