Preparation of thermoresponsive cationic copolymer brush surfaces and application of the surface to separation of biomolecules

We have prepared poly( N-isopropylacrylamide (IPAAm)- co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) brush-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) using the CuCl/CuCl 2/Me 6TREN catalytic system in 2-propanol at 25 degrees C for 16 h. The prepared temperature-responsive surfaces were characterized by chromatographic analysis using the modified silica beads as stationary phases. Chromatographic retention times for adenosine nucleotides in aqueous mobile phases were significantly increased compared to that previously reported for other cationic hydrogel surfaces, indicating that strong electrostatic cationic copolymer brush interactions occur between the surfaces and nucleotide analytes. Retention times for adenosine nucleotides significantly decreased with increasing column temperature, explained by the decreasing basicity in the copolymer with increasing temperature. Step-temperature gradients from 10 to 50 degrees C shorten ATP retention times. These results indicate that cationic copolymer brush surfaces prepared by ATRP can rapidly alter their electrostatic properties by changing aqueous temperature.     

Crystal Structure of the Cmr2–Cmr3 Subcomplex in the CRISPR–Cas RNA Silencing Effector Complex

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci found in prokaryotes are transcribed to produce CRISPR RNAs (crRNAs) that, together with CRISPR-associated (Cas) proteins, target and degrade invading genetic materials. Cmr proteins (Cmr1–6) and crRNA form a sequence-specific RNA silencing effector complex. Here, we report the crystal structures of the Pyrococcus furiosus Cmr2–Cmr3 subcomplex bound with nucleotides (3′-AMP or ATP). The association of Cmr2 and Cmr3 forms an idiosyncratic crevasse, which binds the nucleotides. Cmr3 shares structural similarity with Cas6, which cleaves precursor crRNA for maturation, suggesting the divergent evolution of these proteins. Due to the structural resemblance, the properties of the RNA binding surface observed in Cas6 are well conserved in Cmr3, indicating the RNA binding ability of Cmr3. This surface of Cmr3 constitutes the crevasse observed in the Cmr2–Cmr3 complex. Our findings suggest that the Cmr2–Cmr3 complex uses the crevasse to bind crRNA and/or substrate RNA during the reaction.     

Design and synthesis of antiangiogenic/heparin-binding arginine dendrimer mimicking the surface of endostatin

We designed and synthesized the antiangiogenic arginine-rich dendrimers, TX-1943 and TX-1944, which mimic the surface structure of endostatin. TX-1944 containing 16 arginine residues is more similar in surface structure to endostatin than TX-1943 with eight arginine residues, and has stronger in vivo antiangiogenic activity at 10 microg/CAM in the chicken embryo chorioallantoic membrane (CAM) assay. TX-1944 also has stronger activity to bind heparin and to inhibit the growth of rat lung endothelial (RLE) cells than TX-1943.     

Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3’s known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.     

Identification and Characterization of CD300H,a New Member of the Human CD300 Immunoreceptor Family

Recruitment of circulating monocytes and neutrophils to infection sites is essential for host defense against infections. Here, we identified a previously unannotated gene that encodes an immunoglobulin-like receptor, designated CD300H, which is located in the CD300 gene cluster. CD300H has a short cytoplasmic tail and associates with the signaling adaptor proteins, DAP12 and DAP10. CD300H is expressed on CD16⁺ monocytes and myeloid dendritic cells. Ligation of CD300H on CD16⁺ monocytes and myeloid dendritic cells with anti-CD300H monoclonal antibody induced the production of neutrophil chemoattractants. Interestingly, CD300H expression varied among healthy subjects, who could be classified into two groups according to “positive” and “negative” expression. Genomic sequence analysis revealed a single-nucleotide substitution (rs905709 (G→A)) at a splice donor site on intron 1 on either one or both alleles. The International HapMap Project database has demonstrated that homozygosity for the A allele of single nucleotide polymorphism (SNP) rs905709 (“negative” expression) is highly frequent in Han Chinese in Beijing, Japanese in Tokyo, and Europeans (A/A genotype frequencies 0.349, 0.167, and 0.138, respectively) but extremely rare in Sub-Saharan African populations. Together, these results suggest that CD300H may play an important role in innate immunity, at least in populations that carry the G/G or G/A genotype of CD300H.     

Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy

Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the biodistribution to develop tumor-selective boron carriers. Integrin αvβ3 is an attractive target for anti-tumor drug delivery because of its specific expression in proliferating endothelial and tumor cells of various origins. We, therefore, selected a c(RGDfK) moiety recognizing αvβ3 as an active tumor-targeting device to conjugate with icosahedral boron-10 clusters, disodium mercaptododecaborate (BSH) or o-carborane as a thermal neutron-sensitizing unit. Preparation of o-carborane derivatives involved microwave irradiation, and resulted in high yields in a short time. An in vitro cell adhesion assay on αvβ3-positive U87MG and SCCVII cells demonstrated the high binding affinity of conjugates to integrin αvβ3 (IC(50)=0.19-2.66 μM). Biodistribution experiments using SCCVII-bearing mice indicated that GPU-201 showed comparable tumor uptake and a significantly longer retention in tumors compared with BSH. These results suggest that GPU-201 is a promising candidate for use in BNCT.     

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5′- and 3′-regions of a target gene. Of the four primers used in amplification of the 5′- and 3′-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5′- or 3′-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5′ and 3′ PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.     

THE SYMMETRY OF CORRELATED SELECTION RESPONSES IN ADAPTIVE EVOLUTION: AN EXPERIMENTAL STUDY USING DROSOPHILA

The relationship between the processes of density-dependent and age-specific selection has been investigated by examining a common phenotype, urea resistance, which has apparently evolved in response to each of these selection mechanisms. Twenty populations that have experienced differing levels of age-specific selection show differences in egg-to-adult viability in environments with high levels of urea. Among this group of populations, it appears that resistance to urea is correlated with longevity, but not development time. Ten populations kept at extreme larval densities for many generations also show responses to urea: those kept at high larval densities appear to be most resistant to urea. However, these populations show no differences in adult longevity. An additional five populations were selected directly for urea resistance by adding this compound to the larval food environment. Again, there was a strong response to this artificial selection, with urea resistance increasing dramatically, but these populations showed no response in adult longevity or resistance to crowding when compared to five control populations. There is clearly no simple relationship between longevity and larval urea resistance. It may be that age-specific and density-dependent selection induce similar changes in this phenotype, but do so through different genetic and physiological pathways. We suggest that these data are not consistent with the view of constant and symmetric genetic variance-covariance matrices. These data support a more prominent role for observations of evolutionary trajectories rather than static measurements of genetic components of variance.     

Design and synthesis of 2,6-diprenyl-4-iodophenol TX-1952 with a novel and potent anti-peroxidative activity

The structure-based elucidation of 2,4,6-tri-substituted phenols for their antioxidative and anti-peroxidative effects has been investigated using TX-1952 (2,6-diprenyl-4-iodophenol), TX-1961, TX-1980, BTBP and BHT. In the inhibition of mitochondrial lipid peroxidation, the inhibitory activity of 2,6-di-tert-butyl-4-bromophenol (BTBP) (IC(50)=0.17 microM) was twice as high as that of 2,6-di-tert-butyl-4-methylphenol (BHT) (IC(50)=0.31 microM). This result shows that the 4-halogen group increases inhibitory activity for mitochondrial lipid peroxidation. Besides, TX-1952 (IC(50)=0.60 microM) was the highest inhibitor among 2,6-diprenyl-4-halophenols, followed by TX-1961 (IC(50)=0.93 microM) and TX-1980 (IC(50)=1.2 microM). In 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging experiments, the activity of TX-1952 (IC(0.200)=53.1 microM) was lower than that of BHT (IC(0.200)=33.7 microM) and BTBP (IC(0.200)=16.0 microM), but TX-1952 and BHT showed the same HOMO energy (-8.991 eV). These results suggest that the two prenyl groups at ortho position hinder the phenolic hydrogen abstraction by DPPH radical. These findings demonstrated that TX-1952 was a novel and potent inhibitor for lipid peroxidation.