The angiotensin‐(1‐7)/Mas receptor axis protects from endothelial cell senescence via klotho and Nrf2 activation

Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin–angiotensin system (RAS) heptapeptide angiotensin (Ang)‐(1‐7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence‐associated galactosidase (SA‐β‐gal+) positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein‐coupled receptor Mas, Ang‐(1‐7) inhibited the pro‐senescence action of Ang II, but also of a non‐RAS stressor such as the cytokine IL‐1β. Moreover, Ang‐(1‐7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti‐senescence action of the heptapeptide. Indeed, both Ang‐(1‐7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase‐1 (HO‐1) pathway. The HO‐1 inhibitor tin protoporphyrin IX prevented the anti‐senescence action evoked by Ang‐(1‐7) or recombinant klotho. Overall, the present study identifies Ang‐(1‐7) as an anti‐senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO‐1. Ang‐(1‐7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications.     

Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Variation in Sulfur and Selenium Accumulation Is Controlled by Naturally Occurring Isoforms of the Key Sulfur Assimilation Enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis Species Range

Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored.Sulfur is one of the essential mineral nutrients for all organisms and is required for the biosynthesis of sulfur-containing amino acids, lipids, vitamins, and secondary metabolites, the catalytic and regulatory activity of enzymes, and the stabilization of protein structures. However, animals can only utilize sulfur that has been incorporated into biomolecules primarily originating from plants. Sufficient sulfur fertilization is also important to maximize yields of various crops, including rapeseed (Brassica napus) and wheat (Triticum aestivum; Bloem et al., 2004; Dubousset et al., 2010; Steinfurth et al., 2012). Interestingly, sulfur fertilization can also be important for food quality, with well-fertilized wheat crops producing flour with improved bread-making qualities (Shahsavani and Gholami, 2008).Over the last 50 years, the levels of sulfur in soils have been impacted significantly by the release of sulfur dioxide into the atmosphere as a result of the combustion of fossil fuels (Menz and Seip, 2004). In the atmosphere, sulfur dioxide is oxidized to sulfuric acid and thereafter deposited on soils as acid rain. Regulations controlling the release of sulfur dioxide from power stations introduced in the 1970s have caused significant declines in sulfate deposition due to this acid rain (Menz and Seip, 2004). These reductions in sulfate deposition are significant enough to have increased the need for sulfur fertilization of agricultural crops such as rapeseed and wheat (Dubousset et al., 2010; Steinfurth et al., 2012). The essentiality of sulfur for plants and the relatively recent major fluctuations in soil sulfate deposition make the study of natural variation in sulfur homeostasis by plants attractive. Such studies not only have the potential to identify new molecular mechanisms involved in sulfur homeostasis but also could provide a platform for probing at the genetic level interactions between natural plant populations and a changing soil environment.The sulfur analog selenium is also widely distributed in soil and is incorporated into biomolecules in plants in place of sulfur via sulfur assimilatory processes. Although selenium is not required by plants, it is essential for animals, and plant-based selenium is the primary source of this important nutrient for humans. In animals, selenium plays a vital role in numerous different selenoproteins involved in redox reactions, selenium storage, and hormone biosynthesis (Underwood, 1981; Rayman, 2012; Roman et al., 2014). In these proteins, selenium is incorporated as seleno-Cys. Unlike plants where selenium is incorporated into biomolecules nonspecifically as a sulfur analog, seleno-Cys in animals is biosynthesized by the action of a specific seleno-Cys synthase that converts Ser-tRNA to seleno-Cys-tRNA (Roman et al., 2014). To help prevent the negative health effects of selenium deficiency in the diet, fortification of various foods with selenium is already practiced, and biofortification of crops such as wheat, through the addition of selenium to fertilizers, is being proposed (Lyons et al., 2003). Moreover, there is a growing body of studies suggesting that supraoptimal dietary levels of selenium in the form of seleno-Met and seleno-methylseleno-Cys may be helpful in preventing certain cancers, although the efficacy of these supplements is still debated (Rayman, 2012; Steinfurth et al., 2012). Therefore, the identification of genes that control variation in the uptake and metabolism of sulfur and selenium in plants is not only an essential task for understanding the molecular mechanisms of plant nutrition but also important for crop yield, food quality, and human health.Plants mainly take up sulfur from the soil in the form of sulfate. After uptake of sulfate via the sulfate transporters SULTR1;1 and SULTR1;2 in the root (Yoshimoto et al., 2007; Barberon et al., 2008), sulfate needs to be reduced to sulfide before it can be incorporated into Cys, the first sulfur-containing compound in the assimilatory process (for review, see Takahashi et al., 2011). Sulfate is first activated through adenylation by ATP SULFURYLASE (ATPS) to form adenosine 5′-phosphosulfate (APS) in both plastids and the cytosol (Rotte and