Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen.     

Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism

Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) has the unique ability to hydrolyze cyclodextrins (CDs), with various sized cavities, as well as starch. To understand the relationship between structure and substrate specificity, x-ray structures of a TVAII-acarbose complex and inactive mutant TVAII (D325N/D421N)/alpha-, beta- and gamma-CDs complexes were determined at resolutions of 2.9, 2.9, 2.8, and 3.1 A, respectively. In all complexes, the interactions between ligands and enzymes at subsites -1, -2, and -3 were almost the same, but striking differences in the catalytic site structure were found at subsites +1 and +2, where Trp(356) and Tyr(374) changed the conformation of the side chain depending on the structure and size of the ligands. Trp(356) and Tyr(374) are thought to be responsible for the multiple substrate-recognition mechanism of TVAII, providing the unique substrate specificity. In the beta-CD complex, the beta-CD maintains a regular conical structure, making it difficult for Glu(354) to protonate the O-4 atom at the hydrolyzing site as a previously proposed hydrolyzing mechanism of alpha-amylase. From the x-ray structures, it is suggested that the protonation of the O-4 atom is possibly carried out via a hydrogen atom of the inter-glucose hydrogen bond at the hydrolyzing site.     

An association between oxidative stress and radiation-induced lymphomagenesis

It is generally thought that reactive oxygen species (ROS) play an important role in carcinogenesis. However, direct evidence supporting this idea is still lacking. In the present study, we measured ROS in thymocytes at the thymic prelymphoma stage in C57BL/6 mice. Mice (n = 20) were irradiated at 1.6 Gy/week for 4 consecutive weeks and the levels of ROS were measured 8 to 11 weeks later by dehydrorhodamine 123, which accumulated in mitochondria and became fluorescent dye upon oxidation. Unirradiated littermates (n = 17) served as controls. Thymic prelymphoma cells were diagnosed by the aberrant CD4/CD8 staining profile and monoclonal or oligoclonal T-cell receptor gene rearrangement. A significant fraction of mice (11/13) bearing thymic prelymphoma cells exhibited elevated levels of ROS in thymocytes (P < 0.001). The result is consistent with the hypothesis that ROS may play an important role in radiation carcinogenesis.     

X-ray structures of NADPH-dependent carbonyl reductase from Sporobolomyces salmonicolor provide insights into stereoselective reductions of carbonyl compounds

The X-ray structures of red yeast Sporobolomyces salmonicolor carbonyl reductase (SSCR) and its complex with a coenzyme, NADPH, have been determined at a resolution of 1.8A and 1.6A, respectively. SSCR was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=54.86 A, b=83.49 A, and c=148.72 A. On its cocrystallization with NADPH, isomorphous crystals of the SSCR/NADPH complex were obtained. The structure of SSCR was solved by a single wavelength anomalous diffraction measurement using a selenomethionine-substituted enzyme, and that of the SSCR/NADPH complex was solved by a molecular replacement method using the solved structure of SSCR. The structures of SSCR and the SSCR/NADPH complex were refined to an R-factor of 0.193 (R(free)=0.233) and 0.211 (R(free)=0.238), respectively. SSCR has two domains, an NADPH-binding domain and a substrate-binding domain, and belongs to the short-chain dehydrogenases/reductases family. The structure of the NADPH-binding domain and the interaction between the enzyme and NADPH are very similar to those found in other structure-solved enzymes belonging to the short-chain dehydrogenases/reductases family, while the structure of the substrate-binding domain is unique. SSCR has stereoselectivity in its catalytic reaction, giving rise to excessive production of (S)-alcohols from ethyl 4-chloro-3-oxobutanoate. The X-ray structure of the SSCR/NADPH complex and preliminary modeling show that the formation of the hydrophobic channel induced by the binding of NADPH is closely related to the stereoselective reduction by SSCR.     

The radioprotective 105/MD-1 complex links TLR2 and TLR4/MD-2 in antibody response to microbial membranes

Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105(-/-) B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105(-/-) mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105(-/-) spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was approximately 3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.     

Escape from the interferon response associated with RNA interference using vectors that encode long modified hairpin-RNA

In mammalian cells, siRNAs have been used to induce RNA interference (RNAi) in an attempt to prevent nonspecific effects (including the interferon (IFN) response) which are caused by long double-stranded RNAs (dsRNAs) of more than 30 bp. In this report, we describe a novel and simple strategy for avoiding activation of the IFN response by dsRNA. We show that modified hairpin-RNAs (mhRNAs) of more than 100 bp, with multiple specific point-mutations within the sense strand and transcribed from the U6 or tRNA(Val) promoters, can cause RNAi without inducing the IFN pathway genes. Moreover, we demonstrate that the 50-bp mhRNA vector could effectively suppress the replication of multiple hepatitis C viruses (the genomes of which differ slightly, thus the 21-bp siRNA vector failed to suppress one of them). Our findings should enhance the exploitation of RNAi in mammalian cells, especially in the field of RNAi therapy against pathogenic viruses.     

Identification of Cardiomyocyte-Fated Progenitors from Human-Induced Pluripotent Stem Cells Marked with CD82

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Natural Selection and the Frequency Distributions of ``silent' DNA Polymorphism in Drosophila

In Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster, codon bias may be maintained by a balance among mutation pressure, genetic drift, and natural selection favoring translationally superior codons. Under such an evolutionary model, silent mutations fall into two fitness categories: preferred mutations that increase codon bias and unpreferred changes in the opposite direction. This prediction can be tested by comparing the frequency spectra of synonymous changes segregating within populations; natural selection will elevate the frequencies of advantageous mutations relative to that of deleterious changes. The frequency distributions of preferred and unpreferred mutations differ in the predicted direction among 99 alleles of two D. pseudoobscura genes and five alleles of eight D. simulans genes. This result confirms the existence of fitness classes of silent mutations. Maximum likelihood estimates suggest that selection intensity at silent sites is, on average, very weak in both D. pseudoobscura and D. simulans (|N(e)s| & 1). Inference of evolutionary processes from within-species sequence variation is often hindered by the assumption of a stationary frequency distribution. This assumption can be avoided when identifying the action of selection and tested when estimating selection intensity.     

Coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in mitochondria of a liverwort Marchantia polymorpha.

The liverwort Marchantia polymorpha mitochondrial DNA encodes almost all tRNAs required for mitochondrial translation except for the isoleucine (AUU, AUC) and threonine (ACA, ACG) codons, while the missing tRNAs are supplied in part by the nucleus and imported in mitochondria. In this paper, we report a finding of two radically different nuclear tRNAVal(AAC) genes and import of the corresponding tRNA isoacceptors in M.polymorpha mitochondria. This finding is surprising since the mtDNA encodes the gene for tRNAVal(UAC), which alone was considered sufficient for translating all four valine codons GUN by the U/N wobble mechanism. The present results suggest for the first time that the import of ncDNA-encoded tRNAs may result in decoding overlaps in plant mitochondria. The coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in liverwort mitochondria and the significance for the decoding mechanism as well as evolution of tRNA import are discussed.