Age-dependent variations of cell response to oxidative stress: proteomic approach to protein expression and phosphorylation

We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2-exposure of cells at 100 microM for 30 min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2-exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S-adenosyl-L-homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2-exposure in astrocytes from variously aged rats. Using the Pro-Q Diamond staining, heat shock protein 60 kDa (Hsp 60) and alpha-tubulin were observed to be phosphorylated upon H2O2-exposure. While phosphorylation of alpha-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules.     

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H(2)O(2) induced tyrosine phosphorylation of PLD1, protein kinase C-alpha (PKC-alpha), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-alpha in VSMCs. Stimulation of the cells by H(2)O(2) and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H(2)O(2)-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H(2)O(2)-stimulated PLD activation. The cells stimulated by oxidative stress (H(2)O(2)) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.     

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.     

Patterns of protein synthesis in a growth delay mutant (nuv) of Escherichia coli after treatment by near-UV radiation or hydrogen peroxide

When Escherichia coli cells are stressed by hydrogen peroxide (H2O2), synthesis of a large number of proteins is repressed, while several other proteins are induced. Since there is evidence that some lethal effects of near-UV (NUV) radiation may be directly or indirectly due to hydrogen peroxide generated by NUV light, treatment of cells with NUV radiation or H2O2 might be expected to repress and induce the same set of proteins. In this study, we compared the effects of H2O2 and NUV irradiation on patterns of protein induction and/or repression which were separate from the 4-thiouridine-dependent response using growth delay mutants (nuv). Concentrating initially on the proteins that ceased synthesis following NUV irradiation in an nuv mutant, we observed that these were not the same as those that ceased synthesis following H2O2 treatment. Inspection of two-dimensional polyacrylamide gel electrophoresis proteins indicated that NUV irradiation repressed synthesis of a different set of proteins, although there was some overlap between the two (45%). It was also observed that the new proteins which appeared after each of the two treatments were different. This suggests that the induction and/or repression of new proteins following NUV irradiation is not triggered solely via oxidative stress, although there is some overlap between the proteins that are induced or repressed following the two treatments.     

Gel-based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

We examined responses of cultivated bean (Phaseolus vulgaris L. cv. IDIAP R-3) and maize (Zea mays L. cv. Guarare 8128) plants exposed to ozone (O(3)) using a leaf injury assessment and proteomics approach. Plants grown for 16 days in greenhouse were transferred to an O(3) chamber and exposed continuously to 0.2 ppm O(3) or filtered pollutant-free air for up to 72 h. CBB-stained gels revealed changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein. By Western analysis changes in marker proteins for O(3) damage in leaves by 1-DE were checked. In bean leaves, two superoxide dismutase (SOD) protein (19 and 20 kDa) were dramatically decreased, while ascorbate peroxidase (APX, 25 kDa), small heat shock protein (HSP, 33 kDa), and a naringenin-7-O-methyltransferase (NOMT, 42 kDa) were increased by O(3). In maize leaves, expression levels of catalase (increased), SOD (decreased), and APX (increased) were drastically changed by O(3) depending on the leaf stage, whereas crossreacting HSPs (24 and 30 kDa) and NOMT (41 kDa) proteins were strongly increased in O(3)-stressed younger leaves. These results indicated a clear modulation of oxidative stress-, heat shock-, and secondary metabolism-related proteins by O(3). Finally, 2-DE at 72 h after O(3) exposure revealed changes (induction/suppression) in expression levels of 25 and 12 protein spots in bean and maize leaves, respectively. Out of these, ten and nine nonredundant proteins in bean and maize, respectively, were identified by MS. A novel pathogenesis-related protein 2 may serve as a potential marker for O(3) stress in bean.     

Centrosome amplification and multinuclear phenotypes are Induced by hydrogen peroxide

Multinucleated cells resulted from mitosis defect have been noted in pathophysiological states of the cells such as inflammation, senescence and cancer. Since oxidative stress has been known to correlate with these pathophysiological conditions, we tested the effect of H2O2 on the cell cycle progression and formation of multinucleated cells. H2O2 induced a significant delay in cell cycle progression in Chang liver cells. Interestingly, H2O2 actively induced hyperamplification of centrosomes (n>or=3) and multipolar spindle formation during mitosis and subsequently increased the generation of multinucleated cells. A significant increase of the phospho-ERK level was observed upon H2O2 treatment but PD98059, an MEK1/2 inhibitor, didn't reduce the frequency of cells with hyperamplified centrosomes. On the other hand, treatment of either H2O2 or adriamycin increased intracellular ROS levels and multinucleated cells, which were significantly suppressed by antioxidants, N-acetylcysteine and PDTC. Thus, our results suggest that oxidative stress can trigger centrosome hyperamplification and multinucleated cell formation, which may promote pathophysiological progression.     

Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAi- transfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated β-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.     

ATR/Chk1 Pathway is Activated by Oxidative Stress in Response to UVA Light in Human Xeroderma Pigmentosum Variant Cells

The crucial role of DNA polymerase eta in protecting against sunlight‐induced tumors is evidenced in Xeroderma Pigmentosum Variant (XP‐V) patients, who carry mutations in this protein and present increased frequency of skin cancer. XP‐V cellular phenotypes may be aggravated if proteins of DNA damage response (DDR) pathway are blocked, as widely demonstrated by experiments with UVC light and caffeine. However, little is known about the participation of DDR in XP‐V cells exposed to UVA light, the wavelengths patients are mostly exposed. Here, we demonstrate the participation of ATR kinase in protecting XP‐V cells after receiving low UVA doses using a specific inhibitor, with a remarkable increase in sensitivity and γH2AX signaling. Corroborating ATR participation in UVA‐DDR, a significant increase in Chk1 protein phosphorylation, as well as S‐phase cell cycle arrest, is also observed. Moreover, the participation of oxidative stress is supported by the antioxidant action of N‐acetylcysteine (NAC), which significantly protects XP‐V cells from UVA light, even in the presence of the ATR inhibitor. These findings indicate that the ATR/Chk1 pathway is activated to control UVA‐induced oxidatively generated DNA damage and emphasizes the role of ATR kinase as a mediator of genomic stability in pol eta defective cells.     

Biochemical, molecular genetic, and structural analyses of the staphylococcal nucleoid.

The nucleoid structure of an important human pathogen, Staphylococcus aureus, was dissected by atomic force microscopy (AFM). The nucleoids dispersed on a cover glass consisted of fibrous units with two different widths of 40 and 80 nm, a feature shared with those of Escherichia coli. On the other hand, cells exposed to an oxidative stress exhibited clogged nucleoids. A knock-out of mrgA (metallo regulated genes A) encoding a staphylococcal homolog of the nucleoid compaction factor (E. coli Dps) eliminated the compaction response to the oxidative stress and reduced the susceptibilities to H2O2 and UV irradiation. We also observed that the negative supercoiling of plasmids is increased by the oxidative stress. A possible interrelation between the helical density and the nucleoid compaction is discussed in relation to the oxidative stress response.     

UV released factors induce antioxidant defense in A375 cells

UV light leads to release of different secretory factors from irradiated cells of which some of them have been characterized. We have reported earlier that cells exposed to the supernatant medium from irradiated cells were resistant to killing by some genotoxic agents. In this study, we present our finding that demonstrates DNA damage induced by UV or H(2)O(2) is lowered on prior exposure to the UV released factors (UVRF). Production of ROS in cells and lipid peroxidation was also lowered. It was found that treatment of unexposed cells with UVRF present in the supernatant medium altered the antioxidant defense activity in cells. Significant was the increase in catalase (CAT) and Cu-Zn superoxide dismutase (SOD) activity, whereas glutathione peroxidase (GPx) and reduced glutathione (GSH) levels remained unaffected. Cells exposed to UVRF prior to UV or H(2)O(2) treatment also experienced such upregulation; however, the remarkable increase in the GPx activity exhibited by these cells was not observed in cells exposed to H(2)O(2) or UV alone. It appears that exposure to UVRF tinkered with antioxidant defense in cells to facilitate its proliferation upon assault by an agent that can produce oxidative damage.     

Dual effect of oxidative stress on NF-kappakB activation in HeLa cells

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H(2)O(2) leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)- induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF- kappakB activation, HeLa cells were incubated with H(2)O(2) or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H(2)O(2) and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H(2)O(2) alone, nuclear NF-kappaB was induced after 2 h without detectable degradation of cytosolic IkappaBalphaa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappakB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.     