Leustek, 2000). After activation, sulfate as APS is reduced to sulfite by APS REDUCTASE (APR) using reduced glutathione (GSH) as the electron donor (Gutierrez-Marcos et al., 1996; Setya et al., 1996). Alternatively, APS can be phosphorylated by APS kinase to form 3′-phosphoadenosine 5′-phosphosulfate (PAPS), which acts as the sulfate donor for sulfotransferases to incorporate sulfate directly into saccharides and secondary metabolites such as glucosinolates (Mugford et al., 2009). Sulfite produced by APR is further reduced to sulfide by sulfite reductase (Khan et al., 2010). In the final step, sulfide is combined with O-acetyl-Ser to form Cys, catalyzed by O-acetyl-Ser(thiol)lyase (Wirtz and Hell, 2006). In plants, selenium is taken up as selenate via sulfate transporters (Shibagaki et al., 2002). After uptake, it is thought that selenate is reduced to selenite via the action of ATP sulfurylase and APS reductase (Shaw and Anderson, 1972; Pilon-Smits et al., 1999; Sors et al., 2005a, 2005b). Selenite is most likely nonenzymatically reduced to selenide and combined with O-acetyl-Ser to form seleno-Cys catalyzed by O-acetyl-Ser(thiol)lyase (Ng and Anderson, 1978).Sulfate uptake and reduction represent the two control points for sulfur homeostasis (Kopriva et al., 2009). Based on sequence similarity, there are 14 genes annotated as sulfate transporters in the Arabidopsis (Arabidopsis thaliana) genome, and at least six of them have evidence supporting their functions (Kopriva et al., 2009; Takahashi, 2010). Among these, SULTR1;1 and SULTR1;2 encode high-affinity sulfate transporters in the root responsible for sulfate uptake from the soil solution (Rouached et al., 2008; Takahashi, 2010). Genes encoding both ATPS and APR also form gene families in Arabidopsis, with ATPS being encoded by four genes and APR by three (Kopriva et al., 2009). ATPS enzymes localize to both the cytosol and plastids, whereas APR enzymes localize solely to the plastids (Lunn et al., 1990; Rotte and Leustek, 2000). ATPS1 and APR2 are responsible for the majority of ATP sulfurylase and APS reductase activity of seedlings.In a quantitative trait locus (QTL) analysis of sulfate content in Arabidopsis leaves using recombinant inbred lines generated by crossing Bayreuth (Bay-0) and Shahdara (Sha), APR2 was identified as a locus controlling variation in sulfate accumulation between the Bay-0 and Sha accessions (Loudet et al., 2007). The causal quantitative trait nucleotide in the Sha allele of APR2 results in a substitution of Ala-399 to Glu-399. The substitution localizes in the thioredoxin active site, which reduces the affinity of APR2 for GSH and results in a loss of 99.8% of enzyme activity. The loss-of-function Sha APR2 allele leads to a significant decrease in sulfate reduction and a concomitant increase in leaf sulfate accumulation. In the same analysis, a second QTL for leaf sulfate accumulation was described (Loudet et al., 2007). This second QTL was recently established to be driven by an ATPS1 expression-level polymorphism between Bay-0 and Sha, with Bay-0 showing reduced expression of ATPS1 compared with Sha (Koprivova et al., 2013).Both Loudet et al. (2007) and Koprivova et al. (2013) focused their studies on the genetic architecture of natural variation for leaf sulfate using a single recombinant population created by crossing the Bay-0 and Sha accessions. This recombinant inbred population has proved a powerful tool for the identification of novel alleles controlling several different traits (Loudet et al., 2008; Jiménez-Gómez et al., 2010; Jasinski et al., 2012; Pineau et al., 2012; Anwer et al., 2014). However, because this set of recombinant inbred lines is composed of genotypes from only two accessions, its value is limited when trying to understand allelic diversity across the Arabidopsis species as a whole. To address this limitation, we performed experiments using a set of 349 Arabidopsis accessions collected from across the species range. These 349 accessions were selected from a worldwide collection of 5,810 accessions sampled to minimize redundancy and close family relatedness (Baxter et al., 2010; Platt et al., 2010). To allow us to further probe Arabidopsis species-wide allelic diversity, we also utilized the complete genome sequences of 855 Arabidopsis accessions from the 1,001 Genomes Project (http://signal.salk.edu/atg1001/index.php).Since sulfate uptake and accumulation is only one step in the complex process of sulfur assimilation in plants, uncovering the genetic architecture of sulfate accumulation, as performed by Loudet et al. (2007) and Koprivova et al. (2013), could potentially overlook genetic variation in other important aspects of sulfur metabolism. To enable us to identify natural genetic variation in sulfur homeostasis beyond just sulfate accumulation, we chose to quantify total leaf sulfur. This potentially allowed us to capture variation in the accumulation of both sulfate and other important sulfur-containing metabolites such as GSH and glucosinolates.Furthermore, to test genetically the long-held assumption that selenium in plants is metabolized as a sulfur analog, we also phenotyped the same set of plants for total leaf selenium. Through a combination of high-throughput elemental analysis, linkage mapping, genetic and transgenic complementation, reciprocal grafting, and protein haplotype analysis, we have established that the natural variation in both leaf sulfur and selenium content in Arabidopsis is controlled by several rare APR2 variants across the whole of the Arabidopsis species.     

