A Simple Screening Method for Extra Virgin Olive Oil Adulteration by Determining Squalene and Tyrosol.

A simple screening method for discrimination between commercial extra virgin olive oils and their blends with other vegetable oils was developed. Squalene, which was contained relatively high amounts in virgin olive oil, was determined by HPLC after a simple pretreatment that was carried out by dilution of oil samples with 2-propanol. Tyrosol, which was contained at relatively high concentration in virgin olive oil among phenolic compounds, was determined by HPLC after a simple liquid-liquid extraction. When using squalene and tyrosol contents as axes, extra virgin olive oils could be discriminated from pure olive oils, blended oils (extra virgin olive oils with sunflower oil or grapeseed oil) and other vegetable oils. These results suggest that determining squalene and tyrosol in seed oil samples could be useful in distinguishing between extra virgin olive oil and blended oils as a screening method.

ß-Catenin-dependent transcription is central to Bmp-mediated formation of venous vessels.

ß-catenin regulates the transcription of genes involved in diverse biological processes, including embryogenesis, tissue homeostasis and regeneration. Endothelial cell (EC)-specific gene-targeting analyses in mice have revealed that ß-catenin is required for vascular development. However, the precise function of ß-catenin-mediated gene regulation in vascular development is not well understood, since ß-catenin regulates not only gene expression but also the formation of cell-cell junctions. To address this question, we have developed a novel transgenic zebrafish line that allows the visualization of ß-catenin transcriptional activity specifically in ECs and discovered that ß-catenin-dependent transcription is central to the bone morphogenetic protein (Bmp)-mediated formation of venous vessels. During caudal vein (CV) formation, Bmp induces the expression of aggf1, a putative causative gene for Klippel-Trenaunay syndrome, which is characterized by venous malformation and hypertrophy of bones and soft tissues. Subsequently, Aggf1 potentiates ß-catenin transcriptional activity by acting as a transcriptional co-factor, suggesting that Bmp evokes ß-catenin-mediated gene expression through Aggf1 expression. Bmp-mediated activation of ß-catenin induces the expression of Nr2f2 (also known as Coup-TFII), a member of the nuclear receptor superfamily, to promote the differentiation of venous ECs, thereby contributing to CV formation. Furthermore, ß-catenin stimulated by Bmp promotes the survival of venous ECs, but not that of arterial ECs. Collectively, these results indicate that Bmp-induced activation of ß-catenin through Aggf1 regulates CV development by promoting the Nr2f2-dependent differentiation of venous ECs and their survival. This study demonstrates, for the first time, a crucial role of ß-catenin-mediated gene expression in the development of venous vessels.

Simultaneous determination of water-soluble vitamins in beverages and dietary supplements by LC-MS/MS.

An LC-MS/MS method was developed for the simultaneous determination of 15 water-soluble vitamins that are widely used as additives in beverages and dietary supplements. This combined method involves the following simple pre-treatment procedures: dietary supplement samples were prepared by centrifugation and filtration after an extraction step, whereas beverage samples were diluted prior to injection. Chromatographic analysis in this method utilised a multi-mode ODS column, which provided reverse-phase, anion- and cation-exchange capacities, and therefore improved the retention of highly polar analytes such as water-soluble vitamins. Additionally, the multi-mode ODS column did not require adding ion pair reagents to the mobile phase. We optimised the chromatographic separation of 15 water-soluble vitamins by adjusting the mobile phase pH and the organic solvent. We also conducted an analysis of a NIST Standard Reference Material (SRM 3280 Multi-vitamin/Multi-element tablets) using this method to verify its accuracy. In addition, the method was applied to identify the vitamins in commercial beverages and dietary supplements. By comparing results with the label values and results obtained by official methods, it was concluded that the method could be used for quality control and to compose nutrition labels for vitamin-enriched products.

Simultaneous LC-MS/MS analysis of glyphosate, glufosinate, and their metabolic products in beer, barley tea, and their ingredients.

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H2O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.

A connexin40 mutation associated with a malignant variant of progressive familial heart block type I.

BACKGROUND: Progressive familial heart block type I (PFHBI) is a hereditary arrhythmia characterized by progressive conduction disturbances in the His-Purkinje system. PFHBI has been linked to genes such as SCN5A that influence cardiac excitability but not to genes that influence cell-to-cell communication. Our goal was to explore whether nucleotide substitutions in genes coding for connexin proteins would associate with clinical cases of PFHBI and if so, to establish a genotype-cell phenotype correlation for that mutation. METHODS AND RESULTS: We screened 156 probands with PFHBI. In addition to 12 sodium channel mutations, we found a germ line GJA5 (connexin40 [Cx40]) mutation (Q58L) in 1 family. Heterologous expression of Cx40-Q58L in connexin-deficient neuroblastoma cells resulted in marked reduction of junctional conductance (Cx40-wild type [WT], 22.2±1.7 nS, n=14; Cx40-Q58L, 0.56±0.34 nS, n=14; P<0.001) and diffuse localization of immunoreactive proteins in the vicinity of the plasma membrane without formation of gap junctions. Heteromeric cotransfection of Cx40-WT and Cx40-Q58L resulted in homogenous distribution of proteins in the plasma membrane rather than in membrane plaques in ≈50% of cells; well-defined gap junctions were observed in other cells. Junctional conductance values correlated with the distribution of gap junction plaques. CONCLUSIONS: Mutation Cx40-Q58L impairs gap junction formation at cell-cell interfaces. This is the first demonstration of a germ line mutation in a connexin gene that associates with inherited ventricular arrhythmias and emphasizes the importance of Cx40 in normal propagation in the specialized conduction system.