Probing chemical induced cellular stress by non-Faradaic electrochemical impedance spectroscopy using an Escherichia coli capacitive biochip

A new capacitive biochip was developed using carboxy-CNT activated gold interdigitated (GID) capacitors immobilized with E. coli cells for the detection of cellular stress caused by chemicals. Here, acetic acid, H(2)O(2) and NaCl were employed as model chemicals to test the biochip and monitored the responses under AC electrical field by non-Faradaic electrochemical impedance spectroscopy (nFEIS). The electrical properties of E. coli cells under different stresses were studied based on the change in surface capacitance as a function of applied frequency (300-600 MHz) in a label-free and noninvasive manner. The capacitive response of the E. coli biochip under normal conditions exhibited characteristic dispersion peaks at 463 and 582 MHz frequencies. Deformation of these signature peaks determined the toxicity of chemicals to E. coli on the capacitive biochip. The E. coli cells were sensitive to, and severely affected by 166-498 mM (1-3%) acetic acid with declined capacitance responses. The E. coli biochip exposed to H(2)O(2) exhibited adaptive responses at lower concentrations (<2%), while at a higher level (882 mM, 3%), the capacitance response declined due to oxidative toxicity in cells. However, E. coli cells were not severely affected by high NaCl levels (513-684 mM, 3-4%) as the cells tend to resist the salt stress. Our results demonstrated that the biochip response at a particular frequency enabled the determination of the severity of the stress imposed by chemicals and it can be potentially applied for monitoring unknown chemicals as an indicator of cytotoxicity.     

Identification of stress-inducible proteins in Lactobacillus delbrueckii subsp. bulgaricus

Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a homofermentative bacterium that produces lactic acid during growth. We adapted the two-dimensional electrophoresis (2-DE) technique to study the response of this bacterium to acidity. De novo protein synthesis was monitored by [35S]methionine labeling of exponentially growing cultures under standard (pH 6) and acidic (pH 4.75) conditions. After 2-DE separation, the protein patterns were compared. The protein spots showing increased radioactivity levels under acid conditions were considered acid-induced. We determined the N-terminal amino acid sequence of three highly induced proteins; comparing these proteins to databases we identified them to be the well-known heat shock proteins GroES, GroEL, and DnaK. Their induction levels were measured and compared. This is the first study by 2-DE of stress response in L. bulgaricus. We established the method and present a protein map which will be useful for future studies.     

Regulation of bcl-2 family in hydrogen peroxide-induced apoptosis in human leukemia HL-60 cells

Numerous types of cells have been shown to undergo apoptosis when exposed to oxidant agent such as hydrogen peroxide. In order to understand the functional relationship between the anti- and pro-apoptotic regulatory proteins in the cells under oxidant stress, we have studied the level of expression of apoptosis regulatory proteins, bcl-2 and bax, in human leukemia HL-60 cells. The exposure of HL-60 cells to different concentrations of H2O2 for 6 h resulted in a typical apoptosis of the cells as characterized by flow cytometry, cell cycle analysis, and DNA fragmantation. There was a block in G1 to S transition and apoptotic cells were mainly derived from S and G2 cells. Kinetic study demonstrated that the levels of both bcl-2-mRNA and -protein expression were decreased with the progression of cellular apoptosis whereas the level of bax-mRNA was unchanged but the expressed bax-protein was not detectable. Cycloheximide, a nonspecific translation inhibitor, did not prevent the hydrogen peroxide-mediated apoptosis in HL-60 cells. These results suggest that the regulation of bcl-2, but not of bax are important factor in the oxidative stress-induced apoptosis in HL-60 cells.     

2-D PAGE analysis of pesticide-induced stress proteins of E. coli

Logarithmically growing batch cultures of Escherichia coli were exposed to sublethal concentrations of pyrethroid and carbamate pesticides of four different technical grades. This induced 17–20 stress proteins, as observed using two-dimensional polyacrylamide gel electrophoresis. An E. coli culture growing exponentially in Luria Bertani medium (cell density ~2.3×10⁹ cells/ml) was exposed to predetermined sublethal doses of individual pesticides. The cells were harvested after 30 minutes of induction and the stress response was developed in fresh LB medium for three hours under the same growth conditions. Cell pellets were obtained and stored in sonication buffer. Two-dimensional polyacrylamide gel electrophoresis was performed to resolve the proteins. Visualization of the protein spots by rapid silver staining showed 17–20 stress proteins which were absent in the standard protein profile of E. coli. On average 29% of these stress proteins were unique to the pollutant, while the remaining stress proteins overlapped with those of other pesticides. The iso-electric points (PIs) and molecular weights of the proteins were determined by comparing with protein markers with known PIs and molecular weights. Furthermore, upon comparing the pesticide-induced proteins within the same class and between the two different classes (pyrethroid and carbamate), it was apparent that the general nature of the stress remained the same throughout, which indirectly proved that the gene or set of genes responsible for stress expression are also the same, irrespective of the chemical nature of the substituents of the pesticides.     

