DsRNA-free transmissible hypovirulence associated with formation of intra-hyphal hyphae in Botrytis cinerea

A spontaneous mutant CanBc-3HV and its parental strain CanBc-3 of Botrytis cinerea were investigated in terms of pathogenicity, colony morphology, hypovirulence transmissibility, presence of double-stranded RNA (dsRNA), and formation of intra-hyphal hyphae (IH). Results showed that inoculation of CanBc-3HV on detached leaves of Brassica napus did not produce any visible necrotic lesions (20°C, 72h), whereas inoculation of CanBc-3 caused necrotic leaf lesions. Compared to CanBc-3, CanBc-3HV grew slowly, formed numerous mycelial sectors, sporulated sporadically and failed to produce sclerotia on potato dextrose agar (PDA) (20°C, 15d). Hypovirulence and the abnormal cultural characteristics of CanBc-3HV were transmissible from CanBc-3HV to CanBc-3 in pair cultures on PDA. However, the transmission was unsuccessful from CanBc-3HV to another virulent strain CanBc-2 of B. cinerea. These results suggest that transmission of the hypovirulence and the abnormal cultural characteristics of CanBc-3HV are strain-specific. No dsRNA was detected in mycelia of either CanBc-3HV or CanBc-3, implying that the hypovirulence of CanBc-3HV is caused by a transmissible element (TE) of non-RNA mycoviral origin. Formation of IH through self-infection was observed in CanBc-3HV, CanBc-3T1 (a hypovirulent derivative of CanBc-3 trans-infected by TE in CanBc-3HV), but was not observed in CanBc-3, suggesting that IH formation is associated with the hypovirulence of CanBc-3HV. To our knowledge, this is the first report of dsRNA-free transmissible hypovirulence associated with IH formation in B. cinerea.     

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.     

Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO

The possible existence of transepithelial bicarbonate transport across the isolated bovine ciliary body was investigated by employing a chamber that allows for the measurement of unidirectional, radiolabeled fluxes of CO2 + HCO. No net flux of HCO was detected. However, acetazolamide (0.1 mM) reduced the simultaneously measured short-circuit current (I(sc)). In other experiments in which (36)Cl- was used, a net Cl- flux of 1.12 microeq. h(-1). cm(-2) (30 microA/cm(2)) in the blood-to-aqueous direction was detected. Acetazolamide, as well as removal of HCO from the aqueous bathing solution, inhibited the net Cl- flux and I(sc). Because such removal should increase HCO diffusion toward the aqueous compartment and increase the I(sc), this paradoxical effect could result from cell acidification and partial closure of Cl- channels. The acetazolamide effect on Cl- fluxes can be explained by a reduction of cellular H+ and HCO (generated from metabolic CO2 production), which exchange with Na+ and Cl- via Na+/H+ and Cl-/HCO exchangers, contributing to the net Cl- transport. The fact that the net Cl- flux is about three times larger than the I(sc) is explained with a vectorial model in which there is a secretion of Na+ and K+ into the aqueous humor that partially subtracts from the net Cl- flux. These transport characteristics of the bovine ciliary epithelium suggest how acetazolamide reduces intraocular pressure in the absence of HCO transport as a driving force for fluid secretion.     

Glandular trichomes and essential oil characteristics of in vitro propagated <Emphasis Type="Italic">Micromeria pulegium</Emphasis> (Rochel) Benth. (Lamiaceae)

Main conclusion

In vitro conditions and benzyladenine influenced both content and composition of micropropagated Micromeria pulegium essential oils, with pulegone and menthone being the main essential oil components. The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal segments cultured on solid MS medium supplemented with N⁶–benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 μM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.

     

Two new flavones from Eupatorium coelestinum

Besides coumarin, nobiletin, lucidin dimethyl ether and 5,6,7,3′,4′-pentamethoxyflavone, two new highly oxygenated flavones were isolated from Eupatorium coelestinum. Their structures were determined by spectroscopic methods and alkaline degradations as 5,6,7,8,3′,4′,5′ -heptamethoxyflavone and 5,6,7,8,5′-pentamethoxy-3′,4′-methylenedioxyflavone.     

Differential immunogenicity of a DNA vaccine containing the Streptococcus mutans wall-associated protein A gene versus that containing a truncated derivative antigen A lacking in the hydrophobic carboxyterminal region

Two plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region.     

Spectrin-level modeling of the cytoskeleton and optical tweezers stretching of the erythrocyte

We present a three-dimensional computational study of whole-cell equilibrium shape and deformation of human red blood cell (RBC) using spectrin-level energetics. Random network models consisting of degree-2, 3, ..., 9 junction complexes and spectrin links are used to populate spherical and biconcave surfaces and intermediate shapes, and coarse-grained molecular dynamics simulations are then performed with spectrin connectivities fixed. A sphere is first filled with cytosol and gradually deflated while preserving its total surface area, until cytosol volume consistent with the real RBC is reached. The equilibrium shape is determined through energy minimization by assuming that the spectrin tetramer links satisfy the worm-like chain free-energy model. Subsequently, direct stretching by optical tweezers of the initial equilibrium shape is simulated to extract the variation of axial and transverse diameters with the stretch force. At persistence length p = 7.5 nm for the spectrin tetramer molecule and corresponding in-plane shear modulus mu(0) approximately 8.3 microN/m, our models show reasonable agreement with recent experimental measurements on the large deformation of RBC with optical tweezers. We find that the choice of the reference state used for the in-plane elastic energy is critical for determining the equilibrium shape. If a position-independent material reference state such as a full sphere is used in defining the in-plane energy, then the bending modulus kappa needs to be at least a decade larger than the widely accepted value of 2 x 10(-19) J to stabilize the biconcave shape against the cup shape. We demonstrate through detailed computations that this paradox can be avoided by invoking the physical hypothesis that the spectrin network undergoes constant remodeling to always relax the in-plane shear elastic energy to zero at any macroscopic shape, at some slow characteristic timescale. We have devised and implemented a liquefied network structure evolution algorithm that relaxes shear stress everywhere in the network and generates cytoskeleton structures that mimic experimental observations.     

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.     

Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.