Prion‐associated proteins in yeast: comparative analysis of isogenic [PSI+] and [psi−] strains

A large group of prion‐associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Two‐dimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI⁺] and [psi^?] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prion‐containing aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion‐associated, but we cannot exclude the possibility that several proteins may propagate as prions. Copyright © 2009 John Wiley & Sons, Ltd.     

On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes

In the last years several reports have reported the capacity of the yeast Dekkera (Brettanomyces) bruxellensis to survive and adapt to the industrial process of alcoholic fermentation. Much of this feature seems to relate to the ability to assimilate limiting sources of nutrients, or somehow some that are inaccessible to Saccharomyces cerevisiae, in particular the sources of nitrogen. Among them, amino acids (AA) are relevant in terms of beverage musts, and could also be important for bioethanol. In view of the limited knowledge on the control of AA, the present work combines physiological and genetic studies to understand how it operates in D. bruxellensis in response to oxygen availibility. The results allowed separation of the AA in three groups of preferentiality and showed that glutamine is the preferred AA irrespective of the presence of oxygen. Glutamate and aspartate were also preferred AA in anaerobiosis, as indicated by the physiological data. Gene expression experiments showed that, apart from the conventional nitrogen catabolic repression mechanism that is operating in aerobiosis, there seems to be an oxygen‐independent mechanism acting to overexpress key genes like GAP1, GDH1, GDH2 and GLT1 to ensure adequate anaerobic growth even in the presence of non‐preferential nitrogen source. This could be of major importance for the industrial fitness of this yeast species.     

URE3] prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast

[URE3] and [PSI(+)] are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively. We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain [URE3]. Designated ssa2-10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide-binding domain. This allele also unexpectedly destabilizes [URE3] in newly formed heterozygotes: [URE3] is either absent in heterozygotes formed by crossing wild-type [URE3] cells with ssa2-10 mutants, or present and fully stable. SSA2 deletion mutants are weakly capable of maintaining [URE3]. The ssa2-10 allele is compatible with propagation of [PSI(+)]. However, in combination with a deletion of SSA1, ssa2-10 eliminates the nonsense-suppression phenotype of [PSI(+)] cells.     

Nitrogen starvation induces expression of Lg‐FLO1 and flocculation in bottom‐fermenting yeast

When exponentially growing cells of bottom‐fermenting yeast were starved for nitrogen or were grown on proline (a non‐preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg‐FLO1, which is required for flocculation of bottom‐fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom‐fermenting yeast is under the control of a nitrogen catabolite repression (NCR)‐like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae.

The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.     

Cadmium concentration in durum wheat grain (Triticum turgidum) as influenced by nitrogen rate,seeding date and soil type

BACKGROUND: Cadmium (Cd) is a trace element that has been associated with various human health problems. Cd enters plants, either by direct absorption through leaves or by uptake from soils, allowing Cd into the food chain. Nitrogen (N) fertilizer management is important in optimizing crop yield and protein content of durum wheat, but may influence Cd availability and hence Cd concentration in crops, with the effects being strongly influenced by environmental conditions and crop cultivar. RESULTS: In field studies, Cd and protein concentration in durum wheat grain differed between cultivars and were strongly affected by N application, with only minor effects of N occurring on concentration and uptake of P and Zn. Protein content increased significantly with N application in five of six site‐years, with the response being generally independent of cultivar and seeding data. Cd concentration also increased with N application in five of six seeding dates, with the response being greater in AC Melita than Arcola in three of the six site‐years. There were large differences in Cd concentration from year to year and with seeding date, indicating a strong environmental influence. CONCLUSIONS: This study shows that different cultivars accumulate different levels of Cd in the grain and that seeding date and nitrogen fertilizer management can influence grain Cd concentration, with the magnitude of effects varying with environmental factors. In the future we may be able to manipulate management practices to optimize protein concentration and minimize Cd concentration in durum wheat, which could help to address the health and safety concerns of consumers. © Society of Chemical Industry and Her Majesty the Queen in right of Canada     

Synthesis of quaternary ammonium bromides of 5-[4-(ω-dialkylaminoalkoxy)phenyl-methylene]-1, 3, 3-trimethyl-2-oxabicyclo [2.2.2]octan-6-ones as potential UV sunscreens

Materials and methods for the synthesis of eight quaternary ammonium bromides of 5-[4-(ω-dialkylaminoalkoxy)phenylmethylene]-1,3,-trimethyl-2-oxabicyclo[2.2.2]octan-6-ones are illustrated. They were routinely prepared starting from cineole aminoethers by reaction with primary alkyl bromides and their physico-chemical data are reported. These substances have been tested for UV filtering and/or microbiological activity. The substances have their UV absorption maxima at 315–322 nm. Tests on antimicrobial activity were performed using benzalkonium chloride as reference standard. All quaternary ammonium bromides were totally inactive against Escherichia coli (Gram —) and partially active on Staphylococcus aureus (Gram +). These preliminary findings seem to indicate that these new quaternary ammonium bromides could be considered as potential UV sunscreens.     

