Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation

We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Mc or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively). Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.     

Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae,S. uvarum and a new species,S. douglasii

We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 10⁴ to 10⁵ asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of ‘homeologous’ chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non-switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by chance, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80–90% spore viability, thus establishing a second member of the S. douglasii species.     

Saccharomyces spores are born prepolarized to outgrow away from spore–spore connections and penetrate the ascus wall

How nonspore haploid Saccharomyces cells choose sites of budding and polarize towards pheromone signals in order to mate has been a subject of intense study. Unlike nonspore haploids, sibling spores produced via meiosis and sporulation by a diploid cell are physically interconnected and encased in a sac derived from the old cell wall of the diploid, called the ascus. Nonspore haploids bud adjacent to previous sites of budding, relying on stable cortical landmarks laid down during prior divisions, but because spore membranes are made de novo, it was assumed that, as is known for fission yeast, Saccharomyces spores break symmetry and polarize at random locations. Here, we show that this assumption is incorrect: Saccharomyces cerevisiae spores are born prepolarized to outgrow, prior to budding or mating, away from interspore bridges. Consequently, when spores bud within an intact ascus, their buds locally penetrate the ascus wall, and when they mate, the resulting zygotes adopt a unique morphology reflective of repolarization towards pheromone. Long‐lived cortical foci containing the septin Cdc10 mark polarity sites, but the canonical bud site selection programme is dispensable for spore polarity, thus the origin and molecular composition of these landmarks remain unknown. These findings demand further investigation of previously overlooked mechanisms of polarity establishment and local cell wall digestion and highlight how a key step in the Saccharomyces life cycle has been historically neglected.     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.     

Functional interrelationships between carbohydrate and lipid storage,and mitochondrial activity during sporulation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae under conditions of nutrient stress, meiosis precedes the formation of spores. Although the molecular mechanisms that regulate meiosis, such as meiotic recombination and nuclear divisions, have been extensively studied, the metabolic factors that determine the efficiency of sporulation are less understood. Here, we have directly assessed the relationship between metabolic stores and sporulation in S. cerevisiae by genetically disrupting the synthetic pathways for the carbohydrate stores, glycogen (gsy1/2Δ cells), trehalose (tps1Δ cells), or both (gsy1/2Δ and tps1Δ cells). We show that storage carbohydrate-deficient strains are highly inefficient in sporulation. Although glycogen and trehalose stores can partially compensate for each other, they have differential effects on sporulation rate and spore number. Interestingly, deletion of the G₁ cyclin, CLN3, which resulted in an increase in cell size, mitochondria and lipid stores, partially rescued meiosis progression and spore ascus formation but not spore number in storage carbohydrate-deficient strains. Sporulation efficiency in the carbohydrate-deficient strain exhibited a greater dependency on mitochondrial activity and lipid stores than wild-type yeast. Taken together, our results provide new insights into the complex crosstalk between metabolic factors that support gametogenesis.     

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

Antioxidant activities and functional properties of grass carp (Ctenopharyngodon idellus) protein hydrolysates

BACKGROUND: Grass carp, with an annual production exceeding 4 × 10⁶ t in China in 2009, has not been developed into a high‐value product. In this study the antioxidant activities and functional properties of grass carp protein hydrolysates prepared with Alcalase 2.4L (HA) and papain (HP) were investigated. The hydrolysate with strongest radical‐scavenging activity and reducing power was assessed further for changes in its antioxidant activity during simulated gastrointestinal digestion. RESULTS: As the degree of hydrolysis (DH) increased, the metal‐chelating activity of both HA and HP increased while their reducing power and 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH^?)‐scavenging activity decreased (P < 0.05). At the same DH, HP possessed higher DPPH^?‐scavenging activity and reducing power than HA (P < 0.05). The metal‐chelating activity of HP with 10% DH was significantly increased after in vitro gastrointestinal metabolism (P < 0.05). Regarding their functional properties, all hydrolysates were more than 81% soluble over a wide range of pH (3–8). At the same DH, HP showed higher emulsion activity index but lower solubility and foaming capacity than HA. CONCLUSION: Grass carp protein hydrolysates showed high solubility over a wide pH range and could be used as natural antioxidants in food systems. Copyright © 2011 Society of Chemical Industry     

Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: Ultrastructural analysis of spo mutants

In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation-deficient (spo) mutants (spo4, spo5, spo14 and spo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild-type cells. However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18-h incubation. The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. The spo5 mutant produced anucleate spore-like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore-wall precursors, instead of anucleate spore-like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts.     

Polyphenols bioaccessibility and bioavailability assessment in ipecac infusion using a combined assay of simulated in vitro digestion and Caco-2 cell model

In this report, we investigated for the first time the total polyphenols content (TPC) and antioxidant activity before and after digestion of Carapichea ipecacuanha root infusion, better known as ipecac, prepared at different concentrations. An in vitro digestion system coupled to a Caco-2 cell model was applied to study the bioavailability of antioxidant compounds. The ability of ipecac bioaccessible fractions to inhibit reactive oxygen species (ROS) generation at cellular level was also evaluated. The findings revealed that water volume of 50 mL g⁻¹ of sample provided the maximum yield of extraction of TPC and antioxidant activity. Polyphenols increased in content and activity after digestion and they were highly bioavailable (75% of intestinal absorption). Polyphenols were also present in the residual parts which indicate a possible local activity. Results also suggest that ipecac infusion could represent a promising source of effective bioavailable antioxidants to be exploited in functional foods field.     

