Oxidative stress-dependent inhibition of yeast cell growth by farnesylamine and its possible relation to amine oxidase in the mitochondrial fraction

Among various analogs of the isoprenoid farnesol (FOH), farnesylamine (FNH2) inhibited the growth of the budding yeast Saccharomyces cerevisiae by accelerating cellular reactive oxygen species (ROS) generation. Unlike the case with FOH, however, FNH2 did not cause mitochondrial transmembrane potential (mtDeltaPsi) hyperpolarization so that FNH2-treated cells were not protected against ROS production by inhibiting the proton pumping function of mitochondrial F(O)F1-ATPase. FNH2 promoted ROS generation even in cells of a respiration-deficient mutant, indicating a yeast metabolic pathway other than mitochondrial electron transport as the origin of ROS. FNH2 oxidase activity was detected in the yeast mitochondrial fraction, which produces hydrogen peroxide (H2O2) in the reaction with either FNH2 or geranylgeranylamine (GGNH2), in addition to polyamine oxidase activity specific for spermine. GGNH2 also exhibited the growth inhibitory effect with the accompanying induction of ROS generation, while such an activity was not detected with any of the polyamines tested or geranylamine. FNH2 oxidase, which was sensitive to a typical copper-chelating agent, diethyldithiocarbamic acid (DDC), could be solubilized with Triton X-100, and detected as a single band upon activity staining with FNH2 but not with spermine in polyacrylamide gel electrophoresis. FNH2-treated cells were partly protected against ROS production by the additional supplementation of DDC in the medium. Our results suggest the involvement of H2O2 production due to direct oxidation of FNH2 by copper amine oxidase in oxidative stress-dependent inhibition of yeast cell growth.     

Different response to acetic acid stress in Saccharomyces cerevisiae wild‐type and l‐ascorbic acid‐producing strains

Biotechnological processes are of increasing significance for industrial production of fine and bulk chemicals, including biofuels. Unfortunately, under operative conditions microorganisms meet multiple stresses, such as non‐optimal pH, temperature, oxygenation and osmotic stress. Moreover, they have to face inhibitory compounds released during the pretreatment of lignocellulosic biomasses, which constitute the preferential substrate for second‐generation processes. Inhibitors include furan derivatives, phenolic compounds and weak organic acids, among which acetic acid is one of the most abundant and detrimental for cells. They impair cellular metabolism and growth, reducing the productivity of the process: therefore, the development of robust cell factories with improved production rates and resistance is of crucial importance. Here we show that a yeast strain engineered to endogenously produce vitamin C exhibits an increased tolerance compared to the parental strain when exposed to acetic acid at moderately toxic concentrations, measured as viability on plates. Starting from this evidence, we investigated more deeply: (a) the nature and levels of reactive oxygen species (ROS); (b) the activation of enzymes that act directly as detoxifiers of reactive oxygen species, such as superoxide dismutase (SOD) and catalase, in parental and engineered strains during acetic acid stress. The data indicate that the engineered strain can better recover from stress by limiting ROS accumulation, independently from SOD activation. The engineered yeast can be proposed as a model for further investigating direct and indirect mechanism(s) by which an antioxidant can rescue cells from organic acid damage; moreover, these studies will possibly provide additional targets for further strain improvements. Copyright © 2013 John Wiley & Sons, Ltd.     

Protein-conjugated acrolein as a biochemical marker of brain infarction

The relationship between acrolein (CH(2) =CH-CHO) and brain infarction is the focus of this review. It has been found that acrolein is produced mainly within cells from polyamines by polyamine oxidases (PAOs), especially from spermine by spermine oxidase during cell damage, and that acrolein is more toxic than reactive oxygen species (ROS) in a cell culture system. Thus, the possibility that acrolein and PAOs are good biochemical markers of stroke was tested because there are no other reliable biochemical markers at the early stage of stroke. Levels of protein-conjugated acrolein (PC-Acro) and PAOs (acrolein-producing enzymes) were significantly increased in the plasma of stroke patients. The multiplied value of PC-Acro by PAOs was nearly parallel with the size of stroke. Furthermore, when the combined measurements of PC-Acro, interleukin-6 (IL-6) and C-reactive protein (CRP) were evaluated along with age using a receiver operating characteristic (ROC) curve, even silent brain infarction (SBI), which is a small brain infarction, was indicated with approximately 84% sensitivity and specificity. These findings clearly indicate that acrolein is strongly correlated with cell damage during brain infarction.     

Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough

During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking.     

Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well‐established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10^?5 M ) or spermine (10^?5 M ) compared to a control culture containing 10^?8 M spermidine. 247 genes were upregulated > two‐fold and 11 genes were upregulated >10‐fold after spermidine addition. Functional categorization of the genes showed induction of transport‐related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two‐fold, and six genes were downregulated > eight‐fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.     

Liposome-assisted activity of superoxide dismutase under oxidative stress

A biological membrane is the front line of defense for cells against various environmental stresses such as heat and reactive oxygen species (ROS) and is expected to play an important role in the antioxidant system with antioxidant enzymes, similarly to its chaperone-like function in cooperation with heat shock proteins. The oxidative stress response of superoxide dismutase (SOD), which is known to catalyze the dismutation of O(2)(-) to H(2)O(2), was investigated in the presence of artificial membranes, liposomes, in order to obtain fundamental information on the biological ROS scavenging system. SOD lost its activity in the presence of H(2)O(2) and was found to have two loops including one which contains an alpha-helix which presents the substrate O(2)(-) to the activity center of SOD (Cu(II)). From circular dichroism analysis of SOD in the presence of H(2)O(2), the contents of the alpha-helix in SOD were found to decrease in correspondence with the inactivation and conformational change of SOD, suggesting that the conformation of the alpha-helix loops affects SOD activity. In the presence of liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), SOD was not inactivated in the presence of H(2)O(2) although the contents of its alpha-helix structure were decreased. The oxidized SOD was found to interact with the liposome surface under oxidative stress using dielectric dispersion analysis. Based on these results, a possible mechanism of SOD protection against ROS on liposomes was presented.     

Soy isoflavone genistein induces cell death in breast cancer cells through mobilization of endogenous copper ions and generation of reactive oxygen species

Scope: Worldwide geographical variation in cancer incidence indicates a correlation between dietary habits and cancer risk. Epidemiological studies have suggested that populations with high isoflavone intake through soy consumption have lower rates of breast, prostate, and colon cancer. Isoflavone genistein in soybean is considered a potent chemopreventive agent against cancer. Although several mechanisms have been proposed, a clear anticancer action mechanism of genistein is still not known. Methods and results: Here, we show that the cytotoxic action of genistein against breast cancer cells involves mobilization of endogenous copper. Further, whereas the copper specific chelator neocuproine is able to inhibit the apoptotic potential of genistein, the molecules which specifically bind iron (desferroxamine mesylate) and zinc (histidine) are relatively ineffective in causing such inhibition. Also, genistein‐induced apoptosis in these cells is inhibited by scavengers of reactive oxygen species (ROS) implicating ROS as effector elements leading to cell death. Conclusions: As copper levels are known to be considerably elevated in almost all types of cancers, in this proof‐of‐concept study we show that genistein is able to target endogenous copper leading to prooxidant signaling and consequent cell death. We believe that such a mechanism explains the anticancer effect of genistein as also its preferential cytotoxicity towards cancer cells.     

Depletion of glutathione as a cause of the promotive effects of polygodial, a sesquiterpene on the production of reactive oxygen species in Saccharomyces cerevisiae

The pungent sesquiterpenoid unsaturated dialdehyde, polygodial, exhibited a strong yeastcidal activity against the cells of Saccharomyces cerevisiae, in which production of reactive oxygen species (ROS) at a significant level could be detected with a fluorescent probe. The production of ROS in polygodial-treated cells was further confirmed by its elimination and the accompanying protection against yeastcidal effects in the presence of antioxidants such as L-ascorbate and alpha-tocopherol (alpha-TOH). Polygodial could accelerate ROS production only in cells of the wild-type grande strain but not in those of the respiratory-deficient petite mutant (rho0), indicating the role of the mitochondrial electron transport chain in the production of ROS. Unlike the case with antimycin A which accelerates ROS production by directly targeting the mitochondrial electron flow, polygodial caused depletion of cytoplasmic and mitochondrial glutathione which functions in estiminating ROS inevitably generated during aerobic growth. Polygodial-mediated depletion of intracellular glutathione was possibly dependent on a direct interaction between its enal moiety and the sulfhydryl group of the cysteine in glutathione by a Michael-type reaction.     