An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence and appearance of the [PSI+] prion in Saccharomyces cerevisiae

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to read-through of certain nonsense mutations in a few auxotrophic markers, e.g. ade1-14. This read-through results in growth on adenine-deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants, and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoroorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi-] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods.     

Cyberlindnera jadinii yeast as a protein source in early- to mid-lactation dairy cow diets: Effects on feed intake,ruminal fermentation,and milk production

We examined the effects of substituting soybean meal with either yeast protein from Cyberlindnera jadinii or barley in concentrate feeds on feed intake, ruminal fermentation products, milk production, and milk composition in Norwegian Red (NRF) dairy cows. The concentrate feeds were prepared in pellet form as soy-based (SBM; where soybean meal is included as a protein ingredient), yeast-based (YEA; soybean meal replaced with yeast protein), or barley-based (BAR; soybean meal replaced with barley). The SBM contained 7.0% soybean meal on a dry matter (DM) basis. This was replaced with yeast protein and barley in the YEA and BAR concentrate feeds, respectively. A total of 48 early- to mid-lactation [days in milk ± standard deviation (SD): 103 ± 33.5 d] NRF cows in their first to fourth parity and with initial milk yield of 32.6 kg (SD = 7.7) were allocated into 3 groups, using a randomized block design, after feeding a common diet [SBM and good-quality grass silage: crude protein (CP) and neutral detergent fiber (NDF) content of 181 and 532 g/kg of DM, respectively] for 14 d (i.e., covariate period). The groups (n = 16) were then fed one of the dietary treatments (SBM, YEA, or BAR) for a period of 56 d (i.e., experimental period). The concentrate feeds were offered in split portions from 3 automatic feeders using electronic identification, with ad libitum access to the same grass silage. Dietary treatments had no effect on daily silage intake, total DM intake, or total NDF intake. Dietary CP intake was lower and starch intake was higher in the BAR group compared with the other groups. Ruminal fluid pH, short-chain volatile fatty acid (VFA) concentrations, acetate-to-propionate ratio, and non-glucogenic to glucogenic VFA ratio were not affected by dietary treatments. No effects of the dietary treatments were observed on body weight change, body condition score change, milk yield, energy-corrected milk yield, milk lactose and fat percentages, or their yields. In conclusion, yeast protein can substitute conventional soybean meal in dairy cow diets without adverse effect on milk production and milk composition, given free access to good-quality grass silage.     

杯[4]间苯二酚芳烃类酸增殖剂的合成及性能表征

以间苯二酚为原料,分别与正十一醛和月桂醛,在盐酸、乙醇回流条件下进行缩合成环反应,得到中间体杯[4]间苯二酚芳烃衍生物;中间体再与对甲苯磺酰氯经过酯化反应制备了两种杯[4]间苯二酚芳烃衍生物类酸增殖剂,其结构经1HNMR、IR等分析确认,同时对其基本物性进行了研究。结果表明,两种酸增殖剂都有较高的热稳定性,在常用有机溶剂中都具有较好的溶解性。     

Preparation of [13 C 3]-melamine and 13 C 3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry

For the determination of melamine and cyanuric acid the labelled internal standards [¹³ C ₃]-melamine and [¹³ C ₃]-cyanuric acid were synthesized using the common substrate [¹³ C ₃]-cyanuric chloride by reaction with ammonia and acidified water, respectively. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution liquid chromatography/mass spectrometry (LC/MS) method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid–liquid extraction and ion exchange solid phase clean-up, with normal phase high-performance liquid chromatography (HPLC) in the so-called hydrophilic interaction mode. The method had a limit of detection (LOD) of 10 µg kg^?1 for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation (CV) of less than 10%. The recovery of the method at this level was in the range of 87–110% and 96–110% for melamine and cyanuric acid, respectively.     

[Psmim]HSO4酸性离子液体催化油酸制备生物柴油及其工艺优化

以1-磺酸丙基-3-甲基咪唑硫酸氢盐离子液体为催化剂,油酸与甲醇经酯化反应制备生物柴油。考察了反应时间、反应温度、醇油摩尔比、催化剂用量对油酸转化率的影响,并对制备工艺进行了响应面优化。结果表明:各因素对转化率的影响与硫酸为催化剂的制备工艺基本一致;该离子液体催化活性优越,重复使用4次后,油酸转化率仍达90%以上;响应面法回归拟合的数学模型准确有效、拟合度高;优化工艺条件为反应时间3.5 h、反应温度60℃、醇油摩尔比9∶1、催化剂用量10%,此时油酸转化率为98.3%。     

Amino acid-dependent formation pathways of 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone in the Maillard reaction

The formation pathways of two furanoids, 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone (DMHF) were studied by GC–MS in the Maillard-type model system based on glucose and selected amino acids. The reaction was performed in 0.01 M phosphate buffer by heating a 1:1 mixture of [¹³C6] glucose and [¹²C6] glucose with amino acid. There is only one major formation pathway for DMHF in which the glucose carbon skeleton stayed intact. Formation pathways for 2-acetylfuran were more complicated. They formed either from glucose or from glucose and glycine. In the presence of glycine, the [C-5] unit of glucose combined with formaldehyde from glycine leads to 2-acetylfuran. For other amino acids, either cyclisation of intact glucose or recombination of glucose fragments can lead to 2-acetylfuran formation. These results indicate a competitive trend in controlling Maillard reaction. Therefore, besides changing Miallard reaction impact factors (temperature, time, pH etc.), inhibiting or preventing the competitive reaction cascade may direct desired pathways of Maillard reaction.     

An 11.4 kb DNA segment on the left arm of yeast chromosome II carries the carboxypeptidase Y sorting gene PEP1, as well as ACH1, FUS3 and a putative ARS.

We report the nucleotide sequence of an 11.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains a typical structure of a functional ARS as well as five open reading frames (ORFs) longer than 300 bp. One is PEP1, a gene encoding a transmembrane protein of 1579 amino acids which transits through the secretory pathway and is involved in vacuolar protein sorting. Two genes were previously sequenced: ACH1 (Lee et al., 1990) and FUS3 (Elion et al., 1990), which encode an acetyl-CoA hydrolase and a protein kinase involved in the cell division cycle, respectively. The last two ORFs localized on the complementary strand of ACH1 are not likely to be expressed.     

Chromatographic separation of clenbuterol by bonded phases bearing nano-baskets of p-tert-calix[4]-1,2-crown-3, -crown-4, -crown-5 and -crown-6

BACKGROUND: Clenbuterol (salbutamol), which increases muscle mass and decreases adipose tissue, is misused as a nutrient‐repartitioning agent in livestock. In this paper a new sensitive method for determining clenbuterol in livestock is presented. The novelty of this approach is the separation and determination of salbutamol enantiomers with marked quantitative merits. RESULTS: Four proton di‐ionisable Nano‐baskets, namely 5,11,17,23‐tetrakis(1,1‐dimethylethyl)‐25,26‐bis(carboxy‐methoxy)calix[4]arene‐27,28‐crown‐3, ‐crown‐4, ‐crown‐5 and ‐crown‐6 in the cone conformation, were synthesised and used to prepare bonded phases for high‐performance liquid chromatography separation. The new synthesised bonded phases were characterised and optimised. The bonding interactions of solute/bonded phases were examined and the main interactions are reported. CONCLUSION: The clenbuterol levels in six samples of livestock meat (pork, pork casing, beef, beef casing, mutton and mutton casing) were analysed and the results revealed that for the best bonded phases the limits of detection and quantitation were 0.06 and 0.2 µg mL^?1 respectively. Copyright © 2012 Society of Chemical Industry     

Luminescence spectroscopic observation of singlet oxygen formation in extra virgin olive oil as affected by irradiation light wavelengths, 1,4-diazabicyclo[2.2.2]octane, irradiation time, and oxygen bubbling

A spectrofluorometer equipped with a highly sensitive near-IR InGaAs detector was used for the direct visualization of singlet oxygen emission at 1268 nm in olive oil during light irradiation with various different wavelengths. The virgin olive oil in methylene chloride (20% w/v, oxygen saturated) was irradiated at the 301, 417, 454, 483, and 668 nm, then the emission at 1268 nm, singlet oxygen dimole decaying was observed. The result showed the highest production of (1)O(2) with light irradiation at 417 nm, and followed by at 668 nm in virgin olive oil, indicating that pheophytin a and chlorophyll a were the most responsible components for the production of singlet oxygen. The UV light irradiations at the wavelength of 200, 250, and 300 nm did not induce any detectable luminescence emission at 1268 nm, but 350 nm produced weak emission at 1269 nm. The quantity of (1)O(2) produced with excitation at 350 nm was about 1/6 of that of irradiation at 417 nm. Addition of an efficient (1)O(2) quencher, 1,4-diazabicyclo[2.2.2]octane, in virgin olive oil in methylene chloride greatly decreased the luminescence emission at 1268 nm, confirming the singlet oxygen production in olive oil. Singlet oxygen production was more efficient in oxygen-purged virgin olive oil than in oxygen non-purged olive oil. This represents first report on the direct observation of singlet oxygen formation in olive oil as well as in real-food system after visible light illumination. Practical Application: The present results show the positive evidence of the singlet oxygen involvement in rapid oxidative deterioration of virgin olive oil under visible light. This paper also shows the effects of different wavelength of light irradiation on the formation of singlet oxygen in olive oil. The present results would provide important information for the understanding of the mechanism involved in rapid oxidative quality deterioration of virgin olive oil under light illumination and for searching the preventive methods of deterioration of olive oil quality under light.