Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

Effect of consuming a grape seed supplement with abundant phenolic compounds on the oxidative status of healthy human volunteers.

BACKGROUND: Diverse enzymatic and non-enzymatic antioxidants provide protection against reactive oxygen species in humans and other organisms. The nonenzymatic antioxidants include low molecular mass molecules such as plant-derived phenols. AIM OF STUDY: This study identified the major phenolic compounds of a grape seed extract by HPLC and analyzed the effect of consumption of biscuits enriched with this extract on the urinary oxidative status of healthy subjects by measurement of urine redox potential. METHODS: The major phenolic compounds were characterized in a red grape seed extract separated by HPLC with detection by a photodiode array (PDA), fluorescence (FL) and quadrupole mass spectrometer (MS). A nutritional study in a healthy volunteers group was done. Each volunteer ate eight traditional biscuits with no red grape seed extract supplementation. The second day each volunteer ate eight traditional biscuits supplemented with 0.6% (wt/wt) of grape seed extract. An overnight urine sample was obtained for each treatment. The redox potential was measured at 25 °C using a potentiometer in each urine sample. RESULTS: Epicatechin, catechin, procyanidin dimers B1 to B4, and the procyanidin trimer C2 were the major phenolic components in the extract. Epicatechin gallate and procyanidin dimers B1-3-G and B2-3'-G were the major galloylated flavan-3-ols. The forty-six healthy volunteers each shown a reduction of the urine redox potential after the treatment by traditional biscuits supplemented with the grape seed extract. CONCLUSIONS: This simple dietary intervention significantly reduced (33%) the urine redox potential, reflecting an overall increase in antioxidant status. Incorporation of plant-derived phenols in the diet may increase anti-oxidative status.

Preservation of saffron floral bio-residues by hot air convection.

Large amounts of floral bio-residues are wasted in saffron spice production, which need to be stabilized because of how quickly they deteriorate. These bio-residues are rich in phenolic compounds, and the aim of this study was to evaluate the effect of drying temperatures and air flows on their color and phenolic composition. Anthocyanins and flavonols were degraded at 110 and 125°C. The best drying temperatures were 70 and 90°C for maintaining their physicochemical quality. The duration at 70°C was double than that of 90°C. Anthocyanins and flavonols were stable at 70 and 90°C with 2, 4, 6 and 8ms(-1). Dehydrations at 90°C with 2, 4 and 6ms(-1) were the most appropriate, due to a better color and greater similarity to control samples for their flavonols and anthocyanins.

Increasing the applications of Crocus sativus flowers as natural antioxidants.

UNLABELLED: Large amounts of floral bio-residues (92.6 g per 100 g of flowers) are generated and wasted in the production of saffron (Crocus sativus) spice. Progress in mechanization of saffron crop offer the opportunity to expand the uses of C. sativus flowers, beyond the spice (dried stigmas). The antioxidant potential of flowers of saffron, their separate parts (tepals, stamens, styles, and stigmas) and floral bio-residues were evaluated by 4 in vitro assays: lipid peroxidation, deoxyribose assay, Rancimat test, and Trolox equivalent antioxidant capacity. Phenolic content and crocetin ester composition were also determined. All the samples studied showed to be potential antioxidants. The highest phenolic, flavonoid, and anthocyanin contents were observed in tepals. Stamens showed lower phenolic, flavonoid, and anthocyanin contents than those of whole flowers, tepals, and floral bio-residues. Crocetin esters were not found in tepals or stamens. Stamens exhibited the most potent LOO(•) and OH(•) radicals scavenging activity, being higher than those of food antioxidant propyl gallate. Flowers of saffron, tepals, stamens, styles, and floral bio-residues showed LOO(•), OH(•), and ABTS(•-) radicals scavenging activity, while stigmas showed LOO(•) and ABTS(•-) radicals scavenging activity. All samples studied improved the oxidative stability of sunflower oil in Rancimat test. These antioxidant properties could suggest the application of this floral material as functional ingredients with the subsequent added value. PRACTICAL APPLICATION: Saffron spice, the most valuable spice worldwide, is the dried stigma that only represents 7.4% of Crocus sativus flowers. Other parts of the flowers different to stigmas are discarded. Flower harvest and all the postharvest steps to produce saffron spice are performed manually. Mechanization of flower collection, stigma separation, and dehydration process is a revolution in saffron spice production, which increases the productive capacity making it possible to extend the uses of C. sativus flowers, beyond the production of saffron spice. Flowers possessed high-phenolic content and excellent antioxidant properties that could contribute to their application as functional ingredients.

Picrocrocin kinetics in aqueous saffron spice extracts (Crocus sativus L.) upon thermal treatment.

The kinetics of picrocrocin degradation in aqueous extracts of saffron upon thermal treatment from 5 to 70 °C have been studied, together with the degradation of purified picrocrocin in water at 100 °C. The best fits to experimental data were found for a second-order kinetics model. Picrocrocin showed high stability with half-life periods (t(1/2)) ranging from >3400 h at 5 °C in saffron extracts to 9 h in the experiments with purified picrocrocin at 100 °C. In saffron extracts, the evolution of the rate constant (k) with temperature showed maximum values at 35 °C, and filtration of the extracts contributed to picrocrocin stability. In the case of purified picrocrocin, the generation of safranal in the first 5 h (yield up to 7.4%) was confirmed. Spectrometric parameters used in saffron quality control (E(1cm)(1%) 257 nm and ΔΕ(pic)) were not appropriate for documenting the evolution of picrocrocin.

Effect of centrifugal ultrafiltration on the composition of aqueous extracts of saffron spice (Crocus sativus L.).

The purpose of this research was to study the effect of centrifugal ultrafiltration (UF) on the composition of aqueous extracts of saffron spice. The contents of seven crocetin esters, picrocrocin, and two kaempferol glycosides were analyzed by UV-vis and HPLC in the filtrate and retentate fractions from 16 centrifugal filter devices with regenerated cellulose (RC) and polyethersulfone (PES) membranes ranging from 1-100 kDa nominal molecular weight cutoff (MWCO). The separation of crocetin esters from picrocrocin and their concentration with centrifugal UF have been demonstrated. A great heterogeneity of results regarding devices with equal MWCO was found that could not be related to the membrane material or manufacturer. Four devices of 5 and 10 kDa MWCO, three of which had RC membranes, showed the best results. The device having the lowest MWCO also showed a potential to obtain picrocrocin without crocetin esters and could be considered in successive UF steps. The less polar crocetin esters were rejected better than the others.