Inhibition of the Prostaglandin Transporter PGT Lowers Blood Pressure in Hypertensive Rats and Mice

Inhibiting the synthesis of endogenous prostaglandins with nonsteroidal anti-inflammatory drugs exacerbates arterial hypertension. We hypothesized that the converse, i.e., raising the level of endogenous prostaglandins, might have anti-hypertensive effects. To accomplish this, we focused on inhibiting the prostaglandin transporter PGT (SLCO2A1), which is the obligatory first step in the inactivation of several common PGs. We first examined the role of PGT in controlling arterial blood pressure blood pressure using anesthetized rats. The high-affinity PGT inhibitor T26A sensitized the ability of exogenous PGE₂ to lower blood pressure, confirming both inhibition of PGT by T26A and the vasodepressor action of PGE₂ T26A administered alone to anesthetized rats dose-dependently lowered blood pressure, and did so to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto control rats. In mice, T26A added chronically to the drinking water increased the urinary excretion and plasma concentration of PGE₂ over several days, confirming that T26A is orally active in antagonizing PGT. T26A given orally to hypertensive mice normalized blood pressure. T26A increased urinary sodium excretion in mice and, when added to the medium bathing isolated mouse aortas, T26A increased the net release of PGE₂ induced by arachidonic acid, inhibited serotonin-induced vasoconstriction, and potentiated vasodilation induced by exogenous PGE₂. We conclude that pharmacologically inhibiting PGT-mediated prostaglandin metabolism lowers blood pressure, probably by prostaglandin-induced natriuresis and vasodilation. PGT is a novel therapeutic target for treating hypertension.     

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.     

Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.     

Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.     

Effects of plant odor on oviposition by the black swallowtail butterfly,Papilio polyxenes (Lepidoptera: Papilionidae)

Black swallowtail females laid more eggs on plant models treated with contact stimulants and volatiles from carrot leaves than on models treated only with contact stimulants. The volatiles enhanced landing rates and females alighted more frequently on artificial leaves treated with host volatiles than on adjacent control leaves. Volatiles from cabbage, a nonhost, inhibited landing rates on artificial leaves treated with carrot contact stimulants. Examination of antennae revealed two major types of sensilla, believed to be olfactory in function. Electroantennogram preparations responded more strongly to carrot volatiles than to cabbage volatiles and several shared responses at particular retention times to carrot volatile components eluting from a gas chromatograph. Our results are consistent with a long-standing hypothesis that behavioral responses to essential oil components characteristic of the larval food plants have facilitated host shifts in the genus Papilio.