DJ-1 mutation decreases astroglial release of inflammatory mediators.

Mutations in DJ-1, reactive gliosis and concomitant inflammatory processes are implicated in the pathogenesis and progression of Parkinson's disease (PD). To study the physiological consequences of DJ-1 mutation in the context of neuroinflammatory insult, primary cortical astrocytes were isolated from DJ-1 knockout mice. Astrocytes were exposed to 1µg/mL lipopolysaccharide (LPS) for 24h following 2h pre-exposure to inhibitors of MEK (U0126), JNK (JNK inhibitor II) or p38 (SB203580). Real-time PCR was used to assess the LPS-induced expression of pro-inflammatory mediators cyclooxygenase 2 (COX2), inducible nitric oxide synthetase (NOS2), and tumor necrosis factor α (TNFα). LPS-induced expression of COX2 decreased similarly in DJ-1(+/+) and DJ-1(-/-) astrocytes in response to inhibition of p38, but was unaffected by inhibition of MEK or JNK. No significant alterations in NOS2 expression were observed in any inhibitor-treated cells. The inhibitors did not affect expression of TNFα; however, DJ-1(-/-) astrocytes had consistently lower expression compared to DJ-1(+/+) counterparts. Secretion of TNFα and prostaglandin E2 (PGE2) into the culture medium was significantly decreased in DJ-1(-/-) astrocytes, and inhibition of p38 decreased this secretion in both genotypes. In conclusion, DJ-1(-/-) astrocytes may provide decreased neuroprotection to surrounding neurons due to alterations in pro-inflammatory mediator expression.

Proteomic analysis of diaminochlorotriazine adducts in wistar rat pituitary glands and LbetaT2 rat pituitary cells.

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LbetaT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LbetaT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LbetaT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LbetaT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LbetaT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.

Fluorescent nanosensors for intracellular chemical analysis: decyl methacrylate liquid polymer matrix and ion-exchange-based potassium PEBBLE sensors with real-time application to viable rat C6 glioma cells.

Fluorescent spherical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), in the 500 nm-1 microm size range have been developed using decyl methacrylate as a matrix. A general scheme for the polymerization and introduction of sensing components creates a matrix that allows for the utilization of the highly selective ionophores used in poly(vinyl chloride) and decyl methacrylate ion-selective electrodes. We have applied these optically silent ionophores to fluorescence-based sensing by using ion-exchange and highly selective pH chromoionophores. This allows the tailoring of selective submicrometer sensors for use in intracellular measurements of important analytes for which selective enough fluorescent probes do not exist. The protocol for sensor development has been worked out for potassium sensing. It is based on the BME-44 ionophore (2-dodecyl-2-methyl-1,3-propanediylbis[N-[5'nitro(benzo-15-crown-5)-4'-yl]carbamate]). The general scheme should work for any available ionophore used in PVC or decyl methacrylate ion-selective electrodes, with minor adjustments to account for differences in ionophore charge and analyte binding constant. The reversible and highly selective sensors developed have a subsecond response time and an adjustable dynamic range. Applications to live C6 glioma cells demonstrate their utility; the intracellular potassium activity is followed in real time upon extracellular administration of kainic acid.

Irradiation of mitochondria initiates apoptosis in a cell free system.

The ability to modulate the sensitivity of mammalian cells to ionizing radiation (IR) (e.g. using chemotherapeutics) is dependent on our understanding of the primary target and biochemical pathway that leads to IR-induced apoptosis. We demonstrate using a cell free assay that irradiation of mitochondria is a primary event that initiates IR-induced apoptosis. IR results in loss of mitochondrial membrane potential, opening of the permeability transition pore (PTP) and the release of cytochrome c (cyto c). Apaf-1 and ATP were required to initiate apoptosis upon release of cyto c from mitochondria. The importance of mitochondrial events in the initiation of IR-induced apoptosis was also supported by the observation that inhibition of caspase-9 by the over-expression of dominant negative mutants resulted in the inhibition of IR-induced apoptosis. In contrast, inhibition of caspase-8 had only a minor impact on IR-induced apoptosis. Over-expression of Bcl-X(L) inhibited the initiation of IR-induced apoptosis due to its ability to prevent the loss of mitochondrial membrane potential, PTP opening and cytochrome c release. In a cell free assay for apoptosis, mitochondria as well as cytosol derived from Bcl-X(L) over-expressing cells were less efficient at supporting apoptosis in response to IR suggesting multiple roles for Bcl-X(L) in the regulation of apoptosis.

