Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction.

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis.

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and ß-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.

LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ET(B)) receptors in cultured endothelium-intact rat coronary arteries.

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM-0.3 µM) and ET-1 (1 pM-0.3 µM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.

Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein.

Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM H+ -ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM H+ -ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM H+ -ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM H+ -ATPase regulation.

Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes.

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae).

We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant.