Identification of TLR2/4-mediated phagocytosis and immune response activation pathways by vacuoles isolated from Saccharomyces cerevisiae

The vacuoles of the yeast Saccharomyces cerevisiae are closely related to mammalian lysosomes and play a role in macromolecular degradation due to the hydrolytic enzymes present inside. The vacuoles also regulate osmotic pressure and control cellular homeostasis. In previous results, vacuoles were shown to activate the immune response of macrophages by promoting the production of immune-mediated transporters nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. In this study, the effects of vacuoles on the phagocytosis activity of RAW264.7 cells and their potential as immune enhancers were evaluated, and receptors capable of recognizing vacuoles were examined. An investigation using the phagocytes assay showed that phagocytosis activity increased by the vacuole. Besides, after treatment with TLR2/4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced and the inducible nitric oxide synthase (iNOS) protein was also significantly reduced. However, treatment with a TLR2 inhibitor did not reduce the production of interleukin-6 (IL)-6, a pro-inflammatory cytokine. As a result of confirming the activation of TLR2/4 using Western blot and immunofluorescence (IF), the TLR2/4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Yeast vacuoles significantly upregulate protein expression of p-p65/p-p38 MAPKs. In summary, the vacuoles isolated from S. cerevisiae in macrophages have increased phagocytic ability at a concentration of 20 (µg/ml) and can function as immune-enhancing agent suggesting that TLR2/4 mediated the p38 MAPK/nuclear factor kappa B signaling pathway.     

Biological activity of Brassica rapa L. polysaccharides on RAW264.7 macrophages and on tumor cells

Polysaccharides are a type of natural macromolecule widely existing in nature, and its pharmacological activity has attracted wide research attention. In this study, Brassica rapa L. polysaccharides were taken as the research object, and a preliminary study of the immune activity and mechanism of the antitumor activity of these polysaccharides in vitro was carried out. Five polysaccharides, namely, BRP, BRNP-1, BRNP-2, BRAP-1, and BRAP-2, were compared in terms of their ability to inhibit the growth of three types of cancer cells, namely, A549, AGS, and HepG2. The most effective polysaccharides were screened out, and their mechanism was studied. Immunoassay results showed that the five polysaccharides not only promoted the growth of RAW264.7 cells but also stimulated their endocytic/pinocytosis activity and released NO, TNF, IL-6 cytokines, especially BRP. In vitro antitumor experiments showed that BRP has a significant inhibitory effect (*P < 0.05) on the growth of A549 cells, especially at high concentrations (500–2000 μg/mL). BRP can also induce A549 cells to release reactive oxygen species, cause mitochondrial membrane potential, and effect the expression of Bax, caspase-9, caspase-3, p53, and B-cell lymphoma 2. Immunological experiments showed that the five groups of polysaccharides are not cytotoxic to normal cells and have immunostimulatory effects. Mitochondria represent one of the more important endogenous pathways in the apoptotic process. The results suggested that BRP participates in mitochondria mediated apoptosis and induces A549 cell apoptosis. This study lays a theoretical foundation for further research on the mechanisms of BRP immunoregulation and antitumor activity in vitro and in vivo.     

Anti-inflammatory and antioxidant activities of Sargassum horneri extract in RAW264.7 macrophages

[Purpose]In this study, we investigated whether a 70% ethanolic (EtOH) extract of Sargassum horneri had antioxidant and anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophage-like RAW 264.7 cells.[Methods]The proximate composition, fatty acids, amino acids, and dietary fiber of S. horneri, various biologically active compounds, and antioxidant activity were analyzed.[Results]The DPPH and ABTS free radical scavenging activities, as well as the reduction power, of the S. horneri extract used here were significantly increased in a concentration-dependent manner. This indicates that S. horneri contains bioactive compounds, such as phenols and flavonoids, that have excellent antioxidant activity. The cellular viability and metabolic activity results confirmed that the extract had no discernible toxicity at concentrations up to 100 μg/mL. The levels of nitrites and cytokines (PGE₂, TNF-α and IL-6), which mediate pro-inflammatory effect, were significantly inhibited by treatment with either 50 or 100 μg/mL S. horneri extract, whereas that of IL-1β was significantly inhibited by treatment with 100 μg/mL of the extract. Similarly, the expression of iNOS and COX-2 proteins also decreased according to 50 or 100 μg/mL extract concentrations. NF-κB binding to DNA was also significantly inhibited by treatment with 100 μg/mL of extract.[Conclusion]These results suggest that 70% EtOH extracts of S. horneri can relieve inflammation caused by disease or high intensity exercise.     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264.7 macrophage cells

Inflammation is an essential host defense system particularly in response to infection and injury; however, excessive or undesirable inflammatory responses contribute to acute and chronic human diseases. A high-throughput screening effort searching for anti-inflammatory compounds from medicinal plants deduced that the methanolic extract of Juniperus rigida S. et L. (Cupressaceae) inhibited significantly nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Activity-guided fractionation and isolation yielded 13 phenolic compounds, including one new phenylpropanoid glycosides, 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1). Among the isolated compounds, phenylpropanoid glycosides with p-hydroxy group (2, 4) and massoniaside A (7), (+)-catechin (10), amentoflavone (11) effectively inhibited LPS-induced NO production in RAW264.7 cells.     

