A role for diacylglycerol kinase 4 in signalling crosstalk during Arabidopsis pollen tube growth.

Diacylglycerol kinases (DGKs) play a major role in the production of phosphatidic acid (PtdOH) and were implicated in endomembrane trafficking and signalling cascades. In plants, the role of DGKs is less clear, as PtdOH seems to arise mostly from phospholipase D activity. Here, we investigated the function of the Arabidopsis gene encoding DGK4, which is highly expressed in pollen. In vitro, pollen tubes from homozygous dgk4 plants showed normal morphology, but reduced growth rate and altered stiffness and adhesion properties (revealed by atomic force microscopy). In vivo, dgk4 pollen was able to fertilize wild-type ovules, but self-pollination in dgk4 plants led to fewer seeds and shorter siliques. Phenotypic analysis revealed that the dgk4 mutation affects not only the male germ line but also the vegetative tissue. DGK4-green fluorescent protein fusion imaging revealed a cytosolic localization with a slightly higher signal in the subapical or apical region. dgk4 pollen tubes were found to exhibit perturbations in membrane recycling, and lipid analysis revealed a minor increase of PtdOH concomitant with decreased phosphatidylcholine, compared with wild-type. In vitro, DGK4 was found to exhibit kinase and guanylyl cyclase activity. Quantitative PCR data revealed downregulation of genes related to actin dynamics and phosphoinositide metabolism in mutant pollen, but upregulation of the DGK6 isoform. Altogether, these results are discussed considering a role of DGK4 in signalling cross-talk.

Development of a liposome-based formulation for vitamin K1 nebulization on the skin.

Vitamin K1 (VK1) is a very lipophilic and photosensitive molecule contained in some vegetables. Recently, the use of VK1 on the skin has been proposed for different pharmaceutical or cosmeceutical applications. In this study, an innovative strategy for the administration of VK1 on the skin was proposed. In particular, to overcome the drawbacks associated with a VK1-containing fatty ointment available on the market, an aqueous formulation suitable to be administered by nebulization was developed. The use of liposomes encapsulating VK1 enabled issues due to the lipophilicity of VK1 to be overcome. Thus, different liposomal formulations, with different VK1 concentrations, were prepared and characterized in terms of size, zeta potential, VK1 encapsulation into liposomes, and stability of the formulations during storage. After a first phase of screening, the selected formulation was tested by a portable device for nebulization. No alteration of the vesicle characteristics following the liposome supply through the nebulizer was found. Finally, permeation studies were carried out on pig-excised skin in Franz cells and the newly developed formulation was compared to a marketed VK1-containing ointment. In this test, an enhanced VK1 accumulation into the skin was found when using nebulized liposomes. In conclusion, in order to administer VK1 on the skin, the newly developed formulation could be a valid alternative to the products available on the market today. In particular, the use of liposomes could facilitate the multiple administrations per day by aerosol, but also increase, compared to a semi-solid preparation, the accumulation of VK1 into the epidermis and dermis.