Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.     

Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.     

Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.     

Thermal activation of single kinesin molecules with temperature pulse microscopy

Conventional kinesin is a processive motor protein that keeps "walking" along a microtubule using chemical energy released by ATP hydrolysis. We previously studied the effects of temperature between 15 degrees and 35 degrees C on the moving velocity, force, and processivity of single kinesin molecules using a bead assay [Kawaguchi and Ishiwata, 2000b: Biochem Biophys Res Commun 272:895-899]. However, we could not examine the effects of temperature higher than 35 degrees C because of the thermal damage to proteins. Here, using temperature pulse microscopy (TPM) [Kato et al., 1999: Proc Natl Acad Sci USA 96:9602-9606], we could examine the temperature dependence of the gliding velocity of single kinesin molecules interacting with a microtubule above 35 degrees C up to 50 degrees C (instantaneously, approximately 60 degrees C), where the velocity reached 3.68 microm/s, the highest ever reported. The Arrhenius plot showed no breaks between 15 degrees and 50 degrees C with a unique activation energy of about 50 kJ/mol, suggesting that the molecular mechanism of kinesin motility is common over a broad temperature range including physiological temperature.     

Spatial mosaic and interfacial dynamics in a Müllerian mimicry system

Uncovering why spatial mosaics of mimetic morphs are maintained in a Müllerian mimicry system has been a challenging issue in evolutionary biology. In this article, we analyze the reaction diffusion system that describes two-species Müllerian mimicry in one- and two-dimensional habitats. Due to positive frequency-dependent selection, a local population first approaches the state where one of the comimicking patterns predominates, which is followed by slow movement of boundaries where different patterns meet. We then analyze the interfacial dynamics of the boundaries to find whether a stable cline is maintained and to obtain the wave speed if the cline is unstable. The results are: (1) In a spatially uniform habitat the morph with greater base fitness spreads both in one and two species system. (2) The strength of cross-species interaction determines whether the mimetic morph clines of model and mimic species coalesce into the same geographical region or pass through each other. The joint wave speed of clines decreases by increasing the number of comimicking species in the mimicry ring. (3) In spatial heterogeneous habitats, stable clines can be maintained due to the balance between the base fitness gradient and the biased gene flow by negative curvature of boundary. This allows the persistence of a spatial mosaic even if one of the morphs is in every place advantageous over the other. A balanced cline is also maintained if there is a gradient in the population density. (4) A new advantageous morph occurring at a local region is doomed to go to extinction in a finite time if the "radius" of initial distribution is below a threshold. Possible applications to the heliconiine butterfly mimicry ring, heterozygous disadvantage systems of chromosomal rearrangement and hybrid zone, the third phase of Wright's Shifting Balance theory, and cytoplasmic incompatibility are discussed.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.     

The novel symbiotic phenotype of enhanced-nodulating mutant of Lotus japonicus: astray mutant is an early nodulating mutant with wider nodulation zone

We have isolated a novel enhanced-nodulating mutant astray (Ljsym77) from Lotus japonicus. The name astray derives from the non-symbiotic phenotype of this mutant, agravitropic lateral roots that go "astray" against gravity. In this report we evaluated the symbiotic aspects of this mutant in detail. The astray mutant developed approximately twice the number of nodules on a wider area of roots compared with the wild type. Furthermore, the astray mutant demonstrated early initiation of nodule development, which is an unprecedented symbiotic phenotype. The astray seedlings showed normal sensitivity to the general inhibitors of nodulation such as ethylene and nitrate. These results indicate that the astray mutant is distinct from the hypernodulating mutants reported previously, and that the ASTRAY gene acts as an early and negative regulator in the cascade of nodule development.     

Enzymatic synthesis of a capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives catalyzed by lipases

Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 microl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 A molecular sieves at 25 degrees C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6-C18) were synthesized at 64-86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively.     

In Situ Microspatial Imaging Using Two-Photon and Confocal Laser Scanning Microscopy of Bacteria and Extracellular Polymeric Secretions (EPS) Within Marine Stromatolites

The combination of a hydrophilic embedding resin, Nanoplast, with fluorescent probes, and subsequent imaging using two-photon and confocal laser scanning microscopy (2P-LSM and CLSM) has allowed in imaging of the in situ microspatial arrangements of microbial cells and their extracellular polymeric secretion (EPS) within marine stromatolites. Optical sectioning by 2P-LSM and CLSM allowed imaging of endolithic cyanobacteria cells, Solentia sp., seen within carbonate sand grains. 2P-LSM allowed very clear imaging with a high resolution of bacteria using DAPI, which normally require UV excitation and reduced photo-bleaching of fluorescent probes.     

crinkle,a novel symbiotic mutant that affects the infection thread growth and alters the root hair,trichome, and seed development in Lotus japonicus

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.