Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.     

The Berg-Purcell Limit Revisited

Biological systems often have to measure extremely low concentrations of chemicals with high precision. When dealing with such small numbers of molecules, the inevitable randomness of physical transport processes and binding reactions will limit the precision with which measurements can be made. An important question is what the lower bound on the noise would be in such measurements. Using the theory of diffusion-influenced reactions, we derive an analytical expression for the precision of concentration estimates that are obtained by monitoring the state of a receptor to which a diffusing ligand can bind. The variance in the estimate consists of two terms, one resulting from the intrinsic binding kinetics and the other from the diffusive arrival of ligand at the receptor. The latter term is identical to the fundamental limit derived by Berg and Purcell (Biophys. J., 1977), but disagrees with a more recent expression by Bialek and Setayeshgar. Comparing the theoretical predictions against results from particle-based simulations confirms the accuracy of the resulting expression and reaffirms the fundamental limit established by Berg and Purcell.     

Inhibitor of pyrimidine metabolism from tumor tissues

Inhibitors of normal rat liver 5′-nucleotidase and dUMP kinase $\text{in vitro}$ were found in rapidly proliferating tissues, such as Yoshida sarcoma. Two inhibitors were separated from Yoshida sarcoma by zone electrophoresis, gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography. One inhibited both 5′-nucleotidase and dUMP kinase, while the other inhibited only dUMP kinase. These inhibitors were not detectable in normal rat liver. They were induced in regenerating rat liver and present in rapidly proliferating tissues, such as Yoshida sarcoma and Ehrlich ascites tumor and rat marrow cells. These inhibitors were heat labile. One had a large molecular weight (500,000>) and the other a small molecular weight ($\text{Ca}\text{.}$ 50,000).     

Identification of a caspase 3-independent role of pro-apoptotic factor Bak in TNF-alpha-induced apoptosis

By using our recently developed gene discovery system, we have identified Bak, a member of the Bcl-2 family, as a pro-apoptotic factor in the tumor necrosis factor (TNF)-alpha-induced apoptotic pathway in caspase 3-deficient cells. Unlike Bcl-2, Bak stimulates several apoptotic pathways, however the molecular mechanism(s) of its action remains unclear. For example, it is unclear whether Bak induces apoptosis in caspase 3-deficient cells. In this study, we examined the effects of overexpression of Bak in MCF-7 cells that lack caspase 3. We found that despite the absence of caspase 3 in MCF-7 cells, they were more sensitive to the cell death effects of Bak as compared to caspase 3-expressing HeLa S3 cells. The targeting of Bak function by ribozymes suggests that Bak is required for the TNF-alpha-induced apoptotic pathway in caspase 3-deficient cells. This study demonstrates the caspase 3-independent function of Bak in the TNF-alpha-induced apoptotic pathway.     

Effect of Exogenous Fatty Acids on the Cellular Fatty Acid Composition and Neomycin Formation in a Mutant Strain of Streptomyces fradiae

A mutant of Streptomyces fradiae which requires oleic acid for neomycin formation was isolated and the effects of exogenous fatty acids and other additives on the formation of neomycin were studied. Palmitic acid and high concentration of sodium ions could replace oleic acid in neomycin formation. The fatty acid spectrum of the mutant strain ST–5B was quite different from that of the parent strain 3123. The major fatty acid components of the mutant and the parent were anteiso 15:0 and iso 16: 0, respectively. However the fatty acid composition of the mutant was changed from the anteiso 15: 0-type to the parental iso 16: 0-type by the supplement of oleic acid or high concentration of sodium ions in the medium. In the case of palmitic acid, the major fatty acid component of the mutant cells was changed from anteriso 15: 0 to normal 16:0. The role of these additives in neomycin formation by the mutant is discussed.     

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.     

Techniques of SNP genotyping and remote sensing applied for elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype

We present a conceptual framework for applying techniques of SNP genotyping as a molecular biological approach and remote sensing as an ecological approach to elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype. Additionally, we discuss the obstacles that frustrate our efforts to identify how the human genome encodes the complex phenotype and suggest the use of computational methods designed for knowledge discovery within hereditary database.