Crystallization and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus

A new member of archaeal DNA polymerase from Pyrococcus furiosus was crystallized. Diffraction data to 3.1 A of the selenomethionine-derivatized crystal were collected, and preliminary crystallographic study has been completed. The crystal belongs to the space group C2 with unit cell parameters of a = 93.2 A, b = 124.9 A, c = 87.7 A, alpha = 90 degrees , beta = 109.7 degrees , and gamma = 90 degrees . Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystal is estimated to be 54%, corresponding to a Matthews coefficient V(M) of 2.7A (3) Da(-1).     

Double knockdown of α1,6-fucosyltransferase (<Emphasis Type="Italic">FUT8</Emphasis>) and GDP-mannose 4,6-dehydratase (<Emphasis Type="Italic">GMD</Emphasis>) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

Background  

Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.     

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

Background  

Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs.     

Whole genome analysis of Vietnamese G2P[4] rotavirus strains possessing the NSP2 gene sharing an ancestral sequence with Chinese sheep and goat rotavirus strains

Because imminent introduction into Vietnam of a vaccine against Rotavirus A is anticipated, baseline information on the whole genome of representative strains is needed to understand changes in circulating strains that may occur after vaccine introduction. In this study, the whole genomes of two G2P[4] strains detected in Nha Trang, Vietnam in 2008 were sequenced, this being the last period during which virtually no rotavirus vaccine was used in this country. The two strains were found to be > 99.9% identical in sequence and had a typical DS‐1 like G2‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2 genotype constellation. Analysis of the Vietnamese strains with > 184 G2P[4] strains retrieved from GenBank/EMBL/DDBJ DNA databases placed the Vietnamese strains in one of the lineages commonly found among contemporary strains, with the exception of the NSP2 and NSP4 genes. The NSP2 genes were found to belong to a previously undescribed lineage that diverged from Chinese sheep and goat rotavirus strains, including a Chinese rotavirus vaccine strain LLR with 95% nucleotide identity; the time of their most recent common ancestor was 1975. The NSP4 genes were found to belong, together with Thai and USA strains, to an emergent lineage (VIII), adding further diversity to ever diversifying NSP4 lineages. Thus, there is a need to enhance surveillance of locally‐circulating strains from both children and animals at the whole genome level to address the effect of rotavirus vaccines on changing strain distribution.     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

A radioisotope-nondependent high-sensitivity method for measuring the activity of glioblastoma-related O-methylguanine DNA methyltransferase

O⁶-Methylguanine DNA methyltransferase (MGMT) cancels the anticancer effect of temozolomide (drug for glioblastoma), which introduces methylation to DNA. Therefore, developing an MGMT inhibitor is a promising strategy for the treatment of this cancer. For this purpose, a sensitive detection method that does not depend on the conventional radioisotope (RI) method was developed. This was realized by a fluorescence-based method that measured the amount of cleavable restriction sites demethylated by the action of MGMT; this method was enhanced by introducing a polymerase chain reaction (PCR) amplification step. As an assay of enzyme activity, 20-fold higher sensitivity (subnanomolar) was attained compared with our and others’ fluorescence-based approaches.     

Novel high-affinity Aβ-binding peptides identified by an advanced in vitro evolution, progressive library method

Recent studies have been supporting that the generation of Aβ42 oligomers is responsible for Alzheimer's disease. Therefore, those peptides which bind to Aβ42 are scientifically interesting and can be possible candidates for the diagnosis and therapy of Alzheimer's disease. A systemic in vitro evolution, developed recently and the designated progressive library method (PLM), was applied to obtain Ab42-binding aptamers peptides. As a result, high affinity peptide aptamers made of 8 or 9 amino acids could be identified by this approach, endorsing the methodological effectiveness. Namely, the selection products from the secondary library of diversified peptides, which was constructed based on the information obtained from the primary library selection, were confirmed to be superior to those selected from the primary library as had been reported previously. The affinities of those peptides measured by SPR (surface plasmon resonance) were comparable to or higher than that of those peptides so far reported (K(d) of 10??). The other peptides selected were confirmed of their binding by a novel mode of gel shift assay (fluorescence enhancement caused by the binding). Thus, novel Aβ42-binding peptides with high affinity were provided for the future Alzheimer's disease study. The demonstration of the effectiveness of the systemic in vitro evolution of PLM is very encouraging for the study of identifying novel functional peptides.