Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione (GSH) is a major antioxidant that protects tissues from free radical injury. Glutamine augments host defenses and may be important in GSH synthesis. Acetaminophen toxicity causes hepatic GSH depletion and hepatic necrosis. The authors hypothesized that glutamine-supplemented nutrition would enhance liver GSH stores and diminish hepatic injury and death after acetaminophen overdose. Wistar rats received either a standard total parenteral nutrition (TPN) solution (STD) or an isocaloric, isonitrogenous glutamine-supplemented solution (GLN). On the 5th day of feeding, animals were given acetaminophen (400 mg/kg intraperitoneally) and then killed at various time points. Standard TPN solution animals had a rapid depletion of hepatic glutathione, whereas GLN animals were resistant to this drop and rapidly repleted hepatic GSH stores. Glutamine-supplemented animals maintained higher plasma glutamine concentrations, had lesser elevations in hepatic enzymes, and sustained significantly fewer complications compared with STD animals. The authors conclude that glutamine-supplemented nutrition preserves hepatic glutathione, protects the liver, and improves survival during acetaminophen toxicity. Glutamine may augment host defenses by enhancing antioxidant protection.
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PMID:Glutamine preserves liver glutathione after lethal hepatic injury. 154 97

Experiments were designed to measure cross-link formation following combined treatment of EMT-6/Ro tumor cells with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and misonidazole (MISO) in vitro. To avoid MISO-induced glutathione (GSH) depletion, which might contribute to enhanced monoadduct formation by reducing the protective GSH pools, a post-incubation (i.e. treatment with CCNU for one hour in air followed by MISO treatment in hypoxia) protocol was adopted. Utilizing this treatment scheme, it was possible to significantly enhance CCNU toxicity by post-treating with MISO immediately after exposure to CCNU. Enhanced cross-link formation detected by alkaline elution, at this time, correlated well with the magnitude of cell-kill enhancement, thereby implicating enhanced cross-link formation in the mechanism of potentiation. However, if the cells were allowed to incubate for various intervals between CCNU and MISO treatments, the magnitude of potentiation progressively diminished. Beyond approximately 8-10 hours (corresponding to the time required for maximal cross-link formation after CCNU treatment), treatment with MISO was ineffective at potentiating CCNU cytotoxicity. These experiments suggest that chemopotentiation can be produced by treating with MISO after treatment with CCNU (post-incubation) and that enhanced cross-link formation is involved in the mechanism of MISO chemopotentiation of CCNU activity. The kinetic studies, using the post-incubation protocol, further suggest that the chemopotentiating effect of MISO is exerted subsequent to monoadduct formation and probably does not involve inhibition of DNA-DNA cross-link repair.
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PMID:Cross-link formation and chemopotentiation of EMT-6/Ro cells exposed to MISO after CCNU treatment in vitro. 375 61

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.
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PMID:Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo. 377 30

Depletion of intracellular glutathione (GSH) can enhance misonidazole (MISO) radiosensitizing efficacy both in vivo and in vitro. However, such treatments may also enhance the systemic toxicity in animals. The purpose of the present study was to test various ways of depleting GSH levels in a variety of experimental mouse tumors, to measure the improvement in the efficacy of MISO and its less toxic analog SR 2508 by this depletion, and to determine the effect of daily GSH depletion on the toxicity of MISO and SR 2508. GSH levels were measured daily for 5 days in tumors, livers and brains of mice injected daily with buthionine sulfoximine (BSO), with or without diethylmaleate (DEM). To investigate tumor variability we studied 5 different tumors: EMT-6, RIF-1, KHT, SCC VII, and B16 melanoma. The efficacy of MISO and SR 2508 was evaluated using the KHT and SCC VII tumors either by the regrowth delay assay or by the in vivo/in vitro clonogenic assay. The drug toxicity was evaluated by weight loss and by death. Daily doses of 3 mmole/kg BSO depleted tumor levels of GSH to 20 to 40% of controls by 6 hr after each injection. Injection of DEM (300 mg/kg) 6 hr after BSO further enhanced the depletion. Administration of MISO or SR 2508 at the time of maximum GSH depletion enhanced the MISO efficacy by factors of 2.5 to 8 for depletion to 8% of controls by BSO + DEM, but no enhancement of SR 2508 was seen with tumors at 20% GSH levels achieved with BSO alone in the preliminary experiment. The chronic toxicity of MISO was enhanced not at all or by a factor of up to 2 for BSO and BSO + DEM respectively. Further studies are needed before it can be concluded that GSH depletion by BSO alone may be a useful adjunct to the clinical use of radiosensitizers.
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PMID:Depletion of glutathione in vivo as a method of improving the therapeutic ratio of misonidazole and SR 2508. 623 85