Differential cellular regulation of the mitochondrial permeability transition in an in vitro model of 1,3-dinitrobenzene-induced encephalopathy.

Exposure to 1,3-dinitrobenzene (DNB) is associated with neuropathologic changes in specific brainstem nuclei, mediated by oxidative stress and mitochondrial dysfunction. The expression of Bcl-2-family proteins as a function of sensitivity to 1, 3-dinitrobenzene (DNB)-induced mitochondrial permeability transition (MPT) was examined in C6 glioma and SY5Y neuroblastoma cells. Neuroblastoma cells were 10-fold more sensitive than glioma cells to DNB-induced decreases in mitochondrial reducing potential, measured by reduction of the tetrazolium compound, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The IC(50) values for DNB-related inhibition of MTT reduction were 107+/-25 microM in SY5Y cells and 1047+/-101 microM in C6 cells. Levels of reactive oxygen species (ROS) were increased in both SY5Y and C6 cells following DNB exposure by 4.6- and 6.0-fold above control, respectively. DNB caused abrupt depolarization of mitochondria in both neuroblastoma and glioma cells that was inhibited by trifluoperazine. The first order rate constants for mitochondrial depolarization were: C6, k=0.31+/-0.02 min(-1); SY5Y, k=0.14+/-0.01 min(-1). Onset of MPT occurred at 10-fold lower concentration of DNB in SY5Y cells than in C6 cells. The antioxidants, deferoxamine and alpha-tocopherol, effectively prevented DNB-induced MPT in C6 and SY5Y cells, suggesting involvement of ROS in the initiation of MPT. Exposure to DNB resulted in decreased cellular ATP content in SY5Y cells and efflux of mitochondrial calcium in both SY5Y and C6 cells, concurrent with onset of MPT. The expression of Bcl-2, Bcl-X(L), and Bax was evaluated in both cell types by Western blot analysis. C6 glioma cells strongly expressed Bcl-X(L) and only weakly expressed Bcl-2 and Bax, whereas SY5Y neuroblastoma cells expressed lower levels of Bcl-X(L) and higher levels of both Bcl-2 and Bax. Collectively, these results suggest that higher constitutive expression of Bcl-X(L), rather than Bcl-2, correlates with resistance to DNB-induced MPT in SY5Y and C6 cells and that differential regulation of the permeability transition pore may underlie the cell-specific neurotoxicity of DNB.

Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors.

Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.

Formation and export of the glutathione conjugate of 4-hydroxy-2, 3-E-nonenal (4-HNE) in hepatoma cells.

The cellular metabolism of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic and genotoxic product of oxidative stress-induced lipid peroxidation, was investigated in rat H35 hepatoma cells. Previous studies from our laboratory (1) have characterized the degree to which oxidative, reductive, and conjugative metabolic pathways function simultaneously during hepatocellular metabolism of 4-HNE to rapidly eliminate the compound from suspensions of freshly isolated rat hepatocytes. In the current studies, we have extended the investigation of 4-HNE metabolism to examine the pharmacokinetic parameters of 4-HNE elimination and export in a hepatoma cell line and determined that the ensuing oxidative and conjugative metabolites of 4-HNE are rapidly and efficiently transported out the cell. Low concentrations of 4-HNE (25 microM) were used in an attempt to simulate physiologically relevant conditions. The H35 hepatoma cell line studied was first evaluated for enzymes known to play important roles in the metabolism of 4-HNE and were found to possess activities for glutathione S-transferase, aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase of 24.00 +/- 1.12, 3. 45 +/- 0.17, and 6.44 +/- 0.29 nmol min-1 mg-1 protein, respectively. Hepatoma cells were incubated with 25 microM 4-HNE and metabolites in intra- and extracellular fractions were quantitated by reversed-phase HPLC over the time course of treatment. Reduced glutathione (GSH) and the GSH metabolites of 4-HNE were quantitated by reversed-phase HPLC as the dinitrobenzene derivatives. Uptake of 4-HNE from the extracellular medium occurred with an estimated rate of 0.398 +/- 0.181 min-1 10(6) hepatoma cells-1. The oxidative metabolite of 4-HNE, 4-hydroxy-2-nonenoic acid (HNA), produced by ALDH, appeared rapidly in the intracellular fraction achieving concentrations of 0.28 HNA nmol 10(6) hepatoma cells-1 and was efficiently eliminated with a first-order rate constant of 0.988 min-1. The GST-mediated conjugative metabolite, 3-glutathionyl-4-hydroxy-2-nonanal (4-HNE-SG), rapidly reached maximal intracellular concentrations of 1.88 +/- 0.44 nmol 10(6) hepatoma cells-1 and was eliminated at a rate of 0.101 +/- 0.033 min-1. Extracellular rates of formation, representing export, for HNA and 4-HNE-SG were 0.247 +/- 0.045 and 0.044 +/- 0.009 min-1 10(6) hepatoma cells-1, resulting in maximal extracellular concentrations for HNA and 4-HNE-SG of 0.70 +/- 0.10 and 3.03 +/- 0. 84 nmol 10(6) hepatoma cells-1. Approximately 75% of the administered concentration of 4-HNE was converted to measurable metabolites, with the 4-HNE-GSH conjugate accounting for 61% of total administered 4-HNE and HNA accounting for 14%. Collectively, these results demonstrate that oxidative and conjugative pathways are primarily responsible for elimination of 4-HNE at low concentrations in the hepatoma cell line evaluated and that the 4-HNE metabolites resulting from these pathways are rapidly and efficiently exported out of the cell.

