Structural analysis of a Lotus japonicus genome. II. Sequence features and mapping of sixty-five TAC clones which cover the 6.5-mb regions of the genome.

Sixty-five TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of Lotus japonicus accession MG-20 based on the sequence information of expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined. The average insert size of the TAC clone was approximately 100 kb, and the total length of the sequenced regions in this study is 6,556,100 bp. Together with the nucleotide sequences of 56 TAC clones previously reported, the regions sequenced so far total 12,029,295 bp. By comparison with the sequences in protein and EST databases and by analysis with computer programs for gene modeling, a total of 711 potential protein-encoding genes with known or predicted functions, 239 gene segments and 90 pseudogenes were identified in the newly sequenced regions. The average gene density assigned so far was 1 gene/9140 bp. The average length of the assigned genes was 2.6 kb, which is considerably larger than that assigned in the Arabidopsis thaliana genome (1.9 kb for 6451 genes). Introns were identified in approximately 73% of the potential genes, and the average number and length of the introns per gene were 3.4 and 377 bp, respectively. Simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated based on the nucleotide sequences of the genomic clones obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

New occurrence of microchromosomes B in Moenkhausia sanctaefilomenae (Pisces, Characidae) from the Paraná River of Brazil: analysis of the synaptonemal complex

The Moenkhausia sanctaefilomenae specimens showed a karyotype consisting of 2n = 50 chromosomes with 12 metacentrics, 36 submetacentrics and two subtelocentrics. In addition to the basic karyotype, all the males specimens have cells ranging from zero to two B microchromosomes in mitotic metaphases. These chromosomes were not observed in the female specimens. C-band analysis showed a distribution pattern of characteristic heterochromatin with interstitial and centromeric blocks. However, the B chromosomes were faintly stained with C-banding and were not fluorescent with CMA₃ staining. The meiotic studies showed the formation of bivalents in metaphase I and in pachytene under an optical microscope. Through synaptonemal complex analysis with an electron microscope, the pachytene showed 25 bivalents completely paired and a small bivalent corresponding to the B chromosomes. In the same preparation, one of the B chromosomes was observed in a univalent form. On the basis of pairing behavior and morphology it is assumed that B chromosomes of M. sanctaefilomenae show homology between them and their evolutionary aspects are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The role of sphingosine 1-phosphate in migration of osteoclast precursors; an application of intravital two-photon microscopy

Sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. Through intravital multiphoton imaging of bone tissues, we reveal that the bidirectional function of S1P temporospatially regulates the migration of osteoclast precursors within intact bone tissues. Imaging technologies have enabled in situ visualization of the behaviors of several players in intact tissues. In addition, intravital microscopy has the potential to be more widely applied to functional analysis and intervention.     

Protective Role of Fas-FasL Signaling in Lethal Infection with Herpes Simplex Virus Type 2 in Mice