Diphlorethohydroxycarmalol isolated from Pae (Ishige okamurae) protects high glucose-induced damage in RINm5F pancreatic β cells via its antioxidant effects

Pancreatic β cells are very sensitive to oxidative stress and this may play an important role in β cell death in diabetes. The protective effect of diphlorethohydroxycarmalol (DPHC), one of phlorotannin polyphenol compound purified from pae (Ishige okamurae) against high glucoseinduced oxidative stress was investigated using RINm5F pancreatic β cells. High glucose (30 mM) treatment induced RINm5F pancreatic β cells cell death, but DPHC, at concentration 10 or 50 μg/mL, significantly inhibited the high glucose-induced glucotoxicity and apoptosis. Furthermore, treatment with DPHC dose-dependently decreased thiobarbituric acid reactive substances (TBARS), intracellular reactive oxygen species (ROS) generation and nitric oxide level increased by high glucose. In addition, DPHC treatment increased activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in high glucose pretreated RINm5F pancreatic β cells. DPHC treatment improved the secretory responsiveness following stimulation with glucose. These findings indicate that DPHC might be used as potential nutraceutical agent which will protect the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.     

Use of the synthetic superoxide dismutase/catalase mimetic EUK-134 to compensate for seasonal antioxidant deficiency by reducing pre-existing lipid peroxides at the human skin surface

The generation of reactive oxygen species (ROS) in UV-exposed skin is believed to contribute to the photoaging process. The stratum corneum (SC) contains a variety of enzymatic and non-enzymatic antioxidants to protect against various environmental sources of free radicals. We have previously shown a seasonal variation in SC catalase activity with strong deactivation in sun-exposed skin in the summer, whereas SC superoxide dismutase (SOD) activity remained intact in those conditions. This potentially leads to the local overproduction of H(2)O(2). The oxidized lipid squalene hydroperoxide accumulates at the surface of sun-exposed skin in the summer and upon exposure to ultravoilet A (UVA) doses as low as 0.1 J cm(-2) and adequate protection against excessive lipid peroxidation at times of UV exposure should be aimed for. We have been using the induction of lipid hydroperoxides at the skin surface by a single dose of UVA (1 J cm(-2)) as a model system to evaluate the protective effect of antioxidants in vivo. Topical treatment with the synthetic SOD/catalase mimetic molecule (EUK-134) 1 h before UVA exposure reduced the level of lipid peroxides at the surface of UVA-exposed skin but also baseline peroxide levels on non-irradiated skin were reduced in a dose-dependent fashion. In contrast to alpha-tocopherol, EUK-134 even reduced the level of lipid peroxides at the surface of UVA-exposed skin when it was applied after irradiation. We confirmed that this salen-manganese complex was able to reduce squalene hydroperoxide levels in vitro, suggesting peroxidase-like activity towards organic peroxides. These data support the concept that the synthetic SOD/catalase mimetic EUK-134 might be able to compensate for seasonal deficiencies in antioxidant defense capacity at the skin surface, thereby contributing to an optimal protection of the skin against the accumulation of oxidative damage.     

韩国产芝麻酱油对AAPH诱发LLC-PK1细胞氧化应激的保护作用

以芝麻酿造酱油乙醇提取物(ethanol extract sesame sauce,SSE)为研究对象,探讨其对1 mmol/L 2,2’-偶氮二异丁基脒二盐酸盐(2,2’-azobis(2-methylpropionamidine)dihydrochloride,AAPH)引发的LLC-PK1猪肾近曲小管上皮细胞氧化应激损伤的保护作用。方法:LLC-PK1细胞与不同质量浓度(10~100μg/m L)的SSE预先共同培养24 h后,用含AAPH的DMEM培养液继续培养4 h建立细胞损伤模型。细胞存活率用四甲基偶氮唑蓝法检测,细胞内丙二醛(malondialdehyde,MDA)含量和总活性氧簇(reactive oxygen species,ROS)水平分别以硫代巴比妥酸比色法和2’,7’-二氢二氯荧光黄双乙酸钠探针法测定。细胞内过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化酶(glutathione peroxidase,GSH-Px)、谷胱甘肽巯基转移酶(glutathione S-transferase,GST)、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-GCS)活力和谷胱甘肽(glutathione,GSH)含量按试剂盒说明书以比色法测定。结果表明,SSE预处理可以提高受损细胞存活率,降低细胞内总ROS水平和MDA含量。同时,SSE还能提高受损细胞内抗氧化酶(CAT、SOD、GSH-Px)及γ-GCS活力并提高细胞内GSH含量。实时荧光定量聚合酶链式反应分析也提示SSE能上调细胞内抗氧化物酶(CAT、SOD和GSH-Px)的m RNA表达量。此外,SSE可以通过提高细胞内源性抗氧化系统能力来降低细胞内MDA和ROS水平,并由此缓解AAPH对LLC-PK1细胞所造成的氧化应激损伤。     

