Studies on the Sunlight Flavour of Beer

In order to establish the mechanism of occurrence of sunlight flavour of beer in continuation of the preceding paper, humulone, lupulone, and their analogues and degradation compounds, i.e., cohumulone, adhumulone, 5-acetyl-3-methylfilicinic acid including analogues, tetrahydrohumulone, hexahydrolupulone, humulinic acid and lupuloxinic acid, were tested for the occurrence of sunlight flavour of beer.

As a result, among the above-mentioned compounds, humulone, lupulone, cohumulone, adhumulone, tetrahydrohumulone and hexahydrolupulone were found to cause the sunlight flavour, but the other compounds did not cause typical sunlight flavour. This fact shows that some specific structural components seem requisite for the occurrence of sunlight flavour of beer.

It was also revealed that isomerization caused by boiling accelerates the occurrence of the sunlight flavour of beer. Finally, the result of the experiment conducted by the gas chromatographic procedure showed that any new component is not detected by the exposure of beer to sunlight but, two components somewhat increased.     

mGluR1-Mediated Excitation of Cerebellar GABAergic Interneurons Requires Both G Protein-Dependent and Src–ERK1/2-Dependent Signaling Pathways

Stimulation of type I metabotropic glutamate receptors (mGluR1/5) in several neuronal types induces slow excitatory responses through activation of transient receptor potential canonical (TRPC) channels. GABAergic cerebellar molecular layer interneurons (MLIs) modulate firing patterns of Purkinje cells (PCs), which play a key role in cerebellar information processing. MLIs express mGluR1, and activation of mGluR1 induces an inward current, but its precise intracellular signaling pathways are unknown. We found that mGluR1 activation facilitated spontaneous firing of mouse cerebellar MLIs through an inward current mediated by TRPC1 channels. This mGluR1-mediated inward current depends on both G protein-dependent and -independent pathways. The nonselective protein tyrosine kinase inhibitors genistein and AG490 as well as the selective extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors PD98059 and SL327 suppressed the mGluR1-mediated current responses. Following G protein blockade, the residual mGluR1-mediated inward current was significantly reduced by the selective Src tyrosine kinase inhibitor PP2. In contrast to cerebellar PCs, GABA_B receptor activation in MLIs did not alter the mGluR1-mediated inward current, suggesting that there is no cross-talk between mGluR1 and GABA_B receptors in MLIs. Thus, activation of mGluR1 facilitates firing of MLIs through the TRPC1-mediated inward current, which depends on not only G protein-dependent but also Src–ERK1/2-dependent signaling pathways, and consequently depresses the excitability of cerebellar PCs.     

Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.     

Metabolic diversity in tuber tissues of native Chiloé potatoes and commercial cultivars of <Emphasis Type="Italic">Solanum tuberosum</Emphasis> ssp. <Emphasis Type="Italic">tuberosum</Emphasis> L.

Introduction

The native potatoes (Solanum tuberosum ssp. tuberosum L.) cultivated on Chiloé Island in southern Chile have great variability in terms of tuber shape, size, color and flavor. These traits have been preserved throughout generations due to the geographical position of Chiloé, as well as the different uses given by local farmers.

Objectives

The present study aimed to investigate the diversity of metabolites in skin and pulp tissues of eleven native accessions of potatoes from Chile, and evaluate the metabolite associations between tuber tissues.

Methods

For a deeper characterization of these accessions, we performed a comprehensive metabolic study in skin and pulp tissues of tubers, 3 months after harvesting. Specific targeted quantification of metabolites using 96 well microplates, and high-performance liquid chromatography combined with non-targeted metabolite profiling by gas chromatography time-of-flight mass spectrometry were used in this study.

Results

We observed differential levels of antioxidant activity and phenolic compounds between skin and pulp compared to a common commercial cultivar (Desireé). In addition, we uncovered considerable metabolite variability between different tuber tissues and between native potatoes. Network correlation analysis revealed different metabolite associations among tuber tissues that indicate distinct associations between primary metabolite and anthocyanin levels, and antioxidant activity in skin and pulp tissues. Moreover, multivariate analysis lead to the grouping of native and commercial cultivars based on metabolites from both skin and pulp tissues.

Conclusions

As well as providing important information to potato producers and breeding programs on the levels of health relevant phytochemicals and other abundant metabolites such as starch, proteins and amino acids, this study highlights the associations of different metabolites in tuber skins and pulp, indicating the need for distinct strategies for metabolic engineering in these tissues. Furthermore, this study shows that native Chilean potato accessions have great potential as a natural source of phytochemicals.

