G alpha12 interaction with alphaSNAP induces VE-cadherin localization at endothelial junctions and regulates barrier function

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified alphaSNAP as an interactive partner of G alpha12 using yeast two-hybrid screening. Glutathione S-transferase pull-down assays showed the selective interaction of alphaSNAP with G alpha12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G alpha12 (1-37 amino acids) was necessary and sufficient for its interaction with alphaSNAP. G alpha13 with its N-terminal extension replaced by that of G alpha12 acquired the ability to bind to alphaSNAP, whereas G alpha12 with its N terminus replaced by that of G alpha13 lost this ability. Using four point mutants of alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alphaSNAP was involved in its interaction with G alpha12. Co-transfection of human umbilical vein endothelial cells with G alpha12 and alphaSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alphaSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G12 and alphaSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.     

Electrochemical synthesis and X-ray crystal structures of beta-D-2-phenylselenyl-1,3,4,6-tetra-O-acetylglucopyranose and alpha-D-2-phenylselenyl-1,3,4,6-tetra-O-acetylmannopyranose

Electrochemical acetoxyphenylselenation of 3,4-dihydro-2H-pyran and D-3,4,6-tri-O-acetylglucal was studied. The constant current electrolysis (50mA) of dihydropyran and diphenyl diselenide in an acetic acid solution of tetramethylammonium chloride was performed at room temperature in an undivided cell using a graphite anode and an aluminum cathode and yielded trans-DL-2-acetoxy-3-phenylselenyltetrahydropyran (27%), in agreement with Markovnikov's rule. The analysis of the 1H NMR spectral data showed that the acetoxy and phenylselenyl groups adopt axial positions in the most stable conformation of this compound due to the anomeric effect. Under the same conditions D-3,4,6-tri-O-acetylglucal afforded D-2-phenylselenyl-1,3,4,6-tetra-O-acetylglucopyranose and D-2-phenylselenyl-1,3,4,6-tetra-O-acetylmannopyranose, which were separated by column chromatography and isolated in 87% overall yield (isomer ratio 60:40). The structures of these compounds were established by spectral data. Single crystal X-ray structure determinations of the diastereomers are reported.     

Use of digital photographs for artificial tooth selection

Digital photography has become available to everybody. The aim of this study was to examine possibility of calculating the width of a missing central incisor using digital photographs. Digital photographs were obtained from 51 dentate subjects using a 3.1 Megapixel digital camera from various distances: 35 cm, 70 cm, 1 m and 1.5 m. For the calculation of the width of maxillary left central incisor (MLI), the following equation was used: MLI(calculated) = Photographic width of MLI x IPD / photographic IPD. Statistical analysis was made (Kolmogorov-Smirnov test, dependent sample t-test, correlation and frequencies) using SPSS 10.0 for Windows. Results revealed no significant difference between the calculated MLI (70 cm, 1 m and 1.5 m distance) and actual MLI, however calculated MLI from 35 cm distance was significantly different from the actual MLI value (p < 0.01). The highest correlation was between calculated MLI (70 cm distance) and actual MLI. However, the highest percentage of results from a distance of 70 cm also fitted within +/- 0.3 mm; +/- 0.5 mm and +/- 1 mm difference from the actual MLI values. However, the results obtained from 1 m distance were also satisfactory. The technique of use of digital photography is of proven value in calculating the width of a maxillary central incisor. The photographs using a simple digital camera should be taken from a distance from 70 cm to 1 m. Saving such photographs in a dental office may eventually be helpful for calculating dimensions of artificial teeth.     