Accumulation and intracellular compartmentation of lithium ions in Saccharomyces cerevisiae

Abstract Accumulation of Li⁺ in Saccharomyces cerevisiae X2180-1B occured via an apparent stoichiometric relationship of 1: 1 (K⁺/Li⁺) when S. cerevisiae was incubated in the presence of 5 and 10 mM LiCl for 3 h. Other cellular cations (Mg²⁺, Ca²⁺ and Na⁺) did not vary on Li⁺ accumulation, although lithium chemistry dictates a degree of similarity to Group I and II metal cations. Compartmentation of Li⁺ was mainly in the vacuole which accounted for 85% of the Li⁺ accumulated after a 6-h incubation period. The remainder was located in the cytosol with negligible amounts being bound to cell fragments including the cell wall. Transmission electron microscopy of Li⁺-loaded cells revealed enlarged vacuoles compared with control cells. This asymmetric cellular distribution may therefore enhance tolerance of S. cerevisiae to Li⁺ and ensure that essential metabolic processes in the cytosol are not disrupted.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Characterization of killer-resistant strains of Saccharomyces cerevisiae isolated from spontaneous fermentations

A study of 26 killer-resistant wine strains of Saccharomyces cerevisiae, isolated during spontaneous fermentations in three vineyards in NW Spain, was carried out employing several methods that included a spheroplast-killing assay and analysis of chromosomal DNA patterns by pulse-field agarose electrophoresis. The results showed that 92% of the strains were derivatives of K2 killer toxin producing wine strains isolated from the same fermentations, and that they could be grouped into four different karyotypes. The remaining strains were killer-resistant at cell-wall level and were not related to the others, as was demonstrated by the absence of L and M ds-RNAs and by their different karyotypes.     

Hydrostatic pressure promotes the acidification of vacuoles in Saccharomyces cerevisiae

Abstract Application of hydrostatic pressure caused a delay or cessation of cell growth in Saccharomyces cerevisiae The yeast vacuole is an acidic organelle involved in cellular ion homeostasis and degradation of proteins. Hydrostatic pressure promoted the acidification of the vacuoles in the strain IFO 2347. A pressure of 40 to 60 MPa reduced the vacuolar pH, defined using 6-carboxyfluorescein, from 6.05 to 5.88, while a pressure of 20 MPa did not affect the pH. Similar results were obtained with the strain X2180. Bafilomycin A₁, a specific inhibitor of vacuolar H⁺-ATPase (V-H⁺-ATPase), caused a significant alkalization of vacuoles in the strain X2180. The pHs rose to 7.34 and 6.84 at both atmospheric pressure and a pressure of 40 MPa, respectively. Meanwhile, vacuolar accumulation of the weak base quinacrine was increased by a pressure of 40 MPa, suggesting that uptake of the dye was induced by the increased pH gradient across the vacuolar membrane.     

Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H₂O₂ and cDNA microarray technique was used to produce gene expression profiles. We found that H₂O₂ treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF- B, ERK, JNK, p38, PKC and INF- . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.Y. Zhang and C.C. Fong contributed equally to this work.     

Inhibition of nitric oxide production by natural oxyprenylated coumarins and alkaloids in RAW 264.7 cells

A series of naturally occurring 3,3-dimethylallyloxy- and geranyloxycoumarins and alkaloids were chemically synthesized and tested as anti-inflammatory agents for their inhibitory effects on nitric oxide production in LPS-stimulated RAW 264.7 cells. Results indicated that the alkaloid of fungal origin 3-methylbut-2-enyl-4-methoxy-8-[(3-methylbut-2-enyl)oxy]quinoline-2-carboxylate, commonly known as Ppc-1, and coumarins having an unsubstituted 2-benzopyrone ring exhibited the highest activity with IC₅₀ values from 23 to 34 μM without having poor or not detectable cytotoxicity. Indomethacine and L-NAME used as reference drugs provided by far less activities.     

A genetically isolated population of Saccharomyces cerevisiae in Malaysia

A divergent population of Saccharomyces cerevisiae has been identified in Malaysia by molecular and genetic analysis. It has also demonstrated that the yeast S. bayanus may be found in South America. The origin of S. cerevisiae is discussed.     

Production of Extracellular lipases by Saccharomyces cerevisiae

Seven strains of Saccharomyces cerevisiae all produced lipase when grown in shake flask culture. The best strain, DSM 1848, produced 4.0U of lipase in the medium containing olive oil and yeast extract. Production of the lipase was growth-associated.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

Localization of polyphosphate in vacuoles of Saccharomyces cerevisiae

Virtually all of the polyphosphate (PP) present in yeast protoplasts can be recovered in a crude particulate fraction if polybase-induced lysis is used for disrupting the protoplasts. This fraction contains most of the vacuoles, mitochondria and nuclei. Upon the purification of vacuoles the PP is enriched to the same extent as are the vacuolar markers. The amount of PP per vacuole is comparable to the amount of PP per protoplast.The possibility that PP is located in the cell wall is also considered. In the course of the incubation necessary for preparing protoplasts, 20% of the cellular PP is broken down. As this loss of PP occurs to the same extent in the absence of cell wall degrading enzymes, it is inferred that internal PP is metabolically degraded, no PP being located in the cell walls.It is concluded that in Saccharomyces cerevisiae most if not all of the PP is located in the vacuoles, at least under the growth conditions used.Non-Standard Abbreviations PIPES piperazine-N,N-bis-2-ethanolsulfonic acid - DEAE-dextran diethylaminoethyl-dextran     

PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Reaction kinetics of d-glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of d-glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of d-glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.