3-nitrotyrosine and thiazolyl-blue

Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Antifungal Agents; Antimycin A; Calcium; Cardiolipins; Cimicifuga; Coloring Agents; Cyclic AMP Response Element-Binding Protein; Electron Transport Complex IV; Membrane Potential, Mitochondrial; Mice; Mitochondria; Oncogene Protein v-akt; Osteoblasts; Oxidation-Reduction; Oxidative Stress; Phosphatidylinositol 3-Kinases; Saponins; Tetrazolium Salts; Thiazoles; Thioredoxin-Disulfide Reductase; Triterpenes; Tyrosine

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.

Topics: Animals; Antibodies; Antibody Specificity; Antigens; Coloring Agents; Cross Reactions; Eosine Yellowish-(YS); Fixatives; Formaldehyde; Hematoxylin; Hot Temperature; Immunohistochemistry; Paraffin Embedding; Protein Denaturation; Protein Folding; Receptor, ErbB-2; Staining and Labeling; Swine; Tetrazolium Salts; Thiazoles; Tyrosine

Curcumin, the active compound of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. This agent has been shown to regulate numerous transcription factors, cytokines, protein kinases, adhesion molecules, redox status and enzymes that have been linked to inflammation. While curcumin has been identified as an activator of apoptosis in several cell lines, the mechanism by which it initiates apoptosis, however, remains poorly understood. We considered curcumin from the point of view of its ability to protect against oxidative stress, the latter being one factor strongly implicated in the development of Parkinson's disease. Although the etiology of Parkinson's disease remains unknown, epidemiological studies have linked exposure to pesticides such paraquat to an increased risk of developing the condition. Analysis of the neurotoxic properties of these pesticide compounds has been focused on their ability to induce oxidative stress in neural cells. Given curcumin's capacity to protect against oxidative stress, it has been considered as a potential therapeutic agent for neurodegenerative diseases such as Parkinson's disease that involve an oxidative stress component. In the present report we describe the effect of curcumin in paraquat-mediated apoptosis of N27 mesencepahlic cells. We show that subtoxic concentrations of curcumin sensitize N27 mesencephalic cells to paraquat-mediated apoptosis.

Topics: Acetylcysteine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspase 3; Cell Line, Transformed; Curcumin; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; Gene Expression Regulation; Herbicides; Hydrogen Peroxide; Mesencephalon; Neurons; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Paraquat; Rats; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Tyrosine; Vitamin E

Tumor necrosis factor-alpha (TNF-alpha), a ubiquitous pro-inflammatory cytokine, is an important mediator in the immune-neuroendocrine system that affects the CNS. The present study demonstrates that treatment with TNF-alpha activates microglia to increase TNF-alpha production in primary cultures of glial cells isolated from wild-type (WT) mice and mice deficient in the inducible form of nitric oxide synthase (iNOSKO). However, mitochondrial dysfunction in WT neurons occurs at lower concentrations of TNF-alpha when neurons are directly treated with TNF-alpha or co-cultured with TNF-alpha-treated microglia than iNOSKO neurons similarly treated. Immunofluorescent staining of primary neurons co-cultured with TNF-alpha-treated microglia reveals that the antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), is co-localized with nitrotyrosine in WT but not in iNOSKO primary neuronal cells. Importantly, the percentage of surviving neurons is significantly reduced in WT neurons compared with iNOSKO neurons under identical treatment conditions. Together, the results suggest that TNF-alpha activates microglia to produce high levels of TNF-alpha and that production of nitric oxide (NO) in neurons is an important factor affecting MnSOD nitration and subsequent mitochondrial dysfunction.

Topics: Animals; Cell Death; Cell Survival; Cells, Cultured; Coculture Techniques; Immunohistochemistry; Mice; Mice, Knockout; Mitochondria; Neuroglia; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha; Tyrosine

Oxidative stress caused by various stimuli lead to oxidation of glutathione (GSH), the major redox power of the cell. Amyloid beta [Abeta(1-42)] is one of the key components of senile plaques and is involved in the progress initiation and triggers of Alzheimer's disease (AD). Lower GSH levels correlated with the activation of mitogen-activated proteins kinases (MAPK) have been demonstrated in AD, Parkinson's disease (PD) and other neurodegenerative disorders and have been proposed to play a central role in the deterioration of the aging and neurodegenerative brain. In this study, we evaluated the ability of low molecular weight thiol amides, N-acetyl cysteine amide (AD4) that replenishes GSH levels, N-acetyl glycine cysteine amide (AD7) and N-acetyl-Cys-Gly-Pro-Cys-amide (CB4) to protect primary neuronal culture against the oxidative and neurotoxic effects of Abeta(1-42) and to inhibit cisplatin- and hydrogen-peroxide-induced phosphorylation of two MAP kinases (MAPK), p38 and ERK1/2, in NIH3T3 cells. Cell death induced by Abeta(1-42) in primary neuronal cells was reversed by the thiol amides. Likewise, protein oxidation, loss of mitochondrial function and DNA fragmentation all returned to control levels by pretreatment with the three thiol amides. Elevated phosphorylation of ERK1/2 and p38 induced by cisplatin or H2O2 in NIH3T3 cells was lowered by AD4, AD7 and CB4 in a dose-dependent manner. Taken together, these results suggest that the thiol amides AD4, AD7 and CB4 protect neuronal cells against Abeta(1-42) toxicity by attenuating oxidative stress in correlation with inhibiting the MAPK phosphorylation cascade. These results are consistent with the notion that these small molecular thiol amides may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.