Cardiac structural and functional responses to salt loading in SHR

Increased dietary salt intake induces cardiac fibrosis in the spontaneously hypertensive rat (SHR), yet little information details its effects on left ventricular (LV) function. Additionally, young normotensive rats are more sensitive to the trophic effect of dietary sodium than older rats. Thus cardiac responses to salt loading were evaluated at two ages in the SHR; LV collagen content was also examined. SHR (8 or 20 wk of age) were given an 8% salt diet; their age-matched controls received standard chow. Echocardiographic indexes, arterial pressure, and LV hydroxyproline concentration were measured at 16 and 52 wk in the younger and older SHR groups, respectively. In most SHR, salt excess increased arterial pressure, LV mass, and hydroxyproline concentration and impaired LV relaxation manifested by prolonged isovolumic relaxation time, decreased early and atrial filling velocity ratio (V(E)/V(A)), and slower propagation velocity of E wave (V(P)). LV systolic function remained normal. However, one-quarter of the young salt-loaded SHR developed cardiac failure with systolic and diastolic dysfunction associated with greater LV mass and ventricular fibrosis. They also had lower arterial pressure, decreased fractional shortening, and a restrictive pattern of mitral flow. Moreover, the shorter deceleration time of the E wave and increased V(E)/V(P), an index of LV filling pressure, indicated increased LV stiffness in these rats. These findings demonstrated that sodium sensitivity in SHR is manifested not only by further pressure elevation but also by significant LV functional impairment that most likely is related to enhanced ventricular fibrosis. Moreover, the SHR are more susceptible to cardiac damage when high dietary salt is introduced earlier in life.     

The amino terminus of Epstein-Barr virus glycoprotein gH is important for fusion with epithelial and B cells

Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required for entry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells.     

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.     

Cytokinin-induced growth in the duckweeds <Emphasis Type="Italic">Lemna gibba</Emphasis> and <Emphasis Type="Italic">Spirodela polyrhiza</Emphasis>

Duckweeds, quick-growing aquatic plants, have been recently recognized as promising hosts for the large-scale production of recombinant proteins and as an ideal biomass feedstock for biofuel production. These possible wide-spread industrial uses of duckweeds intensified research aimed at understanding the mechanisms that control duckweed growth. Here, we describe how the hormone cytokinin affects growth. We performed a number of standard cytokinin growth- and physiological-response assays using sterile-grown colonies of Lemna gibba and Spirodela polyrhiza. Similar to land plants, cytokinin inhibited root elongation in duckweeds. Surprisingly, and in contrast to land plants, cytokinin promoted growth of aerial organs in both duckweed species, suggesting that the cytokinin growth response fundamentally differs between aquatic and land plants.     

miR‐212 downregulation contributes to the protective effect of exercise against non‐alcoholic fatty liver via targeting FGF‐21

Non‐alcoholic fatty liver disease (NAFLD) is associated with obesity and lifestyle, while exercise is beneficial for NAFLD. Dysregulated microRNAs (miRs) control the pathogenesis of NAFLD. However, whether exercise could prevent NAFLD via targeting microRNA is unknown. In this study, normal or high‐fat diet (HF) mice were either subjected to a 16‐week running program or kept sedentary. Exercise attenuated liver steatosis in HF mice. MicroRNA array and qRT‐PCR demonstrated that miR‐212 was overexpressed in HF liver, while reduced by exercise. Next, we investigated the role of miR‐212 in lipogenesis using HepG2 cells with/without long‐chain fatty acid treatment (±FFA). FFA increased miR‐212 in HepG2 cells. Moreover, miR‐212 promoted lipogenesis in HepG2 cells (±FFA). Fibroblast growth factor (FGF)‐21, a key regulator for lipid metabolism, was negatively regulated by miR‐212 at protein level in HepG2 cells. Meanwhile, FFA downregulated FGF‐21 both at mRNA and protein levels in HepG2 cells. Also, FGF‐21 protein level was reduced in HF liver, while reversed by exercise in vivo. Furthermore, siRNA‐FGF‐21 abolished the lipogenesis‐reducing effect of miR‐212 inhibitor in HepG2 cells (±FFA), validating FGF‐21 as a target gene of miR‐212. These data link the benefit of exercise and miR‐212 downregulation in preventing NAFLD via targeting FGF‐21.     

A new semi-determinate tomato hybrid for early production

By using the knowledge of the genetic constitution and the characteristics of the available tomato lines for selection, there were created several dozens of hybrid combinations characterised by semi-determinate habit, with large fruits and early maturing. These hybrids were tested in preliminary and comparative trials with the hybrids Balkan F₁, Balca F₁, Žar F₁, and some others as standard varieties. These tomato hybrids were grown earlier but they are grown even today in the early production, for the market in green houses and in the open field. The hybrid Marco F₁ was the best of all. Its yield was 25 t/ha in the plastic-house (the same as in the standard), up to 32 t/ha in the open field (5 % more than in the standard). This hybrid was a few days later than Balkan, with the fruits weighting 200 grams (fruits were approximately 60 % heavier than in the standard). It possess the genetic V, F and Tm resistance.