     

Interactions between bradykinin and plasmin in the endothelial Ca<Superscript>2+</Superscript> response

Frequent abnormalities in 7p12 locus in different tumors like lung cancer candidate this region for novel regulatory elements. MiRNAs as novel regulatory elements encoded within the human genome are potentially oncomiRs or miR suppressors. Here, we have used bioinformatics tools to search for the novel miRNAs embedded within human chromosome 7p12. A bona fide stem loop (named mirZa precursor) had the features of producing a real miRNA (named miRZa) which was detected through RT-qPCR following the overexpression of its precursor. Then, endogenous miRZa was detected in human cell lines and tissues and sequenced. Consistent to the bioinformatics prediction, RT-qPCR as well as dual luciferase assay indicated that SMAD3 and IGF1R genes were targeted by miRZa. MiRZa-3p and miRZa-5p were downregulated in lung tumor tissue samples detected by RT-qPCR, and mirZa precursor overexpression in SW480 cells resulted in increased sub-G1 cell population. Overall, here we introduced a novel miRNA which is capable of targeting SMAD3 and IGF1R regulatory genes and increases the cell population in sub-G1 stage.     

Synthesis,antitumor activity,and cytotoxicity of 4-substituted 1-benzyl-5-diphenylstibano-1H-1,2,3-triazoles

Trisubstituted 5-organostibano-1H-1,2,3-triazoles (3a–f) were synthesized by the Cu-catalyzed azide-alkyne cycloaddition of various ethynylstibanes (1) with benzylazide (2) in the presence of CuBr (5 mol%) under aerobic conditions. The reaction of 5-stibanotriazoles with HCl afforded C5-unsubstituted 1,2,3-triazoles (4a–f). The antitumor activity of trisubstituted 5-organostibano-1H-1,2,3-triazoles (3a–f) and their 5-unsubstituted 1,2,3-triazoles (4a–f) were evaluated in several tumor cell lines. All 5-stibanotriazoles (3a–f) exerted an excellent antitumor activity. On the contrary, 5-unsubstituted 1,2,3-triazoles (4a–f) without a diphenylantimony group in the molecule exhibited very low antitumor activity compared with 5-stibanotriazoles (3a–f). In compounds of both the series, the substituted 4-butyl group appeared to decrease antitumor activity. However, results suggested that organometal (antimony) in the molecule was required for greater antitumor activity. In addition, all 5-stibanotriazoles (3a–f), but not all 5-unsubstituted 1,2,3-triazoles (4a–f), exhibited cytotoxicity in normal vascular endothelial cells derived from bovine aorta. Among the compounds (3b–e) that exhibited excellent antitumor activity, those with 4-methylphenyl (3b) and 1-cyclohexenyl (3e) showed relatively low cytotoxicity to vascular endothelial cells. Together, these results suggest that trisubstituted 5-organostibano-1H-1,2,3-triazoles, including compounds 3b and 3e, may serve as potential anticancer therapeutic drugs in the future.     

WBN/Kob-Ht Rats Spontaneously Develop Dermatitis under Conventional Conditions: Another Possible Model for Atopic Dermatitis.

WBN/Kob-Ht rats (Ht-rats) raised under conventional conditions spontaneously developed dermatitis. In this study, we carried out histopathological analysis to elucidate the pathological features of the dermatitis in Ht-rats. We then tried to detect Staphylococcus species recovered from the skin lesions of Ht-rats. We also measured the serum levels of total IgE, IL-4 and IFN-gamma in these rats. The histopathological data indicated that inflammatory cells had infiltrated the skin lesions. Staphylococcus aureus was recovered from the skin lesions, and the serum levels of total IgE and IL-4 were elevated in Ht-rats with dermatitis. These results suggest that dermatitis in Ht-rats is similar to that in the DS-Nh mice, which has recently been proposed as an animal model for human atopic dermatitis.     