Six mercury compounds [HgCl2 (MC), Hg(CH3COO)2 (MA), Hg(NO3)2 (MN), C2H5HgSC6H4COONa (EMT), C6H5HgOCOCH3 (PMA) and CH3CIHg (MMC)] were studied using two kidney cell lines (MDCK and LLC-PK1), primary cultures of human proximal tubular cells (hPTC) and nonrenal cell lines (SAOS and Hep G2). Cell damage was measured with four different tests: neutral red uptake, mitochondrial dehydrogenase activity (MTT conversion), thymidine incorporation and protein content. Relative toxicity was established by the determination of the concentration of test compound inducing a 50% reduction of the parameter considered (EC50 value). Two groups could be distinguished: PMA, EMT and MMC are one order of magnitude more toxic than MC, MN and MA. Cellular uptake was measured by the HPLC-hybrid generation AAS after 24 hours treatment with 1.5 microM MC, MMC, PMA or EMT in MDCK cells, revealing Hg concentrations of 42.8 +/- 2.5 ng/mg protein for MC, 596.9 +/- 87.8 ng/mg protein for MMC, 269.8 +/- 75.7 ng/mg protein for PMA and of 115.9 +/- 25.2 ng/mg protein for EMT. Cytotoxicity was positively correlated with cellular uptake. The effect of the cellular GSH content on the toxicity of mercury was studied using the GSH synthesis inhibitor L-buthionine sulfoximine (BSO). In all cases an enhanced cytotoxicity was observed after BSO treatment. 2-Oxo-4-thiazolidine carboxylic acid (OTC) was used as a substrate for the GSH synthesis. Although OTC did not enhance the GSH content, the cytotoxicity of MC, MN and MA decreased significantly, no changes were observed for the other mercurials.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytotoxicity of mercury compounds in LLC-PK1, MDCK and human proximal tubular cells. 772 29

While cancer drug resistance has been extensively studied in cell culture, little is known about more clinically relevant in vivo resistance. The in vivo resistance of a murine mammary carcinoma EMT-6 to alkylating agents was demonstrated in the present study to be associated with multiple biochemical changes. These included an up to 1.5-fold increase in activity of phase II drug metabolizing enzymes (DMEs), such as glutathione (GSH), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPX) and aldehyde dehydrogenase (ALDH), and an up to 88% decrease of phase I DME activity [7-ethoxycumarin O-deethylase (ECOD), P450 reductase (PR)] in the resistant tumors compared with the parental tumor. Transplant of either parental or resistant tumors to mice was accompanied by a decrease of both phase I and phase II DME activity in the livers of female Balb/C mice compared with the non-tumor mice. Moreover, at the protein level, while cytochrome P450 (CYP) IIB1/2 in the liver of mouse bearing both the sensitive and the resistant tumor was significantly diminished compared to that in the liver of non-tumor control mouse in Western analysis, there was actually an increase of this protein in the liver of the host bearing either of the two resistant tumors compared to that of the sensitive tumor-bearing animal. Although this in vivo resistance phenotype is not expressed in cell culture, the profile of most of the enzyme changes in the resistant tumors remained similar in in vitro culture of the isolated tumor cells. Collectively, these results demonstrate that this in vivo alkylating agent resistance is associated with multiple changes of both phase I and phase II DMEs in the resistant tumors, and some of these, such as CYP IIB1/2 protein are further altered in the resistant tumor-bearing mouse liver, suggesting a potential role of systemic factors in this resistance phenotype.
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PMID:Biochemical characterization of in vivo alkylating agent resistance of a murine EMT-6 mammary carcinoma. Implication for systemic involvement in the resistance phenotype. 992 73

Nitric oxide (NO) is a potential contributor to neurotoxicity following overactivation of N-methyl-D-aspartate (NMDA) receptors. In this work we investigated the effect of Nomega-nitro-L-arginine (L-NARG 25, 50, or 100 microM), a selective inhibitor of nitric oxide synthase (NOS) -the synthetic enzyme of NO- on quinolinic acid (QUIN 100 microM)-induced neurotoxicity (measured as lactate dehydrogenase (LDH) leakage) in rat striatal slices. Oxidative stress was also measured both as lipid peroxidation and as the levels of reduced (GSH) and oxidized (GSSG) glutathione, in an effort to elucidate a possible participation of NO in the toxic mechanisms involved in NMDA receptor-mediated neuronal injury. The action of L-arginine (L-ARG 100 or 200 microM), a well-known NO precursor, was also tested on QUIN-induced neurotoxicity and oxidative stress. Results showed that QUIN produced significant changes in both cell damage (177%) and oxidative injury (203% in lipid peroxidation, 68% in GSH, and 123% in GSSG) as compared to control values. All these effects were antagonized by adding L-NARG to the incubation media, whereas L-ARG alone, or in combination with QUIN, significantly enhanced both lipid peroxidation and LDH leakage. Moreover, the protective effects of L-NARG on QUIN-induced lipid peroxidation were reversed by addition of an excess of L-ARG to the media. These findings indicate that NO is probably mediating the mechanism of neurotoxicity produced by QUIN, which may be of potential value to explain the molecular basis of neurodegenerative processes linked to QUIN-mediated NMDA receptor overactivation.
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PMID:Nomega-nitro-L-arginine, a nitric oxide synthase inhibitor, antagonizes quinolinic acid-induced neurotoxicity and oxidative stress in rat striatal slices. 1040 23

