Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.     

Low-dose lipopolysaccharide pretreatment suppresses choroidal neovascularization via IL-10 induction

Recent studies have suggested that some kinds of microbial infection may have a crucial role in the development of many diseases such as autoimmune diseases and certain types of cancer. It has been reported that some chronic infections, such as Chlamydia pneumoniae, and immunological dysfunctions are associated with age-related macular degeneration (AMD), a leading cause of blindness. To evaluate the association between systemic low-level inflammation induced by infection and AMD pathogenesis, we investigated whether intraperitoneal injection of lipopolysaccharide (LPS) can modulate the development of laser-induced choroidal neovascularization (CNV), a key feature of AMD. Contrary to our expectations, the sizes of CNV in mice with LPS pretreatment were approximately 65% smaller than those of the control mice. After LPS pretreatment, serum IL-10 concentration and IL-10 gene expression in peritoneal macrophages and in the posterior part of the eye increased. Peritoneal injection of anti-IL10 antibody reduced CNV suppression by LPS pretreatment. Moreover, adoptive transfer of the resident peritoneal macrophages from LPS-treated mice into control littermates resulted in an approximately 26% reduction in the size of CNV compared with PBS-treated mice. We concluded that CNV formation was suppressed by low-dose LPS pretreatment via IL-10 production by macrophages.     

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

RTKN-1/Rhotekin shields endosome-associated F-actin from disassembly to ensure endocytic recycling

Cargo sorting and the subsequent membrane carrier formation require a properly organized endosomal actin network. To better understand the actin dynamics during endocytic recycling, we performed a genetic screen in C. elegans and identified RTKN-1/Rhotekin as a requisite to sustain endosome-associated actin integrity. Loss of RTKN-1 led to a prominent decrease in actin structures and basolateral recycling defects. Furthermore, we showed that the presence of RTKN-1 thwarts the actin disassembly competence of UNC-60A/cofilin. Consistently, in RTKN-1–deficient cells, UNC-60A knockdown replenished actin structures and alleviated the recycling defects. Notably, an intramolecular interaction within RTKN-1 could mediate the formation of oligomers. Overexpression of an RTKN-1 mutant form that lacks self-binding capacity failed to restore actin structures and recycling flow in rtkn-1 mutants. Finally, we demonstrated that SDPN-1/Syndapin acts to direct the recycling endosomal dwelling of RTKN-1 and promotes actin integrity there. Taken together, these findings consolidated the role of SDPN-1 in organizing the endosomal actin network architecture and introduced RTKN-1 as a novel regulatory protein involved in this process.     

Nerve Growth Factor is a Potential Treated Target in Tg(SOD1*G93A)1Gur Mice

Nerve growth factor (NGF) is a protective factor of neural cells; the possible relationship between the NGF and the pathogenesis of amyotrophic lateral sclerosis (ALS) hasn’t been completely known. In this study, we observed and analyzed the expression and distribution of NGF, as well as the possible relationship between the NGF expression and distribution and the neural cell death in both SOD1 wild-type (WT) and Tg(SOD1*G93A)1Gur (TG) mice applying the fluorescence immunohistochemistry method. The results showed that the expression and distribution of NGF in the anterior horn (AH), the lateral horn (LH), and the surrounding central canal (CC) significantly increased at the supper early stage of ALS (Pre-onset stage) and the early stage (Onset stage), but the NGF expression and distribution in the AH, the LH, and the surrounding CC significantly reduced at the progression stage. The astrocyte, neuron, and oligodendrocyte produced the NGF and the neural precursor cells (NPCs) produced the NGF. The neural cell death gradually increased accompanying with the reduction of NGF expression and distribution. Our data suggested that the NGF was a protective factor of neural cells, because the neural cells in the AH, the LH, and the surrounding CC produced more NGF at the supper early and early stage of ALS; moreover, the NPCs produced the NGF. It implied that the NGF exerted the protective effect of neural cells, prevented from the neural cell death and aroused the potential of self-repair in the development of ALS.

     

The DNA‐ and RNA‐binding protein FACTOR of DNA METHYLATION 1 requires XH domain‐mediated complex formation for its function in RNA‐directed DNA methylation

Studies have identified a sub‐group of SGS3‐LIKE proteins including FDM1–5 and IDN2 as key components of RNA‐directed DNA methylation pathway (RdDM). Although FDM1 and IDN2 bind RNAs with 5′ overhangs, their functions in the RdDM pathway remain to be examined. Here we show that FDM1 interacts with itself and with IDN2. Gel filtration suggests that FDM1 may exist as a homodimer in a heterotetramer complex in vivo. The XH domain of FDM1 mediates the FDM1–FDM1 and FDM1–IDN2 interactions. Deletion of the XH domain disrupts FDM1 complex formation and results in loss‐of‐function of FDM1. These results demonstrate that XH domain‐mediated complex formation of FDM1 is required for its function in RdDM. In addition, FDM1 binds unmethylated but not methylated DNAs through its coiled‐coil domain. RNAs with 5′ overhangs does not compete with DNA for binding by FDM1, indicating that FDM1 may bind DNA and RNA simultaneously. These results provide insight into how FDM1 functions in RdDM.     

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.     

Effect of feeding frequency on growth, feed utilization, and size variation of juvenile gibel carp (Carassius auratus gibelio)

Juvenile (3.0 ± 0.2 g) gibel carp (Carassius auratus gibelio) were fed to satiation for 8 weeks to investigate the effect of feeding frequency on growth, feed utilization and size variation. Five feeding frequencies were tested: two meals per day (M2), three meals per day (M3), four meals per day (M4), 12 meals per day (M12) and 24 meals per day (M24). The results showed that daily food intake increased significantly with the increase in feeding frequency and there was no significant difference between daily food intakes in M12 and M24 treatments. Growth rate, feed efficiency increased significantly with increasing feeding frequencies. Size variation was not affected by feeding frequency. Apparent digestibility of dry matter was not influenced by feeding frequency, while apparent digestibility of protein and energy increased significantly at high feeding frequencies. The feeding frequency had no significant effect on the moisture, lipid, protein, or energy contents of gibel carp, while the ash content decreased with increased feeding frequency. It was recommended that 24 meals per day was the optimal feeding frequency for juvenile gibel carp.