The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Summary The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.     

Effects of inducible nitric oxide synthase inhibitors on asthma depending on administration schedule

The effectiveness of two inducible nitric oxide synthase (iNOS) inhibitors on allergic airway inflammation was investigated under different administration schedules. Rats sensitized to ovalbumin (OVA) were exposed to OVA for 3 consecutive days. Both iNOS inhibitors showed markedly different effects between two pretreatment schedules: pretreatment before each of three OVA exposures S1 and before the third exposure alone S2. S1 pretreatment resulted in higher pulmonary resistance than triple OVA alone. This potentiation was associated with increased eosinophil infiltration and malondialdehyde levels in the lungs, which were suppressed by superoxide dismutases (SODs) but not by methylprednisolone. However, the S2 administration of both iNOS inhibitors completely suppressed the airway response. Administration by schedule S1 completely suppressed plasma nitrite and nitrate levels, but that by S2 caused only a slight suppression. The triple OVA exposures resulted in the upregulation of iNOS in alveolar macrophages and arginase activity, Mn- and Cu/Zn-SOD expression, and nitrotyrosine and lipid peroxide deposition in the airway. However, inhibitors administered by schedule S1 suppressed this upregulation, but further potentiated nitrotyrosine, which in turn was inhibited by SOD. Although iNOS inhibitors may be beneficial for asthma, repeated administration may be detrimental because of extensive reduction of NO and downregulation of SOD.     

Dynamic state of DNA topology is essential for genome condensation in bacteria

In bacteria, Dps is one of the critical proteins to build up a condensed nucleoid in response to the environmental stresses. In this study, we found that the expression of Dps and the nucleoid condensation was not simply correlated in Escherichia coli, and that Fis, which is an E. coli (gamma-Proteobacteria)-specific nucleoid protein, interfered with the Dps-dependent nucleoid condensation. Atomic force microscopy and Northern blot analyses indicated that the inhibitory effect of Fis was due to the repression of the expression of Topoismerase I (Topo I) and DNA gyrase. In the Deltafis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nucleoid condensation in the presence of Dps. DNA-topology assays using the cell extract showed that the extracts from the Deltafis and Topo I-/DNA gyrase-overexpressing strains, but not the wild-type extract, shifted the population toward relaxed forms. These results indicate that the topology of DNA is dynamically transmutable and that the topology control is important for Dps-induced nucleoid condensation.     

Identification of hepatitis C virus (HCV) 2a-derived epitope peptides having the capacity to induce cytotoxic T lymphocytes in human leukocyte antigen-A24+ and HCV2a-infected patients

Since virus-specific cytotoxic T lymphocytes (CTLs) play a critical role in preventing the spread of hepatitis C virus (HCV), vaccine-based HCV-specific CTL induction could be a promising strategy to treat HCV-infected patients. In this study, we tried to identify HCV2a-derived epitopes, which can induce human leukocyte antigen (HLA)-A24-restricted and peptide-specific CTLs. Peripheral blood mononuclear cells of HCV2a-infected patients or healthy donors were stimulated in vitro with HCV2a-derived peptides, which were prepared based on the HLA-A24 binding motif. As a result, three peptides (HCV2a 576-584, HCV2a 627-635, and HCV2a 1085-1094) efficiently induced peptide-specific CTLs from HLA-A24(+) HCV2a-infected patients as well as healthy donors. The cytotoxicity was exhibited by peptide-specific CD8(+) T cells in an HLA-A24-restricted manner. In addition, the HCV2a 627-635 peptide was frequently recognized by immunoglobulin G of HCV2a-infected patients. These results indicate that the identified three HCV2a peptides might be applicable to peptide-based immunotherapy for HLA-A24(+) HCV2a-infected patients.     

Inhibitory activity of 1-farnesylpyridinium on the spatial control over the assembly of cell wall polysaccharides in Schizosaccharomyces pombe

The modes of actions of 1-farnesylpyridinium (FPy) on yeast cell growth were investigated on the basis of its effects on cell cycle progression, morphogenesis and the related events for construction of cell wall architecture in Schizosacchromyces pombe. FPy predominantly inhibited the growth of the yeast cells after various cycles of cell division so that cells were arrested at the phase of separation into daughter cells accompanying morphological changes to swollen spherical cells at 24 h of incubation. FPy-treated cells were osmotically stable but were susceptible to the lytic action of (1, 3) beta-D-glucanases, and characterized by serious damages to the cell wall architecture as represented by a rough and irregular surface outlook. The isolated cell wall fraction gave a similar hexose composition with or without FPy treatment, suggesting that FPy did not inhibit the synthesis of each cell wall polysaccharide. FPy was permissive for the extracellular accumulation of amorphous cell wall materials and septum development in protoplasts, but absolutely interfered with the following morphogenetic process for construction of the rod-shaped cell wall architecture. Our results suggest the inhibitory activity of FPy on the spatial control over the assembly of cell wall polysaccharides.     

Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.     

Establishment and functional characterization of novel natural killer cell lines derived from a temperature-sensitive SV40 large T antigen transgenic mouse

Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.     

Morphological appearance of epidermal cells cultured on fibroblast-reorganized collagen gels

Summary Human foreskin fibroblasts were used to reorganize hydrated collagen gels into a dermal-like matrix, after which freshly isolated keratinocytes isolated from rabbit ear skin were placed on the surfaces of the matrices and cultured for up to 12 days. Transmission electron microscopy revealed 8–12 cell layers of epidermal cells organized in three distinct strata. The basal stratum consisted of cuboidal to columnar cells with typical complement of organelles, oval nuclei, and prominent tonofilaments inserting into desmosomes. Mitotic cells often were found at this level. There was no well-defined basement membrane region; rather, many of the cells appeared to be in close contact with collagen fibrils. The intermediate stratum of suprabasal cells consisted of elongated cells that had reduced organelles, but still were connected to each other by desmosomes. Finally, the superficial stratum of suprabasal cells contained cells that were completely flattened and often appeared to be sloughing off the apical surfaces of the cultures. Indirect immunofluorescence studies carried out on frozen sections revealed bullous pemphigoid antigen associated with basal epidermal cells; pemphigus vulgaris antigen around the epidermal cells of all strata, and keratin present in the epidermal cells of all strata. Filaggrin was observed in punctate and fibrillar arrangements in suprabasal cells. Fibronectin was found in a linear deposit at the dermal-epidermal junction and around the fibroblasts in the reorganized collagen gels. Type-IV collagen and laminin, however, were not detected.