Topics: Acetylcysteine; Amides; Amyloid beta-Peptides; Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Cerebral Cortex; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Free Radical Scavengers; Glutathione; Lipid Peroxidation; Mice; Mitogen-Activated Protein Kinase Kinases; Molecular Weight; Neurons; Oligopeptides; Oxidation-Reduction; Peptide Fragments; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles; Tyrosine

Green tea polyphenols (GTP) are thought to help prevent oxidative stress-related diseases, such as cancer, cardiovascular disease, neurodegenerative disease, and aging. We here investigate the protective mechanisms of GTP on SH-SY5Y cells against apoptosis induced by the pro-parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). GTP rescued the changes in condensed nuclear and apoptotic bodies, attenuated 6-OHDA-induced early apoptosis, prevented the decrease in mitochondrial membrane potential, and suppressed accumulation of reactive oxygen species (ROS) and of intracellular free Ca(2+). GTP also counteracted the 6-OHDA-induced nitric oxide increase and overexpression of nNOS and iNOS, and decreased the level of protein-bound 3-nitrotyrosine (3-NT). In addition, GTP inhibited the autooxidation of 6-OHDA and scavenged oxygen free radicals in a dose- and time-dependent manner. Our results show that the protective effects of GTP on SH-SY5Y cells are mediated, at least in part, by controlling the ROS-NO pathway.

Topics: Annexin A5; Apoptosis; Blotting, Western; Calcium; Cell Line, Tumor; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Flavonoids; Flow Cytometry; Free Radicals; Guanosine Triphosphate; Humans; Membrane Potentials; Mitochondria; Models, Biological; Neurons; Nitric Oxide; Oxidopamine; Oxygen; Parkinson Disease; Phenols; Polyphenols; Quinones; Reactive Oxygen Species; Tea; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.

Topics: Adolescent; Adult; Blotting, Western; Cell Survival; Chemotaxis, Leukocyte; Cyclic GMP; Eosinophils; Female; Humans; In Vitro Techniques; Male; Middle Aged; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tetrazolium Salts; Thiazoles; Tyrosine

Here we investigate the effects of the stable, water-soluble nitroxyl radical, TEMPONE, on renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the rat kidney in vivo. TEMPONE significantly improved both glomerular and tubular function (serum urea, creatinine, creatinine clearance, and fractional excretion of Na(+)) in a dose-dependent manner and significantly attenuated the reperfusion-injury associated with I/R (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase, assessment of renal histology). TEMPONE also markedly reduced the immunohistochemical evidence of the formation of nitrotyrosine and poly(ADP-ribose), indicating reduction of nitrosative and oxidative stress, respectively. The latter was reflected in vitro, where TEMPONE significantly reduced cellular injury of primary cultures of rat renal proximal tubular (PT) cells caused by hydrogen peroxide in a dose-dependent manner. Importantly, in contrast to its in vivo metabolite TEMPOL (which also provided protective effects against renal I/R and oxidative stress of PT cells), TEMPONE reduced renal dysfunction and injury without causing a significant reduction in blood pressure upon administration. These results suggest, for the first time, that TEMPONE can reduce the renal dysfunction and injury caused by I/R and the injury caused to PT cells by oxidative stress without producing the adverse cardiovascular effects observed when using other nitroxyl radicals.

Topics: Animals; Coloring Agents; Dose-Response Relationship, Drug; Hydrogen Peroxide; Immunohistochemistry; Kidney; Kidney Diseases; Male; Mice; Nitrogen; Oxidative Stress; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Tetrazolium Salts; Thiazoles; Triacetoneamine-N-Oxyl; Tyrosine; Urine

Carbon monoxide causes a perivascular oxidative injury in animals, and we tested the hypothesis that endothelial cells could be a source of the injurious oxidants. Studies were undertaken to assess whether exposure to carbon monoxide would cause cultured bovine pulmonary artery endothelial cells to liberate reactive species. Concentrations of carbon monoxide between 11 and 110 nM caused progressively higher concentrations of nitric oxide to be released by endothelial cells based on measurements of nitrite and nitrate. Intracellular production of peroxynitrite was indicated by elevated concentrations of nitrotyrosine, and extracellular liberation of peroxynitrite was indicated by oxidation of p-hydroxyphenylacetic acid and dihydrorhodamine-123. Carbon monoxide did not disturb mitochondrial function based on the rate of oxygen consumption, intracellular production of hydrogen peroxide, and the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Carbon monoxide also did not alter arginine transport by cells or nitric oxide synthase activity, but it was found to increase steady state levels of nitric oxide by competing for intracellular binding sites. Acute cytotoxicity from carbon monoxide, assessed as radioactive chromium leakage, was due to nitric oxide-derived oxidants. A delayed cell death, whose mechanism is not entirely clear, was also demonstrated by chromium leakage and uptake of vital stain. These findings offer a possible mechanism for adverse health effects caused by carbon monoxide at concentrations ranging from the relatively low levels in polluted environments to levels typically encountered with life-threatening poisoning. Carbon monoxide causes oxidative stress by a novel mechanism involving a competition for intracellular binding sites which increases steady state levels of nitric oxide and allows for generation of peroxynitrite by endothelium.

Topics: Animals; Arginine; Carbon Monoxide; Cattle; Cell Survival; Cells, Cultured; Chromium; Endothelium, Vascular; Nitrates; Nitric Oxide Synthase; Nitrites; Oxidants; Oxidation-Reduction; Oxygen Consumption; Sulfhydryl Compounds; Tetrazolium Salts; Thiazoles; Tyrosine