坛紫菜多酚抗氧化及抑制UVB致HSF细胞氧化损伤作用

从坛紫菜中提取分离制备多酚组分,通过1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除能力、抑制β-胡萝卜素褪色能力和铁还原能力分析法评价坛紫菜多酚的体外抗氧化作用;以紫外线B(ultraviolet radiation B,UVB)致人表皮成纤维细胞(human skin fibroblast cells,HSFs)损伤模型评价坛紫菜多酚抑制紫外损伤表皮作用,考察其用于防护紫外损伤领域的可行性。体外实验结果显示,坛紫菜多酚具有较强的抗氧化作用,其DPPH自由基清除能力与同等质量浓度的二丁基羟基甲苯(butylated hydroxytoluene,BHT)相当;但抑制β-胡萝卜素亚油酸体系褪色能力和铁还原能力弱于同等质量浓度的BHT。细胞实验结果显示,坛紫菜多酚能有效降低UVB辐照所致HSFs的乳酸脱氢酶活力,维护细胞膜的完整性;降低受损细胞的活性氧类水平,增加超氧化物歧化酶、谷胱甘肽过氧化物酶活性,从而降低细胞的氧化应激状态,提升内皮细胞的抵抗氧化应激的能力,显著抑制HSFs凋亡水平。坛紫菜多酚能在细胞水平有效保护HSFs免受UVB的氧化损伤,表明紫菜多酚具有用于紫外损伤防护领域的潜力。     

竹盐酿造酱油对H2O2诱发LLC-PK1细胞氧化损伤的保护作用

目的：研究竹盐酿造酱油乙醇提取物对H2O2诱发猪肾近曲上皮小管细胞（pig proximal tubular cell）LLC-PK1氧化损伤的保护作用。方法：以不同质量浓度（10～200 μg/mL）的竹盐酿造酱油乙醇提取物预培养LLC-PK1细胞24 h后,换用含500 μmol/L H2O2的DMEM细胞培养液继续培养4 h建立细胞氧化损伤模型。四甲基偶氮唑蓝（methyl thiazolyl tetrazolium,MTT）法检测细胞生存率,硫代巴比妥酸比色法测定细胞内丙二醛（malondialdehyde,MDA）含量,比色法测定细胞内过氧化氢酶（catalase,CAT）、超氧化物歧化酶（superoxidedismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活性和谷胱甘肽（glutathione,GSH）含量。结果：经不同质量浓度竹盐酿造酱油乙醇提取物预处理24 h后,受损细胞的生存率上升,细胞内MDA生成量减少,ROS水平显著降低。同时,细胞内抗氧化酶（CAT、SOD和GSH-Px）的活性及其mRNA转录水平,GSH含量也较未经竹盐酿造酱油乙醇提取物处理的受损细胞增加。结论：竹盐酿造酱油乙醇提取物可以通过提高细胞内抗氧化酶系的活性和抗氧化物质GSH的含量而有效地对抗H2O2诱发的LLC-PK1细胞氧化损伤。     

Antioxidant effect derived from bioaccessible fractions of fruit beverages against H2O2-induced oxidative stress in Caco-2 cells

This work evaluates the effect of bioaccessible fractions from fruit beverages against oxidative stress (OS) in Caco-2 cells. A fruit beverage (grape + orange + apricot) (with/without milk and/or iron/zinc) was subjected to in vitro gastrointestinal digestion, and bioaccessible fractions were incubated with Caco-2 cell cultures. Following preincubation, OS was induced with 5 mM H₂O₂. Intracellular reactive oxygen species (ROS), mitochondrial potential (Δψ_m), mitochondrial metabolism (MTT test), intracellular reduced glutathione (GSH) and superoxide dismutase activity (SOD) were measured. The data evidenced viable cultures with increased mitochondrial metabolism and GSH-Rd activities, without alteration in SOD activity. Accordingly, more preserved mitochondrial integrity was also evidenced, allowing the action of antioxidant systems in preincubated cultures. Based on these data, we can conclude that a cytoprotective effect is derived from bioaccessible fractions of fruit beverages, though this effect failed to prevent intracellular ROS accumulation in Caco-2 cell cultures exposed to 5 mM H₂O₂.     

