3,4-Dicaffeoylquinic Acid, a Major Constituent of Brazilian Propolis, Increases TRAIL Expression and Extends the Lifetimes of Mice Infected with the Influenza A Virus

Brazilian green propolis water extract (PWE) and its chemical components, caffeoylquinic acids, such as 3,4-dicaffeoylquinic acid (3,4-diCQA), act against the influenza A virus (IAV) without influencing the viral components. Here, we evaluated the anti-IAV activities of these compounds in vivo. PWE or PEE (Brazilian green propolis ethanol extract) at a dose of 200 mg/kg was orally administered to Balb/c mice that had been inoculated with IAV strain A/WSN/33. The lifetimes of the PWE-treated mice were significantly extended compared to the untreated mice. Moreover, oral administration of 3,4-diCQA, a constituent of PWE, at a dose of 50 mg/kg had a stronger effect than PWE itself. We found that the amount of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA in the mice that were administered 3,4-diCQA was significantly increased compared to the control group, while H1N1 hemagglutinin (HA) mRNA was slightly decreased. These data indicate that PWE, PEE or 3,4-diCQA possesses a novel and unique mechanism of anti-influenza viral activity, that is, enhancing viral clearance by increasing TRAIL

Uncovering of major genetic factors generating naturally occurring variation in heading date among Asian rice cultivars

To dissect the genetic factors controlling naturally occurring variation of heading date in Asian rice cultivars, we performed QTL analyses using F2 populations derived from crosses between a japonica cultivar, Koshihikari, and each of 12 cultivars originating from various regions in Asia. These 12 diverse cultivars varied in heading date under natural field conditions in Tsukuba, Japan. Transgressive segregation was observed in 10 F2 combinations. QTL analyses using multiple crosses revealed a comprehensive series of loci involved in natural variation in flowering time. One to four QTLs were detected in each cross combination, and some QTLs were shared among combinations. The chromosomal locations of these QTLs corresponded well with those detected in other studies. The allelic effects of the QTLs varied among the cross combinations. Sequence analysis of several previously cloned genes controlling heading date, including Hd1, Hd3a, Hd6, RFT1, and Ghd7, identified several functional polymorphisms, indicating that allelic variation at these loci probably contributes to variation in heading date. Taken together, the QTL and sequencing results indicate that a large portion of the phenotypic variation in heading date in Asian rice cultivars could be generated by combinations of different alleles (possibly both loss- and gain-of-function) of the QTLs detected in this study

Identification of a Novel Subpopulation of Tumor-Initiating Cells from Gemcitabine-Resistant Pancreatic Ductal Adenocarcinoma Patients

Pancreatic ductal adenocarcinoma is highly resistant to systemic chemotherapy. Although there are many reports using pancreatic cancer cells derived from patients who did not receive chemotherapy, characteristics of pancreatic cancer cells from chemotherapy-resistant patients remain unclear. In this study, we set out to establish a cancer cell line in disseminated cancer cells derived from gemcitabine-resistant pancreatic ductal adenocarcinoma patients. By use of in vitro co-culture system with stromal cells, we established a novel pancreatic tumor-initiating cell line. The cell line required its direct interaction with stromal cells for its in vitro clonogenic growth and passaging. Their direct interaction induced basal lamina-like extracellular matrix formation that maintained colony formation. The cell line expressed CD133 protein, which expression level changed autonomously and by culture conditions. These results demonstrated that there were novel pancreatic tumor-initiating cells that required direct interactions with stromal cells for their in vitro cultivation in gemcitabine-resistant pancreatic ductal adenocarcinoma. This cell line would help to develop novel therapies that enhance effects of gemcitabine or novel anti-cancer drugs

Concanavalin A disrupts the release of fibrous material necessary for zygote formation of a unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex

The Closterium peracerosum–strigosum–littorale (C. psl.) complex is the best characterized charophycean alga with respect to the processes of sexual reproduction. We examined the effect of concanavalin A (Con A) on physiological and ultrastructural changes during the conjugation of the C. psl. complex. Two heterothallic gametangial cells formed a sexual pair as usual; however, the release of gametes was completely blocked by the addition of Con A. Fluorescein isothiocyanate-labeled Con A bound to the outermost layer of the conjugation papillae of paired cells. In the absence of Con A, the disruption of outer cell walls on the conjugation papillae and the secretion of fibrous materials from the conjugation papillae were observed using a transmission electron microscope, but Con A-treated cells did not show these changes. Instead, a highly electron-dense layer was observed in the outermost papillae, and the excess fibrous materials remained at the inside of the layer. These results suggest that an unknown molecule(s) recognized by Con A is essential for the diffusion of fibrous materials at the conjugation papillae, which is an indispensable step for gamete release during conjugation of the C. psl. complex

CD133 expression in KMC14 cells.