Caveolin-1 is involved in reactive oxygen species-induced SHP-2 activation in astrocytes

Recent evidence supports a neuroprotective role of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) against ischemic brain injury. However, the molecular mechanisms of SHP-2 activation and those governing how SHP-2 exerts its function under oxidative stress conditions are not well understood. Recently we have reported that reactive oxygen species (ROS)-mediated oxidative stress promotes the phosphorylation of endogenous SHP-2 through lipid rafts, and that this phosphorylation strongly occurs in astrocytes, but not in microglia. To investigate the molecules involved in events leading to phosphorylation of SHP-2, raft proteins were analyzed using astrocytes and microglia. Interestingly, caveolin-1 and -2 were detected only in astrocytes but not in microglia, whereas flotillin-1 was expressed in both cell types. To examine whether the H2O2-dependent phosphorylation of SHP-2 is mediated by caveolin-1, we used specific small interfering RNA (siRNA) to downregulate caveolin- 1 expression. In the presence of caveolin-1 siRNA, the level of SHP-2 phosphorylation induced by H2O2 was significantly decreased, compared with in the presence of control siRNA. Overexpression of caveolin- 1 effectively increased H2O2-induced SHP-2 phosphorylation in microglia. Lastly, H2O2 induced extracellular signal-regulated kinase (ERK) activation in astrocytes through caveolin-1. Our results suggest that caveolin-1 is involved in astrocyte-specific intracellular responses linked to the SHP-2-mediated signaling cascade following ROS-induced oxidative stress.     

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.     

Kale extract increases glutathione levels in V79 cells, but does not protect them against acute toxicity induced by hydrogen peroxide

This study aims to evaluate the antioxidant potential of extracts of Brassica oleracea L. var. acephala DC. (kale) and several materials of Pieris brassicae L., a common pest of Brassica cultures using a cellular model with hamster lung fibroblast (V79 cells) under quiescent conditions and subjected to H?O? induced oxidative stress. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and glutathione was determined by the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-oxidized glutathione (GSSG) reductase recycling assay. The phenolic composition of the extracts was also established by HPLC-DAD. They presented acylated and non acylated flavonoid glycosides, some of them sulfated, and hydroxycinnamic acyl gentiobiosides. All extracts were cytotoxic by themselves at high concentrations and failed to protect V79 cells against H?O? acute toxicity. No relationship between phenolic composition and cytotoxicity of the extracts was found. Rather, a significant increase in glutathione was observed in cells exposed to kale extract, which contained the highest amount and variety of flavonoids. It can be concluded that although flavonoids-rich extracts have the ability to increase cellular antioxidant defenses, the use of extracts of kale and P. brassicae materials by pharmaceutical or food industries, may constitute an insult to health, especially to debilitated individuals, if high doses are consumed.     

蛋白质组学技术筛选与鉴定镉诱导下褐云玛瑙螺肝脏的差异蛋白质

比较丙酮/TCA沉淀法和直接裂解法,优化提取与分离褐云玛瑙螺(Achatina fulica, AF)肝脏全蛋白.采用丙酮/TCA沉淀法,可获得约600个蛋白质斑点.用0.5 mg/L CdCl2溶液浸泡后的去梗小白菜喂养AF,并作为镉盐诱导AF肝脏表达应激蛋白质的实验材料.采用蛋白质组学技术筛选出由镉盐诱导AF肝脏表达的14个差异蛋白质斑点,并用肽质量指纹图谱技术(peptide mass fingerprinting, PMF)和数据库比对法初步鉴定出7种差异蛋白质,其中部分为热激蛋白(heat shock protein)、甲基转移酶(Methyltransferase)、三磷酸腺苷结合盒子转运体(ABC transporter)、钼酸盐转运子亚基(molybdate transporter subunit)和磷酸甘油酸变位酶(phosphoglycerate mutase).差异蛋白质均参与镉盐代谢,并适合作为监测土壤或食物中镉污染程度及危害性的指示蛋白质.