B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5⁺ IgM⁺ WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-κB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.     

Nucleoredoxin Negatively Regulates Toll-like Receptor 4 Signaling via Recruitment of Flightless-I to Myeloid Differentiation Primary Response Gene (88)

We previously characterized nucleoredoxin (NRX) as a negative regulator of the Wnt signaling pathway through Dishevelled (Dvl). We perform a comprehensive search for other NRX-interacting proteins and identify Flightless-I (Fli-I) as a novel NRX-binding partner. Fli-I binds to NRX and other related proteins, such as Rod-derived cone viability factor (RdCVF), whereas Dvl binds only to NRX. Endogenous NRX and Fli-I in vivo interactions are confirmed. Both NRX and RdCVF link Fli-I with myeloid differentiation primary response gene (88) (MyD88), an important adaptor protein for innate immune response. NRX and RdCVF also potentiate the negative effect of Fli-I upon lipopolysaccharide-induced activation of NF-κB through the Toll-like receptor 4/MyD88 pathway. Embryonic fibroblasts derived from NRX gene-targeted mice show aberrant NF-κB activation upon lipopolysaccharide stimulation. These results suggest that the NRX subfamily of proteins forms a link between MyD88 and Fli-I to mediate negative regulation of the Toll-like receptor 4/MyD88 pathway.     

Validation and efficacy of transgenerational mass marking of otoliths in viviparous fish larvae

Transgenerational mass marking of viviparous fish larvae in vivo was validated by intra‐muscular injection of elemental strontium chloride (SrCl₂) in gestating females and detection of the Sr in the otoliths of developing larvae. All otoliths of brown rockfish Sebastes auriculatus larvae produced from SrCl₂‐injected females showed enriched Sr:Ca ratios near the otolith edges, and the signatures did not appear to be affected by the anterior, centre and posterior positions of larvae within the ovary. Results from the present study indicate that transgenerational marking is a highly reliable technique for marking large numbers of extremely small viviparous fish larvae.     

Conservation of Toll-Like Receptor Signaling Pathways in Teleost Fish

In mammals, toll-like receptors (TLR) recognize ligands, including pathogen-associated molecular patterns (PAMPs), and respond with ligand-specific induction of genes. In this study, we establish evolutionary conservation in teleost fish of key components of the TLR-signaling pathway that act as switches for differential gene induction, including MYD88, TIRAP, TRIF, TRAF6, IRF3, and IRF7. We further explore this conservation with a molecular phylogenetic analysis of MYD88. To the extent that current genomic analysis can establish, each vertebrate has one ortholog to each of these genes. For molecular tree construction and phylogeny inference, we demonstrate a methodology for including genes with only partial primary sequences without disrupting the topology provided by the high-confidence full-length sequences. Conservation of the TLR-signaling molecules suggests that the basic program of gene regulation by the TLR-signaling pathway is conserved across vertebrates. To test this hypothesis, leukocytes from a model fish, rainbow trout (Oncorhynchus mykiss), were stimulated with known mammalian TLR agonists including: diacylated and triacylated forms of lipoprotein, flagellin, two forms of LPS, synthetic double-stranded RNA, and two imidazoquinoline compounds (loxoribine and R848). Trout leukocytes responded in vitro to a number of these agonists with distinct patterns of cytokine expression that correspond to mammalian responses. Our results support the key prediction from our phylogenetic analyses that strong selective pressure of pathogenic microbes has preserved both TLR recognition and signaling functions during vertebrate evolution.     