<p><b>A</b>. Flow cytometric analyses for CD133⁺ cells in pleural effusions derived from patient 1. The result is representative of three independent experiments. <b>B</b>. CD133 expression in KMC14 cells. Frozen sections of KMC14 colonies were immunostained for human CD133 (hCD133) (brown color). The boxed region (magnification, x100) is shown at higher magnification in the insert (magnification, x400). The result is representative of three independent experiments. <b>C</b>. CD133 expression in KMC14-derived pancreatic tumor. PDAC tissues from the patient 1 (parental PDAC) and KMC14-derived pancreatic tumor tissues in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081283#pone-0081283-g001" target="_blank">Figure 1E</a> were immunostained for hCD133 (brown color). Arrows indicate hCD33⁺ carcinoma cells. Scale bar = 200 µm. The result is representative of three independent experiments. <b>D</b>. <i>CD133</i> mRNA expression analyses of each cell line by qRT-PCR assays. Confluent PA6 cells were cultured with KMC14, BxPC-1, MIAPaCa-2, PANC-1 or AsPC-1 cells in serum-free Stem medium, followed by qRT-PCR assays. Results were normalized to <i>human </i><i>ß-actin</i> mRNA and to the control condition (PANC-1 cells). Columns, mean of three experiments ± SE completed in triplicate, **, <i>p</i> < 0.005. <b>E</b>. Gene expression analyses of each cell line by RT-PCR. Confluent PA6 cells were cultured with KMC14, BxPC-1, MIAPaCa-2, PANC-1 or AsPC-1 cells in serum-free Stem medium, followed by qRT-PCR assays. Control: PA6 cells alone. <i>Human </i><i>ß-actin</i> was used as an endogenous control. The result is representative of three independent experiments. <b>F</b>. Effect of hCD133 expression in KMC14 cells on their clonogenic activity and xenograft tumor formation. (a) Scheme of separation of mPDGFRß⁺ PA6 cells and hCD133⁺ and hCD133^- mPDGFRß^– KMC14 cells from the co-culture system in serum-free Stem medium. (b) Comparison of clonogenic activity between hCD133⁺ and hCD133^- mPDGFRß^– KMC14 cells. mPDGFRß⁺ PA6 cells, and hCD133⁺ and hCD133^- mPDGFRß^– KMC14 cells were co-cultured with PA6 cells in serum-free Stem medium for 2 weeks and the number of colonies were counted. Columns, mean of three independent experiments ± SE completed in triplicate. (c) Comparison of xenograft tumor formation between hCD133⁺ and hCD133^- mPDGFRß^– KMC14 cells. hCD133⁺ and hCD133^- mPDGFRß^– KMC14 cells were subcutaneously injected into the right and left back of nude mice at 1 x 10⁴ cells per injection. After 7 weeks mice were examined for xenograft tumor formation. The result is representative of five independent experiments. (d) Xenograft tumor size. Xenograft tumors formed subcutaneously in Fig. 4Fc were dissected and weighted. Columns, mean of five experiments ± SE; *, <i>p</i> < 0.05. (e) hCD133 expression in xenograft tumors derived from hCD133⁺ and hCD133^- mPDGFRß^– KMC14 cells. The xenograft tumor tissues in Fig. 4Fc were examined by H&E staining and immunostaining for hCD133 (brown color). The result is representative of five independent experiments. Control: PBS instead of anti-hCD133 monoclonal antibody. Arrows indicate hCD133⁺ carcinoma cells. Original magnifications: 400x.</p

Effect of culture conditions on CD133 expression and morphology of KMC14 colonies.

<p><b>A</b>. Scheme of culture medium switch experiments. <b>B</b>. CD133 immunofluorescent staining of KMC14 colonies. KMC14 colonies on PA6 cells were incubated in serum-free Stem medium (SFM) or αMEM containing 10% FCS (FCS), followed by hCD133 immunofluorescent staining. The result is representative of three independent experiments. Scale bar = 200 µm. <b>C</b>. Flow cytometric analyses for hCD133⁺ KMC14 cells in the co-culture system. KMC14 colonies on PA6 cells were incubated in serum-free Stem medium (SFM) or αMEM containing 10% FCS (FCS), followed by flow cytometric analyses. I: hCD133⁺ mPDGFRß^- cells (hCD133⁺ KMC14 cells); II, hCD133^- mPDGFRß^- cells (hCD133^- KMC14 cells); and III, mPDGFRß⁺ cells (PA6 cells). The result is representative of five independent experiments. <b>D</b>. Morphology change of KMC14 colonies. KMC14 colonies co-cultured with PA6 cells were incubated in serum-free Stem medium (SFM) or αMEM containing 10% FCS (FCS), followed by H&E staining and EMA immunostaining (brown color) (transverse sections). Original magnification, 200x. The result is representative of three independent experiments.</p