Alpha,beta-unsaturated aldehydes increase glutathione S-transferase mRNA and protein: correlation with activation of the antioxidant response element.

A series of alpha,beta-unsaturated aldehydes was evaluated to determine if these compounds could mediate inducible expression of glutathione S-transferase (GST) through the 5'-flanking antioxidant response element (ARE). The ARE from rGST A1 was subcloned into a luciferase reporter construct and used to transiently transfect rat Clone 9 hepatoma cells. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4-HNE), trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethacrynic acid (EA), a control compound also containing an alpha,beta-unsaturated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC50 values for growth inhibition were measured using 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. IC50 values in Clone 9 cells were: 4-HNE, 6.3 +/- 0.7 microM; t-2-HE, 16.0 +/- 0.7 microM; 2-PE, 2.2 +/- 0.4 microM; and EA, 38.0 +/- 1.6 microM. A dose-dependent increase in luciferase activity was observed in transfected cells with all four compounds tested, indicating that alpha, beta-unsaturated aldehydes function as direct activators of the ARE. To determine whether or not the observed promoter activation led to increased transcriptional and translational induction of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in Clone 9 cells revealed increased steady-state message for GST A1 and A4, increased GST A4-4 protein by Western blotting, and increased GST activity toward 1-chloro-2,4-dinitrobenzene in response to treatment with all four compounds evaluated. Collectively, these studies demonstrate that EA and certain alpha,beta-unsaturated aldehydes produced as a result of cellular membrane lipid peroxidation are activators of the ARE and efficient inducers of GST A1-1 and A4-4.

Association of glutathione S-transferase isozyme-specific induction and lipid peroxidation in two inbred strains of mice subjected to chronic dietary iron overload.

The alpha-class glutathione S-transferases are proposed to play a prominent role in catalyzing the conjugation of glutathione with electrophilic aldehydic products of lipid peroxidation. The effect of iron-induced lipid peroxidation on induction of glutathione S-transferase (GST) isozymes A1 and A4 in the livers of male C57/BL6Ibg and DBA/J2Ibg mice was studied. C57 and DBA mice were fed for 4 months on a diet supplemented with iron as ferrocene and then were assessed for liver injury, hepatic iron loading, indices of lipid peroxidation, GST activity, and induction of GST isozymes A1 and A4. Iron-treated animals displayed a loss in body weight from pair-fed controls and had large increases in hepatic non-heme iron with concomitant liver injury, as measured by serum alanine aminotransferase. Hepatic lipid hydroperoxides, a direct measure of oxidized membrane lipids, were significantly increased only in C57 mice, but hepatic concentrations of reduced glutathione (GSH) were significantly increased in both inbred strains. Total GST activity toward 1-chloro-2,4-dinitrobenzene was significantly increased in C57 mice but not in DBA. Western blot studies using polyclonal antibodies specific for GST A1 and A4 revealed significant increases of 1.5-2.0-fold in these GST isoforms in both inbred strains. These results in a unique murine model for hepatic iron overload further support recent in vivo studies (Khan et al., Toxicol. Appl. Pharmacol., 131, 63-72, 1995) that have associated induction of GST A4 with protection against oxidative stress-induced lipid peroxidation. The observed increases in lipid hydroperoxides, hepatic GSH, GST activity, and GST A1 and A4 protein strongly support the hypothesis that induction of GST A1 and A4 represents an important protective event in the detoxification of electrophilic products of lipid peroxidation.

Subunit composition and domain structure of the Spo0A sporulation transcription factor of Bacillus subtilis.

The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.