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [¹³C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [¹³C]glutamate and [¹⁵N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the ¹³C/¹⁵N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.Plants are sessile, unable to relocate when exposed to diverse environmental and seasonal stimuli, and hence must be able to respond rapidly to survive stress conditions. Flooding or waterlogging of the soil is a common environmental condition that can greatly affect crop production and quality by blocking the entry of oxygen into the soil so that roots and other belowground organs cannot maintain respiration. In recent decades, the number of extreme floodings has strongly increased, which is especially tragic because most arable land worldwide is located in regions that are threatened by regular flooding events (Voesenek and Bailey-Serres, 2015).In plant heterotrophic tissues, respiratory metabolism is composed of various pathways, including glycolysis, the mitochondrial tricarboxylic acid cycle, and the mitochondrial electron transport chain. Under normal conditions, the conversion of Glc to pyruvate in the cytosol involves an initial input of ATP and produces the reduced cofactor NADH. The reactions of the tricarboxylic acid cycle occur within the mitochondrial matrix and lead to the complete oxidation of pyruvate, moving electrons from organic acids to the oxidized redox cofactors NAD⁺ and FAD, forming the reducing equivalents NADH and FADH₂ and concomitantly releasing carbon dioxide (Tovar-Méndez et al., 2003; Millar et al., 2011). Finally, the reduced cofactors generated during glycolysis and the tricarboxylic acid cycle are subsequently oxidized by the mitochondrial electron transport chain to fuel ATP synthesis by a process known as oxidative phosphorylation (Fernie et al., 2004; Plaxton and Podesta, 2006). The tricarboxylic acid cycle turnover rate depends greatly on the rate of NADH reoxidation by the mitochondrial electron transport chain and on the cellular rate of ATP utilization (Plaxton and Podesta, 2006). Besides supporting ATP synthesis, the reactions of the tricarboxylic acid cycle also contribute to the production of key metabolic intermediates for use in many other fundamental biosynthetic processes elsewhere in the cell (Fernie et al., 2004; Sweetlove et al., 2010; van Dongen et al., 2011; Araújo et al., 2012). Nevertheless, the control and regulation of the carbon flux through the tricarboxylic acid cycle are still poorly understood in plants, and noncyclic modes have been described to operate under certain circumstances (Rocha et al., 2010; Sweetlove et al., 2010; Araújo et al., 2012).Upon hypoxia, respiratory energy (ATP) production via oxidative phosphorylation by the mitochondrial electron transport chain goes down. To compensate for this, the glycolytic flux increases and Glc is consumed faster in an attempt to produce ATP via the glycolytic pathway, a process known as the Pasteur effect. To survive short-term hypoxia during flooding or waterlogging, plants must generate sufficient ATP and regenerate NADP⁺ and NAD⁺, which are required for glycolysis (Narsai et al., 2011; van Dongen et al., 2011). In addition to the accumulation of ethanol and lactate in oxygen-deprived plant tissues, metabolites such as Ala, succinate, and γ-aminobutyric acid (GABA) have also been shown to accumulate (Sousa and Sodek, 2003; Kreuzwieser et al., 2009; van Dongen et al., 2009; Rocha et al., 2010; Zabalza et al., 2011), although hardly anything is known about the fate of these products of hypoxic metabolism. However, the relative abundance of these products of hypoxic metabolism varies between plant species, genotypes, and tissues and can change throughout the course of oxygen limitation stress as well (Narsai et al., 2011).A model describing metabolic changes during hypoxia has been described previously for waterlogged roots of the highly flood-tolerant model crop legume Lotus japonicus (Rocha et al., 2010): upon waterlogging, the rate of pyruvate production is enhanced due to the activation of glycolysis (Pasteur effect) and the concomitant production of ATP via substrate-level phosphorylation. At the same time, the fermentation pathway is activated with the accumulation of lactate via lactate dehydrogenase and ethanol via two subsequent reactions catalyzed by pyruvate decarboxylase and alcohol dehydrogenase (Tadege et al., 1999). The amount of pyruvate produced can be reduced via alanine aminotransferease (AlaAT), which catalyzes the reversible reaction interconverting pyruvate and Glu to Ala and 2-oxoglutarate (2OG). Concomitantly, 2OG was suggested to reenter the tricarboxylic acid cycle to be used to produce another ATP and also succinate, which accumulates in the cell (Rocha et al., 2010). This Ala pathway provides a means for the role of Ala accumulation during hypoxia via reorganization of the tricarboxylic acid cycle. Furthermore, given that the use of this strategy prevents pyruvate accumulation, the continued operation of glycolysis during waterlogging can occur.It should be noted, however, that measurements of metabolite levels alone do not provide information about the actual activity of the metabolic pathways involved. Furthermore, the previous studies did not reveal which enzymes of the tricarboxylic acid cycle change their activity that leads to reorganization of the tricarboxylic acid cycle. To overcome this, analysis of metabolism using isotope-labeled substrates has proven to be essential for understanding the control and regulation of metabolic networks, and it has often been observed that significant changes in carbon flow are sometimes associated with only small adjustments in metabolite abundance (Schwender et al., 2004; Ratcliffe and Shachar-Hill, 2006). Metabolomics studies that require extensive metabolite labeling utilize uniformly labeled stable isotope tracers. Alternatively, detailed analysis of central carbon metabolism can make use of positional labeling as well. Following the extraction of labeled metabolites, the ¹³C label redistribution is measured usually with NMR or gas chromatography-mass spectrometry methods (Jorge et al., 2015). Schwender and Ohlrogge (2002) used both labeling approaches to investigate embryo development in Brassica napus seeds. While uniformly labeled [¹³C₆]Glc and [¹³C₁₂]Suc were applied to determine the metabolic flux through the major pathways of carbon metabolism, positionally labeled [1,2-¹³C]Glc was used to specifically outline the glycolytic/oxidative pentose phosphate pathway network during embryo development (Schwender and Ohlrogge, 2002). Gas chromatography-mass spectrometry analysis was used in this study to evaluate the ¹³C enrichment and isotopomer composition. In earlier studies of hypoxic metabolism, positionally labeled [1-¹³C]Glc was used to specifically investigate energy metabolism and pH regulation in hypoxic maize (Zea mays) root tips (Roberts et al., 1992; Edwards et al., 1998).In this study, we performed stable isotope labeling experiments using wild-type soybean (Glycine max) roots in order to better understand the dynamics of metabolism in operation in plant cells under hypoxic conditions. For this, we used fully labeled ¹³C and ¹⁵N tracers rather than positional labeling, as this allowed us to cover a broad view of the central carbon and nitrogen metabolic network. The labeling pattern of metabolites was subsequently measured with gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). Our studies confirm the activity of Ala metabolism while revealing the parallel activity of the GABA shunt. The results provide evidence that the bifurcation of the tricarboxylic acid cycle results from the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), also known as complex II of the mitochondrial electron transport chain (mETC).