Chemical composition and biological activity of the essential oil of Ballota hispanica (L.) Benth. growing wild in Sicily

Ballota L. comprises several relevant species largely used for their excellent therapeutic properties. Ballota hispanica (L.) Benth. is widely used in herbal medicine, and it is sold in herbalist shops for its sedative and antispasmodic properties. Considering its traditional medicinal use and the lack of scientific studies on the volatile components of this species as well as on its biological activities, in this study the chemical composition of the essential oil from aerial parts of B. hispanica, collected in Sicily, was evaluated by gas chromatography and gas chromatography–mass spectrometry. α-Elemol was the most abundant component of the oil (10.9%), followed by α-ylangene (8.5%), γ-dodecalactone (5.1%), and manoyl oxide (4.8%). A comparison was made of the composition of the different Ballota taxa studied so far showing a peculiar profile of B. hispanica. Futhermore, the antimicrobial and the free radical scavenging activities of the oil were determined.     

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A.?saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A.?saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50?°C, and at 60?°C it had a half-life of approximately 6?h.     

Capsaicin Modulates Proliferation,Migration, and Activation of Hepatic Stellate Cells

Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro.     

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.     

Cell motility measurements with an automated microscope system

The motility of 3T3 cells has been studied using a newly developed automated microscope system which is capable of recognizing live unstained cells growing in tissue culture. A large number of individual cells can be rapidly identified and characterized and their precise positions recorded. All cells can be revisited automatically every few minutes, and the new cell positions can be determined. Quantitative data from up to 1 000 cells can then be obtained, and cell movement parameters like cell speed, distance travelled, direction of movement, etc., can be measured for individual cells and for the whole cell population. In addition, for any number of chosen cells, high-resolution digitized images can be taken for further morphological studies, including acquisition of images of individual cells.     

Patterns of dispersion, density and dispersal in alpine populations of the land snail Arianta arbustorum (L.) (Helicidae)

In studying populations of the land snail Arianta arbustorum in the eastern Swiss Alps, juvenile and subadult snails were found to be aggregated all the time, while adults were mostly aggregated during summer, the main mating period. The mean size of aggregations was 25 cm × 25 cm but varied, according to the area a grass tuft covered, while the intensity of aggregations was influenced by weather conditions. Densities of 10–15 subadults and 3–6 adults m⁻² were found along ditches and streams, 2–25 subadults and 2–20 adults on uncultivated alpine meadows, while on scree-covered alpine grassland the density of subadults and adults was 0.3–0.5 m⁻². The extent of daily movements varied with microclimatic factors and season and depended on the structure of the habitat. The dispersal rate was greatest in June (2.6 m month⁻¹) and least in August (1.1 m month⁻¹). In summer and autumn, active uphill movements were noted, a process which compensates for the loss of altitude by passive downhill displacements. Roiling down on snowfields and being carried away by avalanches and streams were observed.     

Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds

An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild-type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv. tomato and Peronospora parasitica. The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens. Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild-type plants, but this induction was almost completely abolished in esa1. This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.