Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin 'gentiodelphin'. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar 'Polarno White' might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helix-loop-helix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. 'Polarno White' the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.
Transient expression of the maize streak geminivirus virion-sense proteins V1 and V2 (movement protein, MP, and coat protein, CP, respectively) in maize leaves allowed investigation of their roles in inter- and intracellular movement. Bombardment of a construct directing expression of a V1:green fluorescent protein (GFP) fusion product resulted in significantly increased spread of fluorescence from the bombarded cell to adjacent cells compared to that obtained following expression of free GFP. A mutant V1:GFP fusion product exhibited markedly less movement than the V1:GFP protein. Thus, the MSV V1 protein moves from cell to cell in the absence of other viral proteins. However, V1:GFP did not localize to plasmodesmata in maize or tobacco leaves although a tobacco mosaic virus MP:GFP fusion protein was shown to do so in tobacco. The CP:GFP fusion product targeted exclusively to the nucleus and did not move from cell to cell or exit the nucleus when expressed alone. When coexpressed with V1, some CP:GFP fluorescence was seen at the cell periphery in a proportion of cells, but in no case was cell-to-cell movement of CP:GFP detected. The likely roles of V1 and CP in MSV movement are discussed.
Morus alba L., also known as white mulberry or Mhon, has long been used in traditional medicines. This study was aimed to investigate anti-inflammatory activities of mulberry stem ethanolic extract (MSE) in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophage cell line. The MSE was first prepared and then investigated for cell viability using the MTT assay. The anti-inflammatory activities were investigated through the inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase- (COX-) 2 mRNA expression, and iNOS protein expression using reverse transcription-polymerase chain reaction (RT-PCR) assay and immunoblotting analysis, respectively. The inhibition of nitric oxide production of the MSE was also investigated using the Griess reaction assay. The MSE concentration ranging from 10 to 40 µg/ml yielded cell viability higher than 80%. The MSE at concentrations of 20 and 40 µg/ml demonstrated anti-inflammatory activity through the inhibition of nitric oxide production via suppression of both the iNOS mRNA and protein. It was also found to inhibit the expression of COX-2 mRNA in LPS-induced RAW 264.7 cells. This study is the first to report the anti-inflammatory potential of the extract prepared from the stem of mulberry.
Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.
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