Two secopregnane-type steroidal glycosides from Cynanchum stauntonii (Decne.) Schltr.ex Levl.

Two new steroid glycosides, stauntosaponins A (1) and B (2), were isolated from Cynanchum stauntonii (Decne.) Schltr.ex Levl. (Asclepiadaceae) together with five known compounds, anhydrohirundigenin monothevetoside, glaucogenin C mono-d-thevetoside, hirundoside A, cynatratoside A, and glaucogenin C. Stauntosaponins A and B were formulated as 3-O-β-d-oleandropyranosyl-14, 16: 15, 20: 18, 20-triepoxy-14, 15-secopregn-4, 6, 8(14)-triene (1) and 3-O-β-d-thevetopyranosyl-14, 16: 15, 20: 18, 20-triepoxy-14, 15-secopregn-4, 6, 8(14)-triene (2). Compounds 1 and 2 showed moderate inhibitory activities against Na⁺/K⁺-ATPase with IC₅₀ values of 21 and 29 μM, respectively, whereas ouabain as a positive control displayed an IC₅₀ value of 3.5 μM.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Monilinia ssiori sp. nov. in the Sclerotiniaceae, causing leaf blight and young fruit rot of Prunus ssiori in Japan

The leaf blight and mummy fruit disease fungus of Prunus ssiori in northern Japan is newly named M. ssiori, as a fourth member in the M. padi group of section Disjunctoriae of the genus Monilinia (Sclerotiniaceae). It has been misidentified with Monilinia kusanoi, but recent studies show it is different from M. kusanoi as well as other related species on prunaceous hosts in respect to host relation, pathogenicity, morphology, and gene analysis.     

Effect of external torque on the ATP-driven rotation of F1-ATPase

F₁-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F₀ to synthesize ATP in vivo. Therefore elucidation of the behavior of F₁ under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F₁ rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 μM), 120° stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.     

Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers

The cell permeability of hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, hesperetin permeated cells in the apical-to-basolateral direction at the rate (Jap-->bl) of 10.43+/-0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023+/-0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of hesperetin was almost constant irrespective of the TER. Apically loaded NaN3 or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap-->bl of hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap-->bl and Jbl-->ap of hesperetin were almost the same (5.75+/-0.40 and 5.16+/-0.73 nmol/min/mg protein). Jbl-->ap of hesperetin in the presence of a proton gradient was lower than Jbl-->ap in the absence of a proton gradient. Furthermore, Jbl-->ap in the presence of a proton gradient remarkably increased upon addition of NaN3 specifically to the apical side. These results indicate that hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of hesperetin, that is proton-coupled and energy-dependent polarized transport.     

Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease

Excessive production of airway mucus is a cardinal feature of bronchial asthma and chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality in these diseases. IL-13, a Th2-type cytokine, is a central mediator in the pathogenesis of bronchial asthma, including mucus overproduction. Using a genome-wide search for genes induced in airway epithelial cells in response to IL-13, we identified pendrin encoded by the SLC26A4 (PDS) gene as a molecule responsible for airway mucus production. In both asthma and COPD mouse models, pendrin was up-regulated at the apical side of airway epithelial cells in association with mucus overproduction. Pendrin induced expression of MUC5AC, a major product of mucus in asthma and COPD, in airway epithelial cells. Finally, the enforced expression of pendrin in airway epithelial cells in vivo, using a Sendai virus vector, rapidly induced mucus overproduction in the lumens of the lungs together with neutrophilic infiltration in mice. These findings collectively suggest that pendrin can induce mucus production in airway epithelial cells and may be a therapeutic target candidate for bronchial asthma and COPD.     

Adhesion of MRP8/14 to amastigotes in skin lesions of Leishmania major-infected mice

In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that MRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular MRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for MRP14 are expressed commonly in both developmental stages.     

In vivo depletion of endogenous glutathione facilitates trimethyltin-induced neuronal damage in the dentate gyrus of mice by enhancing oxidative stress

Acute treatment with trimethyltin chloride (TMT) produces neuronal damage in the hippocampal dentate gyrus of mice. We investigated the in vivo role of glutathione in mechanisms associated with TMT-induced neural cell damage in the hippocampus by examining mice depleted of endogenous glutathione by prior treatment with 2-cyclohexen-1-one (CHO). In the hippocampus of animals treated with CHO 1h beforehand, a significant increase was seen in the number of single-stranded DNA-positive cells in the dentate gyrus when determined on day 2 after the injection of TMT at a dose of 2.0 mg/kg. Immunoblot analysis revealed that CHO treatment induced a significant increase in the phosphorylation of c-Jun N-terminal kinase in the cytosolic and nuclear fractions obtained from the dentate gyrus at 16 h after the TMT injection. There was also a concomitant increase in the level of phospho-c-Jun in the cytosol at 16 h after the injection. Expectedly, lipid peroxidation was increased by TMT in the hippocampus, and was enhanced by the CHO treatment. Moreover, CHO treatment facilitated behavioral changes induced by TMT. Taken together, our data indicate that TMT-induced neuronal damage is caused by activation of cell death signals induced at least in part by oxidative stress. We conclude that endogenous glutathione protectively regulates neuronal damage induced by TMT by attenuating oxidative stress.