Determination of the freshwater mark in otoliths of Japanese eel elvers using microstructure and Sr/Ca ratios

The microstructure, in particular checks within the otolith edge, of Anguilla japonica glass-eels and elvers and changes in otolith Sr/Ca ratios were examined to ascertain the environmental history of the eels, especially with regard to the time when glass-eels entered the river, and as a benchmark for count daily increments. The percentage of glass eels and elvers with checks and the mean number of checks within the otoliths of glass-eels caught at four localities, Tosa Bay off Tosa City, the mouth of the Gokase River, the mouth of the Saigo River and the dam of the Tsuri River were 0% (0), 15.0% (0.2), 51.6% (1.0) and 100.0% (4.2), respectively. The Sr/Ca ratios and Sr content peaked in the region where checks were formed and the values decreased rapidly towards the edge of the checks; on the other hand, these decreased gradually in the otolith when checks were not formed. These checks were estimated to be formed by stress when the glass-eels were affected by ambient fresh water within the river. The innermost check was called the freshwater mark in the present study and this mark may be useful as a benchmark in studying the growth history of the eel before and after entering freshwater.     

Accumulation of β-tubulin-containing fibres in the cortical domain ofSaccharomyces cerevisiae spheroplasts

Summary The distribution of microtubules inSaccharomyces cerevisiae was studied with the monoclonal antibody (mab) TU-14 against -tubulin. Immunoblotting and immunoprecipitation experiments with a strain overexpressing Tub2p confirmed that the mab TU-14 specifically recognized Tub2p. By immunofluorescence microscopy, the mab TU-14 attached to all known tubulin structures labelled with the standard polyclonal anti--tubulin antibody 206-1. In addition, the mab TU-14 revealed cortical patches in wild-type cells and an abundant network of fibres in the cortex of spheroplasts cultivated in nutrient medium. These cortical fibres seemed to be specific to spheroplasts and suggest that the accumulated Tub2p is predominantly associated with the plasma membrane.     

Phosphomannopentaose sulfate (PI-88) inhibits retinal leukostasis in diabetic rat

Retinal leukostasis, mediated by intracellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF), has been implicated in the pathogenesis of early diabetic retinopathy. Phosphomannopentaose sulfate (PI-88) is a highly sulfonated oligosaccharide which inhibits heparanase activity and competes with heparan sulfate binding to growth factors. In this study, we evaluated whether PI-88 could inhibit retinal leukostasis in strepotzotocin(STZ)-induced diabetic rat and elucidated the possible mechanisms. Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection (i.p.) of STZ. Three months after induction, diabetic rats were administered PI-88 (25 mg/kg body weight) or vehicle solution daily via i.p. for 14 consecutive days. Leukostasis was analyzed on retinal flatmounts by concanavalin A and CD45 immunofluorescence staining. Retinal function was analyzed by electroretinography (ERG). ICAM-1 and VEGF levels in retinas were studied by Western blot and enzyme-linked immunosorbent assay (ELISA) respectively. The systemic administration of PI-88, but not vehicle, significantly decreased the number of adherent leukocytes in retinas by 52.24% (P < 0.001) and led to significant preservation (about 50%, P < 0.001) of scotopic ERG a- and b-wave amplitudes in treated diabetic rats as compared to those of diabetic control rats. These changes were associated with downregulation of ICAM-1 (45%, P < 0.001) and VEGF (26.83 ± 2.01 versus 40.8 ± 3.24 pg/mg, P < 0.01) in retinas of PI-88 treated diabetic rats as compared to those of diabetic control rats. PI-88 significantly inhibited retinal leukostasis and reversed retinal dysfunction by a mechanism that may include decreased ICAM-1 and VEGF expression in diabetic rats. Our data suggests that PI-88 is a promising agent for the treatment of diabetic retinopathy.     

New 2-naphthyloxyacetates for trunk sucker growth control on grapevine (Vitis vinifera L.)

Eighteen new hexyl esters of 2-naphthyloxyacetic acid (-NOA) were synthesized and their efficiency on sucker control of grapevine was investigated. Different solutions (0.5, 1, and 2%, w/w concentrations) of each compound were sprayed on suckers and the wilting effect after 4, 10, and 30 days after treatment was evaluated. Esters derived from primary and secondary alcohols gave a rapid control of suckers, whereas tertiary alcohol derivatives showed milder activity. Moreover, all compounds proved to cause a long-lasting inhibition effect on the growth of new suckers even 1 year after treatment, especially when applied at a 2% concentration.