Relationship of climate and genotype to seasonal variation in the glucosinolate–myrosinase system. II. Myrosinase activity in ten cultivars of Brassica oleracea grown in fall and spring seasons

Myrosinase catalyzes the hydrolysis of glucosinolates found in the Brassicaceae, generating a variety of bioactive reaction products that may aid in the prevention of some cancers and that are suppressive to soil‐borne plant pathogens. Two cultivars each of broccoli (Brassica oleracea L var italica), Brussels sprouts (B oleracea var gemmifera), cabbage (B oleracea var capitata), cauliflower (B oleracea var botrytis), and kale (B oleracea var acephala) were grown during two fall seasons and two spring seasons to determine if myrosinase activity varied by season. Regression models that included mean temperature and photosynthetic photon flux (PPF) during the growing seasons showed that climatic variables explained seasonal differences for myrosinase activity. Activity‐FW (FW = fresh weight; U g^?1) and specific activity (U mg^?1) were significantly (p ≤ 0.05) affected by season, botanical group and group × season. Activity‐FW had a negative linear relationship with temperature, and a positive linear but negative quadratic relationship with PPF. Specific activity had a positive linear and a negative quadratic relationship with both temperature and PPF. Therefore the influence of climatic factors on myrosinase activity in Brassica species may affect the potential benefits of the glucosinolate–myrosinase system. Copyright © 2004 Society of Chemical Industry     

In vitro evaluation of the fermentation of added‐value agroindustrial by‐products: cactus pear (Opuntia ficus‐indica L.) peel and pineapple (Ananas comosus) peel as functional ingredients

Agroindustrial by‐products derived from fruit processing are an important source of biocompounds that can be used as functional food ingredients. The objective of this work was to evaluate cactus pear and pineapple peel flours as an alternative carbon source during fermentation using bacteria with probiotic potential. The total fibre content of both flours was over 60%, with total soluble carbohydrate content around 20%, indicating a good carbon source for lactic acid bacteria. Kinetic parameters indicate that peel flours are a suitable carbon source because the lactic acid bacteria grow (mean growth rate constant, k, values close to glucose, 1.52 h) and acidify the culture media (maximum acidification rate, V_max, approximately 1.60 pH × 10^?3 min^?1). There was no difference in prebiotic potential or prebiotic activity score for both the peel flours. Pediococcus pentosaceus performs better during fermentation. In this respect, cactus pear and pineapple peel flours can be used as functional ingredients due to their fermentable properties.     

Long-Term Cell Suspension Culture and Regeneration of the Single-Leafed Strawberry Fragaria vesca monophylla

Cell suspensions have been used for obtaining somaclonal variability and for the production of secondary metabolites, such as aroma compounds, natural dyes and drugs. Undifferentiated growing cells completely lose their regeneration capacity due to the formation of cells with abnormal DNA content during this phase. Callus culture of Fragaria vesca monophylla was obtained on agarised media supplemented with different growth regulators. Afterwards, a stable cell suspension was obtained on an SH (1972) medium supplemented with BSAA 0·25 mg litre⁻¹ and BA 0·1 mg litre⁻¹. The cells were poured onto an agarised regeneration medium, where it was possible to obtain regenerants. This is the first report of a stable cell suspension of a diploid Fragaria and regeneration after 2 years of unorganised cell culture.     

Proteomic and genomic characterization of a yeast model for Ogden syndrome

Naa10 is an N^α‐terminal acetyltransferase that, in a complex with its auxiliary subunit Naa15, co‐translationally acetylates the α‐amino group of newly synthetized proteins as they emerge from the ribosome. Roughly 40–50% of the human proteome is acetylated by Naa10, rendering this an enzyme one of the most broad substrate ranges known. Recently, we reported an X‐linked disorder of infancy, Ogden syndrome, in two families harbouring a c.109 T > C (p.Ser37Pro) variant in NAA10. In the present study we performed in‐depth characterization of a yeast model of Ogden syndrome. Stress tests and proteomic analyses suggest that the S37P mutation disrupts Naa10 function and reduces cellular fitness during heat shock, possibly owing to dysregulation of chaperone expression and accumulation. Microarray and RNA‐seq revealed a pseudo‐diploid gene expression profile in ΔNaa10 cells, probably responsible for a mating defect. In conclusion, the data presented here further support the disruptive nature of the S37P/Ogden mutation and identify affected cellular processes potentially contributing to the severe phenotype seen in Ogden syndrome. Data are available via GEO under identifier GSE86482 or with ProteomeXchange under identifier PXD004923. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.     

Development and quality characteristics of functional Kulfi fortified with microencapsulated betalains

Fruit and vegetable waste valorisation has opened opportunities for the utilisation of food waste for extraction and development of valuable functional foods. Therefore, the study was designed to develop a functional Kulfi fortified with encapsulated betalains extracted from red beetroot (Beta vulgaris L.) pomace. Moreover, fortified functional Kulfi samples were studied for physico-chemical, antioxidant, microbiological and sensory analyses. Fortified functional Kulfi reported the higher antioxidant activity (64.88% and 75.27%) with reduced melting rate (1.84 mL min⁻¹ and 2.08 mL min⁻¹) and microbial profile (3.77 log CFU g⁻¹ and 3.14 log CFU g⁻¹) compared with control. Sensory analysis showed a no significant (P > 0.05) impact on the overall acceptability of functional Kulfi (7.25) compared with the commercial Kulfi (7.20), which was further confirmed by a bi-plot of principal component analysis. Overall, the encapsulated betalains improved the quality characteristics of functional Kulfi and could be used for the development of frozen dairy products.