Investigation of biologically active components in ginkgo leaf products on the Japanese market.

The rapid and highly separative ultra high-performance liquid chromatography (UHPLC)-UV method was adopted and validated to investigate the flavonol glycoside compositions in ginkgo leaf products on the Japanese market. The result indicates that certain products contained amounts of flavonol glycosides approximately equivalent to the medicinal product. Additionally, we examined the correlations between the total amount of flavonol glycosides and of terpene lactones in various ginkgo leaf products.

Analysis of terpene lactones in a Ginkgo leaf extract by high-performance liquid chromatography using charged aerosol detection.

A new HPLC method using charged aerosol detection was developed for the determination of terpene lactones in a Ginkgo leaf extract. The linearity of the standard curves was excellent (r>0.999). The repeatability of the method was less than 3%, and its reproducibility was less than 5% for each analyte. The limit of detection was between 0.087 and 0.45 microg/ml. The developed method was applied to the analysis of terpene lactones in Ginkgo leaf products distributed in the Japanese market. The results suggest that some health food products contained approximately equivalent amounts of terpene lactones to those in the medical product and that the proportion of terpene lactones varied in each health product.

Determination of ochratoxin a in ready-to-drink coffee by immunoaffinity cleanup and liquid chromatography-tandem mass spectrometry.

We developed a simple and accurate method for determining ochratoxin A (OTA) in ready-to-drink coffee, using an immunoaffinity column for cleanup and liquid chromatography-tandem mass spectrometry (LC/MS/MS) for identification and quantification. When uniformly stable isotope-labeled OTA (U-[(13)C(20)]-OTA) was employed as an internal standard, the recovery rate of the method was 97.3% (the spiked OTA level was 0.10 ng/mL), the repeatability (relative standard deviation) was 1.9%, and the intermediate precision (relative standard deviation) was 4.0%. The limit of quantification was 0.0065 ng/mL based on a signal-to-noise ratio in coffee of 10:1. The developed method was used for the determination of OTA in ready-to-drink coffee. A total of 30 ready-to-drink coffee samples commercially available in Japan were analyzed. OTA was detected in all of the samples at concentrations ranging from trace levels (0.0020-0.010 ng/mL) to 0.037 ng/mL. This method was shown to be useful for accurately evaluating the intake of OTA from coffee beverages.

Metformin prevents progression of heart failure in dogs: role of AMP-activated protein kinase.

BACKGROUND: Some studies have shown that metformin activates AMP-activated protein kinase (AMPK) and has a potent cardioprotective effect against ischemia/reperfusion injury. Because AMPK also is activated in animal models of heart failure, we investigated whether metformin decreases cardiomyocyte apoptosis and attenuates the progression of heart failure in dogs. METHODS AND RESULTS: Treatment with metformin (10 micromol/L) protected cultured cardiomyocytes from cell death during exposure to H2O2 (50 micromol/L) via AMPK activation, as shown by the MTT assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and flow cytometry. Continuous rapid ventricular pacing (230 bpm for 4 weeks) caused typical heart failure in dogs. Both left ventricular fractional shortening and left ventricular end-diastolic pressure were significantly improved in dogs treated with oral metformin at 100 mg x kg(-1) x d(-1) (n=8) (18.6+/-1.8% and 11.8+/-1.1 mm Hg, respectively) compared with dogs receiving vehicle (n=8) (9.6+/-0.7% and 22+/-0.9 mm Hg, respectively). Metformin also promoted phosphorylation of both AMPK and endothelial nitric oxide synthase, increased plasma nitric oxide levels, and improved insulin resistance. As a result of these effects, metformin decreased apoptosis and improved cardiac function in failing canine hearts. Interestingly, another AMPK activator (AICAR) had effects equivalent to those of metformin, suggesting the primary role of AMPK activation in reducing apoptosis and preventing heart failure. CONCLUSIONS: Metformin attenuated oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with activation of AMPK. Therefore, metformin may be a potential new therapy for heart failure.

Cell type-specific regulation of RhoA activity during cytokinesis.

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.

Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo.

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.