RNAi silencing of exogenous and endogenous reporter genes using a macrocyclic octaamine as a "compact" siRNA carrier. Studies on the nonsilenced residual activity

A macrocyclic octaamine 1 having a covalently linked lipid-bundle structure was introduced as a new type of siRNA carrier. Gel electrophoresis, DLS, and SPR results indicate that it strongly binds to a luciferase-targeting 21-mer (42P) siRNA with a ratio of 1/P congruent with 0.3 (1/N congruent with 2.4) to give remarkably compact 1-siRNA complexes with an average size of approximately 10 nm. The 1-mediated siRNA silencing of the exogenous luciferase gene occurs with a 90-95% efficiency. The overall suppression-[siRNA] profile with a 5-10% residual activity in the saturation region is commonly observed irrespective of the cell type (HeLa, HepG2, or HEK293), the order, or timing (stepwise or simultaneous) of supply of the siRNA and that of the luciferase-encoding plasmid, the level of mRNA transcribed, or the type of carriers (1 vs lipofectamine 2000). The silencing of the endogenous DsRed2 gene stably incorporated in the genome of HeLa cells also has a similar overall profile. These results suggest that (1) the cellular uptake of the plasmid and that of the siRNA are basically independent of each other and (2) the incomplete silencing is not due to insufficient siRNA delivery. Implication of item 2 is briefly discussed.     

Simple SNP typing assay using a base-discriminating fluorescent probe

We have developed a new concept involving a single-step homogeneous method for single-nucleotide polymorphism (SNP) typing. In this method, a probe containing base-discriminating fluorescent (BDF) bases is added to a sample solution. BDF base-containing DNA usually shows only a weak fluorescence, but emits a strong blue fluorescence when it recognizes a target base at a specific site in a hybridized strand. By utilizing this feature, a simple mix-and-read SNP typing assay was achieved without any tedious probe-designing or washing processes for exclusion of hybridization error or any addition of DNA-modifying enzymes. This is very different from conventional methods. We simultaneously analyzed a number of samples with ease, with a high accuracy, using our BDF assay.     

Cerasome as an infusible and cell-friendly gene carrier: synthesis of cerasome-forming lipids and transfection using cerasome

Sonication of a pre-agitated aqueous solution of cationic lipid having a (EtO)3SiCH2CH2CH2 group on the quarternized ammonium nitrogen results in partially silica- or ceramic-coated liposome (cerasome), which can be used as an excellent transfection agent. Non-silylated reference lipid, which may represent cationic lipids that are used in conventional lipofection experiments, form a compact liposome, which undergoes DNA-induced fusion to provide transfection-irrelevant and larger (100-300 nm), more toxic particles. The surface-rigidified cerasome is infusible and the monomeric cerasome complex of DNA is of viral size (approximately 70 nm) and exhibits a remarkable transfection performance with a 10(2)-10(3)-fold higher efficiency (relative to the non-silylated reference lipid), minimized cytotoxicity and serum compatibility. The cerasome lipid is obtained by the reaction of 3-bromopropyltriethoxysilane with a tertiary amine derivative of the lipid. Preparation of an aqueous cerasome solution takes 1-2 h. The cerasome-DNA complex and the transfection takes about 3 d to complete.     

Structural basis for the design of novel Schiff base metal chelate inhibitors of trypsin

The crystal structures of the complexes of bovine trypsin with m-guanidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 1), [N,N′-bis(m-guanidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 2), and [N,N′-bis(m-amidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 4) have been determined. The guanidine-containing trypsin-inhibitors (1 and 2) bind to the trypsin active site in a manner similar to that previously reported for amidine-containing inhibitors, for example, m-amidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 3). However, the binding mode of the guanidino groups of inhibitors 1 and 2 to Asp189 in the S1 pocket of trypsin was found to be markedly different from that of the amidino group of inhibitor 3. The present X-ray analyses revealed that the interactions of the metal ion of the inhibitors with the active site residues of trypsin play a crucial role in the binding affinity to the trypsin molecule. These structural information and inhibitory activity data for amidine- and guanidine-containing Schiff base metal chelate inhibitors provide new avenues for designing novel inhibitors against physiologically important trypsin-like serine proteases.     

Walking activity of flightless Harmonia axyridis (Coleoptera: Coccinellidae) as a biological control agent

The use of flightless strains of the multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), established via artificial selection, can be highly effective as a biological control agent for aphids. However, flightless H. axyridis must depend on walking for dispersion. Therefore, data on the walking activity levels in flightless strains are important for the development of effective methods when releasing these agents in the field. Results of measurement of walking activity levels using an infrared actograph showed that walking activity levels during the daytime (but not nighttime) in both sexes of pure flightless strains tended to be lower than those of control strains. We also found that walking activity levels during the daytime for the F1 generation of hybrid strains, produced by reciprocal crossing between two pure flightless strains, were approximately equal to those of pure strains; the reduction in walking activity levels was not recovered by hybrid vigor. Our results indicate that the reduction in walking activity levels in the pure flightless strains was not caused merely by inbreeding depression stemming from the artificial selection process. Instead, potentially flight ability and walking activity levels in this species may be controlled by the pleiotropic effect of a gene.     

Elucidation of the speciation history of three sister species of crown-of-thorns starfish (Acanthaster spp.) based on genomic analysis

The crown-of-thorns starfish (COTS) is a coral predator that is widely distributed in Indo-Pacific Oceans. A previous phylogenetic study using partial mitochondrial sequences suggested that COTS had diverged into four distinct species, but a nuclear genome-based analysis to confirm this was not conducted. To address this, COTS species nuclear genome sequences were analysed here, sequencing Northern Indian Ocean (NIO) and Red Sea (RS) species genomes for the first time, followed by a comparative analysis with the Pacific Ocean (PO) species. Phylogenetic analysis and ADMIXTURE analysis revealed clear divergences between the three COTS species. Furthermore, within the PO species, the phylogenetic position of the Hawaiian sample was further away from the other Pacific-derived samples than expected based on the mitochondrial data, suggesting that it may be a PO subspecies. The pairwise sequentially Markovian coalescent model showed that the trajectories of the population size diverged by region during the Mid-Pleistocene transition when the sea-level was dramatically decreased, strongly suggesting that the three COTS species experienced allopatric speciation. Analysis of the orthologues indicated that there were remarkable genes with species-specific positive selection in the genomes of the PO and RS species, which suggested that there may be local adaptations in the COTS species.     

Effects of combined administration of quercetin, rutin, and extract of white radish sprout rich in kaempferol glycosides on the metabolism in rats

Quercetin, rutin, the extract of white radish sprout rich in kaempferol glycosides, and their combination were intragastrically administered to Wistar rats to investigate the interactive metabolism of these flavonoids. The combined administration of these flavonoids changed the concentrations of the metabolites in plasma as compared with the concentrations after the administration of a single compound.     

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.