Herpes simplex virus type 2 (HSV-2) induces acute local infection followed by latent infection in the nervous system and often leads to the development of lethal encephalitis in immunocompromised hosts. The mechanisms of immune protection against lethal HSV-2 infection, however, have not been clarified. In this study, we examined the roles of Fas-Fas ligand (FasL) signaling in lethal infection with HSV-2 by using mice with mutated Fas (lpr) or FasL (gld) in C57BL/6 background. Both lpr and gld mice exhibited higher mortality than wild-type (WT) C57BL/6 mice after infection with virulent HSV-2 strain 186 and showed significantly increased viral titers in the spinal cord compared with WT mice 9 days after infection, just before the mice started to die. There were no differences in the numbers of CD4⁺ and CD8⁺ T cells infiltrated in the spinal cord or in the levels of HSV-2-specific gamma interferon produced by those cells in a comparison of lpr and WT mice 9 days after infection. Adoptive transfer studies demonstrated that CD4⁺ T cells from WT mice protected gld mice from lethal infection by HSV-2. Furthermore, CD4⁺ T cells infiltrated in the spinal cord of HSV-2-infected WT mice expressed functional FasL that induced apoptosis of Fas-expressing target cells in vitro. These results suggest that FasL-mediated cytotoxic activity of CD4⁺ T cells plays an important role in host defense against lethal infection with HSV-2.Fas-Fas ligand (FasL) signaling-induced apoptotic cell death has pleiotropic roles in T-cell-mediated host defense mechanisms. First, Fas and FasL are expressed on activated T cells and thereby limit their number by inducing suicide or fratricide. It is generally accepted that Fas-mediated activation-induced cell death plays a predominant role during chronic infection, whereas starvation-induced cell death mediated by the proapoptotic BH3-only subgroup of the Bcl-2 protein family is the main mechanism for T-cell death during termination of immune responses in acute infection (30). Fas-FasL signaling might also play a role in T-cell development, as suggested by an accumulation of T-cell receptor αβ-positive (TCR αβ⁺) CD4⁻ CD8⁻ T cells expressing B220 in lymphoid organs of mice with mutated Fas (lpr) or FasL (gld) although the origin and functions of such double-negative T cells are still a matter of debate (21). Lastly, Fas-FasL interaction can be directly involved in host defense by inducing apoptosis of infected cells to facilitate pathogen clearance (23). Therefore, the roles of Fas-FasL signaling in immune responses for host defense might vary depending on the pathogen.Herpes simplex virus type 2 (HSV-2) is an alphaherpesvirus that causes genital herpes, the most common viral sexually transmitted disease (29). After initial infection in the vaginal epithelium, HSV-2 invades local nerve termini, travels via retrograde axonal transport to neuronal cell bodies in sensory ganglia, and establishes latent infection (13). However, especially in neonates and immunocompromised hosts, HSV-2 can cause lethal central nervous system (CNS) infection, which indicates the importance of immune systems in limiting the pathogenicity of HSV-2. Immune responses against HSV-2 have been studied in various murine models using different strains of virus and routes of inoculation, with or without vaccination with an attenuated strain of HSV-2. In such vaccination models, CD4⁺ T cells producing gamma interferon (IFN-γ) predominantly conferred protection against challenge with a virulent strain of HSV-2 (11, 19), whereas various subsets of lymphocytes, including NK cells, NK T cells, and TCR γδ T cells as well as CD4⁺ T cells were reported to be involved in host defense against primary infection with virulent HSV-2 (3, 15, 24), in which IFN-γ also played an important role (9). Fas-FasL signaling was shown to be dispensable for the clearance of an attenuated strain of HSV-2, which lacks thymidine kinase and causes only transient mild vaginal pathologies but not neurologic diseases (6, 16). Similarly Fas-mediated apoptosis was not involved in the vaccination effect of the attenuated HSV-2 (11). However, the roles of Fas-FasL signaling in host defense against a virulent strain of HSV-2 have not been clarified.In this study, we examined the roles of Fas-FasL signaling in a murine model of HSV-2 infection by using a highly virulent HSV-2 strain 186 with lpr and gld mice. We found that FasL-Fas signaling plays an important role in host defense against lethal HSV-2 infection.     

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva

BACKGROUND: It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. METHODS: In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). RESULTS: AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. GENERAL SIGNIFICANCE: AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.     

An NAD(P)H Model Reaction: Reduction of α-Keto Esters by the Hantzsch Ester Accelerated by Zinc Complex in the Reformatsky Mixture

Aiming to get useful steroidal alkaloids by tissue culture of Solanum laciniatum Ait., indefinitely growing callus tissue was prepared from the mother plant. Some nutritional requirements for the growth of the callus tissue were studied. By examining steroidal compounds in callus culture, cholesterol, stigmasterol, β-sitosterol, lanosterol, squalene, diosgenin and a new steroidal alkaloid were found to be formed in the callus culture. The new steroidal alkaloid was found to be solasodine derivative containing rhamnose and other unidentified sugars.     

<Emphasis Type="Italic">Streptomyces</Emphasis> metabolites in divergent microbial interactions

Streptomyces and related bacteria produce a wide variety of secondary metabolites. Of these, many compounds have industrial applications, but the question of why this group of microorganism produces such various kinds of biologically active substances has not yet been clearly answered. Here, we overview the results from our studies on the novel function and role of Streptomyces metabolites. The diverged action of negative and positive influences onto the physiology of various microorganisms infers the occurrence of complex microbial interactions due to the effect of small molecules produced by Streptomyces. The interactions may serve as a basis for the constitution of biological community.