数学分析法评估大蒜素对HeLa细胞氧化还原水平的影响

为探究大蒜素调控典型肿瘤细胞中的氧化还原水平,建立了佛波酯(phorbol-12-myristate-13-acetate,PMA)诱导人宫颈癌细胞HeLa中活性氧(reactive oxygen species,ROS)升高的氧化应激模型,通过借助中心响应面模型,探究发现当HeLa细胞在10 mg/L的大蒜素培养液中...     

Involvement of oxidative stress induction in Na+ toxicity and its relation to the inhibition of a Ca2+ -dependent but calcineurin-independent mechanism in Saccharomyces cerevisiae

Uridine 5'-hexadecylphosphate (UMPC16) inhibited the growth of Saccharomyces cerevisiae under a hypersaline stress condition with Na+ more strongly than the calcineurin inhibitor cyclosporine A (CsA). Additional Ca2+ supplementation similarly suppressed the inhibitory activities of UMPC16 and CsA on yeast cell growth in a medium with Na+. UMPC16, but not CsA, accelerated mitochondrial reactive oxygen species (ROS) generation in combination with Na+, suggesting its inhibition of a Ca2+ -dependent but calcineurin-independent mechanism for protection against Na+ toxicity.     

茶树精油对灰葡萄胞霉生理功能的干扰研究

为探寻新型生物防治方法,揭示茶树精油(TTO)对果蔬采后病原真菌灰葡萄孢霉(Botrytis Cinerea)的抑菌作用机理,以液体培养后收集的B.Cinerea菌丝体为研究对象,进行2倍MIC(最小抑菌浓度)的TTO处理,采用荧光显微镜(FEM)、扫描电镜(SEM)和透射电镜(TEM)研究了处理对细胞膜通透性、菌体形态和超微结构的影响,以及处理过程中菌丝体内活性氧(ROS)积累及相关抗氧化酶类和物质的变化。结果表明:TTO处理后菌丝荧光强度增强,说明其细胞膜通透性明显上升,细胞膜完整性受到破坏;同时菌丝发生严重的皱缩、干瘪,胞浆消化呈碎渣样;TTO导致了菌丝内过氧化氢(H2O2)的大量积累,诱导提高了超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸-谷胱甘肽(As A-GSH)循环的活性,说明TTO是通过提高B.Cinerea细胞膜通透性、破坏菌体结构和诱导ROS爆发来发挥抑菌作用。     

Free radical scavenging and cytoprotective activities of phenolic antioxidants

The free radical scavenging activities of three flavonoids (quercetin, rutin and catechin) and four hydroxycinnamic acids (caffeic, ferulic, sinapic, and chlorogenic acids) were evaluated using both oxygen radical absorbance capacity (ORAC) and lipid peroxidation inhibition capacity (LPIC) assays. The cytoprotective effects of these compounds were also measured by the degree of protection against H(2)O(2)-induced damage of human Jurkat cells. All compounds exhibited protection against H(2)O(2)-mediated cytotoxicity in a dose-dependent manner. The concentrations required to result in a 50% reduction in cell death (EC(50) value) were calculated from their dose-response curves. These ranged from 0.15-2.65 microM. Overall, the four hydroxycinnamic acids tested were less effective than the three flavonoids, and of all compounds tested, quercetin offered the strongest protection against H(2)O(2)-induced cell death. A comparison of the results showed that the ability to inhibit peroxidation of lipids in a liposomal system (LPIC) correlated well with the cytoprotective activities (EC(50)), but not with the ability to protect an aqueous fluorescent substrate in the ORAC assays. The results suggest that the behavior of antioxidants in a liposomal membrane is to some extent similar to the mechanism involved in the protection of living cells from oxidative damage.     

Bacteriocin involved in premature death of Lactobacillus acidophilus NCFM during growth at pH 6

Lactobacillus acidophilus NCFM maintained at pH 6 during growth in lactobacilli MRS broth appeared to exhibit premature death. However, during extended incubation, the culture reinitiated growth. Spent broth collected from the culture when it began the premature death was very toxic to the culture, but growth did occur during extended incubation of the assay tubes. When several successive subcultures with extended incubation were made in MRS broth containing added inhibitory spent broth, a strain (identified as L. acidophilus) was isolated that was resistant to the inhibitor. This strain produced a bacteriocin that was inhibitory to the growth of L. acidophilus NCFM and the predominating strains isolated from it. The producer strain grew more slowly than did the predominating strains in culture NCFM. The results show that when L. acidophilus NCFM, a mixed strain culture, was grown at pH 6, the inhibitory strain was able to grow sufficiently to produce enough bacteriocin to destroy the predominating strain(s) in the culture.