Mitoxantrone changes spectrin-aminophospholipid interactions

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatidylcholine (PE/PC) monolayers. The change in delta pi induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5'-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16'-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.     

RP-HPLC method for quantitative determination of cystathionine, cysteine and glutathione: An application for the study of the metabolism of cysteine in human brain

The RP-HPLC method for a simultaneous separation and quantitation of the dinitrophenyl derivative of cystathionine (N,N'-di-DNP) in biological samples together with GSH, GSSG, cysteine and cystine, provides a very useful tool for investigation of the transsulfuration pathway in biological samples, at the same providing results which reflect the redox status (GSH/GSSG ratio) and the potential of the generation of H?S. An application of the method for the study of the process of transsulfuration in various human brain regions shows the presence of cystathionine in all the investigated regions; it also demonstrates that cystathionine levels vary greatly between particular regions. The highest level in the thalamus and the lowest in the cerebellum were associated with respectively a low or high γ-cystathionase activity, and at the same time, a high cysteine and GSH level in the thalamus and a low value in the cerebellum. Based on the above results, one may suggest a regulatory mechanism responsible for inhibition of the CGL activity at high concentration values of cysteine and/or GSH. Simultaneous determinations of GSH and GSSG levels allow for determining the GSH/GSSG ratio, which reflects tissue redox status. The method may be also employed in determining the activity of γ-cystathionase and cystathionine-β synthase.     

Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione.Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.     

SERS-based sensor for direct L-selectin level determination in plasma samples as alternative method of tumor detection

Selectin ligands are present on the surface of tumor cells, for this reason lowering the L-selectin level in the blood and lymph can indicate presence of the tumor. Therefore the selectin level in the plasma are potential targets for anticancer therapy. We demonstrate the surface enhanced Raman spectroscopy (SERS)-based sensor for the determination of L-selectin level in biological samples that can be used in medical diagnosis. The combination of SERS with the method of multivariate analysis as principle component analysis (PCA) allows to strengthen the presented data analysis. The loadings of PCA permit to indicate those vibration modes, that are the most important for the assumed identification (bands at 1574, 1450, 1292 cm⁻¹). Two bands at 1286 and 1580 cm⁻¹ were selected for the determination of the calibration curve (bands intensities I₁₂₈₆/I₁₅₈₀ ratio). The L-selectin level of biological samples can be read, directly from the calibration curve. The presented sensor is as a sensitive tool with good specificity and selectivity of L-selectin, even in the case of coexistence of P- and E-selectin.     

A chemical screen for modulators of mRNA translation identifies a distinct mechanism of toxicity for sphingosine kinase inhibitors

We here conducted an image-based chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. None of the compounds up-regulated translation, which could be due to the screen being performed in cancer cells grown in full media where translation is already present at very high levels. Regarding translation down-regulators, and consistent with current knowledge, inhibitors of the mechanistic target of rapamycin (mTOR) signaling pathway were the most represented class. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically, this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum (ER), which activates the integrated stress response (ISR) and contributes to the toxicity of SPHK inhibitors. Surprisingly, the toxicity and activation of the ISR triggered by 2 independent SPHK inhibitors, SKI-II and ABC294640, the latter in clinical trials, are also observed in cells lacking SPHK1 and SPHK2. In summary, our study provides a useful resource on the effects of medically used drugs on translation, identified compounds capable of reducing translation independently of mTOR and has revealed that the cytotoxic properties of SPHK inhibitors being developed as anticancer agents are independent of SPHKs.

A chemical screen to evaluate how 4100 drugs modulate translation rates confirms mTOR as the main pathway regulating translation and reveals that sphingosine kinase inhibitors downregulate translation via activation of the ER-stress response. Sphingosine kinase inhibitors, including one in clinical trials, activate stress responses and kill cells independently of the cognate target.