CRYPTIC PRECOCIOUS/MED12 is a novel flowering regulator with multiple target steps in Arabidopsis

The proper timing of flowering is of crucial importance for reproductive success of plants. Regulation of flowering is orchestrated by inputs from both environmental and endogenous signals such as daylength, light quality, temperature and hormones, and key flowering regulators construct several parallel and interactive genetic pathways. This integrative regulatory network has been proposed to create robustness as well as plasticity of the regulation. Although knowledge of key genes and their regulation has been accumulated, there still remains much to learn about how they are organized into an integrative regulatory network. Here, we have analyzed the CRYPTIC PRECOCIOUS (CRP) gene for the Arabidopsis counterpart of the MED12 subunit of the Mediator. A novel dominant mutant, crp-1D, which causes up-regulation of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), FRUITFULL (FUL) and APETALA1 (AP1) expression in a FLOWERING LOCUS T (FT)-dependent manner, was identified in an enhancer screen of the early-flowering phenotype of 35S::FT. Genetic and molecular analysis of both crp-1D and crp loss-of-function alleles showed that MED12/CRP is required not only for proper regulation of SOC1, FUL and AP1, but also for up-regulation of FT, TWIN SISTER OF FT (TSF) and FD, and down-regulation of FLOWERING LOCUS C (FLC). These observations suggest that MED12/CRP is a novel flowering regulator with multiple regulatory target steps both upstream and downstream of the key flowering regulators including FT florigen. Our work, taken together with recent studies of other Mediator subunit genes, supports an emerging view that the Mediator plays multiple roles in the regulation of flowering.     

Site-directed mutagenesis of the <Emphasis Type="Italic">Arabidopsis</Emphasis> heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins

Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins.     

A new validated mathematical model of the Wnt signalling pathway predicts effective combinational therapy by sFRP and Dkk

The Wnt signalling pathway controls cell proliferation and differentiation, and its deregulation is implicated in different diseases including cancer. Learning how to manipulate this pathway could substantially contribute to the development of therapies. We developed a mathematical model describing the initial sequence of events in the Wnt pathway, from ligand binding to β-catenin accumulation, and the effects of inhibitors, such as sFRPs (secreted Frizzled-related proteins) and Dkk (Dickkopf). Model parameters were retrieved from experimental data reported previously. The model was retrospectively validated by accurately predicting the effects of Wnt3a and sFRP1 on β-catenin levels in two independent published experiments (R(2) between 0.63 and 0.91). Prospective validation was obtained by testing the model's accuracy in predicting the effect of Dkk1 on Wnt-induced β-catenin accumulation (R(2)≈0.94). Model simulations under different combinations of sFRP1 and Dkk1 predicted a clear synergistic effect of these two inhibitors on β-catenin accumulation, which may point towards a new treatment avenue. Our model allows precise calculation of the effect of inhibitors applied alone or in combination, and provides a flexible framework for identifying potential targets for intervention in the Wnt signalling pathway.     

Effect of liposomes on energy-dependent uptake of the antioxidant SkQR1 by isolated mitochondria

The mitochondria-targeted antioxidant SkQR1 composed of a plastoquinone part covalently bound to a cationic rhodamine 19 moiety via a decane linker was previously shown to effectively protect brain and kidney from ischemia injury accompanying generation of reactive oxygen species. In the present paper the energy-dependent SkQR1 uptake by isolated rat liver mitochondria was studied by fluorescence correlation spectroscopy peak intensity analysis (FCS PIA). This approach can be used to measure the number of fluorescent molecules per single mitochondrion. A large portion of SkQR1 appeared to be taken up by mitochondria in an energy-independent fashion because of its high affinity to membranes. Liposomes were found to compete effectively with mitochondria for the energy-independent SkQR1 binding, thereby facilitating, as an "SkQR1-buffer", observation of energy-dependent SkQR1 accumulation in mitochondria. The rate of energy-dependent SkQR1 uptake by mitochondria observed in the presence of liposomes was rather low (minutes) which was apparently due to slow redistribution of SkQR1 between liposomal and mitochondrial membranes. This can explain the low rate of staining of mitochondria by SkQR1 in living cells containing, besides mitochondria, other membrane components (endoplasmic reticulum, Golgi membranes, endosomes, lysosomes, etc.) which can compete with mitochondria for the energy-independent SkQR1 binding.     

The remote sensing of mental stress from the electromagnetic reflection coefficient of human skin in the sub-THz range

Recent work has demonstrated that the reflection coefficient of human skin in the frequency range from 95 to 110 GHz (W band) mirrors the temporal relaxation of stress induced by physical exercise. In this work, we extend these findings to show that in the event of a subtle trigger to stress, such as mental activity, a similar picture of response emerges. Furthermore, the findings are extended to cover not only the W band (75-110 GHz), but also the frequency band from 110 to 170 GHz (D band). We demonstrate that mental stress, induced by the Stroop effect and recorded by the galvanic skin response (GSR), can be correlated to the reflection coefficient in the aforementioned frequency bands. Intriguingly, a light physical stress caused by repeated hand gripping clearly showed an elevated stress level in the GSR signal, but was largely unnoted in the reflection coefficient in the D band. The implication of this observation requires further validation.     

Halloysite clay nanotubes for resveratrol delivery to cancer cells

Halloysite is natural aluminosilicate clay with hollow tubular structure which allows loading with low soluble drugs using their saturated solutions in organic solvents. Resveratrol, a polyphenol known for having antioxidant and antineoplastic properties, is loaded inside these clay nanotubes lumens. Release time of 48?h is demonstrated. Spectroscopic and ζ-potential measurements are used to study the drug loading/release and for monitoring the nanotube layer-by-layer (LbL) coating with polyelectrolytes for further release control. Resveratrol-loaded clay nanotubes are added to breast cell cultures for toxicity tests. Halloysite functionalization with LbL polyelectrolyte multilayers remarkably decrease nanotube self-toxicity. MTT measurements performed with a neoplastic cell lines model system (MCF-7) as function of the resveratrol-loaded nanotubes concentration and incubation time indicate that drug-loaded halloysite strongly increase of cytotoxicity leading to cell apoptosis.     

Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

Kaposi''s Sarcoma (KS), caused by Kaposi''s Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.