To evaluate the oxidative stress-related parameters and to determine their order of appearance in the brain aging process, radionuclide experiments were carried out on male DBF1 mice at 3, 12, 24 and 30 months of age. The content of nonprotein sulfhydryl compounds, mainly glutathione, was estimated with technetium-99m meso-hexamethyl propyleneamine oxime ([99mTc]meso-HMPAO) tissue sampling. Glucose transport and metabolism was examined with [1-14C]2-deoxy-D-glucose (2-DG) tissue sampling. Mitochondrial electron transport function was estimated with [15O]O2 gas-tissue ARG. [99mTc]Meso-HMPAO uptake in brain expressed as standardized uptake value (SUV), (radioactivity in brain tissue/tissue weight)/(total administered radioactivity/body weight), reached maximum at 12 months of age and decreased at 24 and 30 months of age in every region examined. The pattern of 2-DG, expressed as SUV, showed a tendency to increase rather than decrease with aging. [15O]O2 fixation in brain slices remained constant until 24 months, while it decreased significantly at 30 months of age. The results suggested the possibility of using imaging techniques in vivo for longitudinal evaluation of the aging process and indicated reduction of nonprotein sulfhydryl compounds including GSH at the early stages of aging may also accelerate the dysfunction of mitochondrial electron transport and neurodegeneration.
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PMID:Age-related changes of glutathione content, glucose transport and metabolism, and mitochondrial electron transfer function in mouse brain. 1118 61

Interaction between neutrophils and endothelial cells is one of the first steps in the functional response of polymorphonuclear neutrophils (PMN), and is necessary for their migration toward damaged tissues. PMN activation, leading to their adhesion to and migration between endothelial cells, is part of a complex phenomenon that can be altered in pathological situations such as the ischemia-reperfusion syndrome, in which large numbers of PMN are recruited to the tissue and release reactive oxygen species (ROS) near the vessel wall. ROS have been implicated in the pathogenesis of various inflammatory diseases. The increased adhesion of PMN to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of a tyrosine kinase focal adhesion kinase (p125FAK) and several cytoskeleton proteins, including paxillin and p130 cas. We examined the role of glutathione (GSH) in the regulation of this adhesion phenomenon and in the increased tyrosine phosphorylation induced by ROS. For this purpose we used anethole dithiolthione (ADT), which increases the glutathione synthesis by activating gamma-glutamyl-cysteine synthetase. We found that ADT reduced both PMN adhesion to ROS-stimulated human umbilical vein endothelial cells (HUVEC) and tyrosine phosphorylation of p125FAK and paxillin. ADT increased redox status by increasing intracellular GSH content in oxidized cells. These results show that GSH can reverse the effect of oxidation on tyrosine kinase activation and phosphorylation, and thus plays an important role in cell signaling. They also confirm the antioxidant activity of ADT.
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PMID:Anethole dithiolethione regulates oxidant-induced tyrosine kinase activation in endothelial cells. 1121 83

A speciation technique for arsenic has been developed using an anion-exchange high-performance liquid chromatography/inductively coupled argon plasma mass spectrometer (HPLC/ICP MS). Under optimized conditions, eight arsenic species [arsenocholine, arsenobetaine, dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), arsenite (As(III)), and arsenate (As(V))] can be separated with isocratic elution within 10 min. The detection limit of arsenic compounds was 0.14-0.33 microg/L. To validate the method, Standard Reference Material in freeze-dried urine, SRM-2670, containing both normal and elevated levels of arsenic was analyzed. The method was applied to determine arsenic species in urine samples from three arsenic-affected districts of West Bengal, India. Both DMA(III) and MMA(III) were detected directly (i.e., without any prechemical treatment) for the first time in the urine of some humans exposed to inorganic arsenic through their drinking water. Of 428 subjects, MMA(III) was found in 48% and DMA(III) in 72%. Our results indicate the following. (1) Since MMA(III) and DMA(III) are more toxic than inorganic arsenic, it is essential to re-evaluate the hypothesis that methylation is the detoxification pathway for inorganic arsenic. (2) Since MMA(V) reductase with glutathione (GSH) is responsible for conversion of MMA(V) to MMA(III) in vivo, is DMA(V) reductase with GSH responsible for conversion of DMA(V) to DMA(III) in vivo? (3) Since DMA(III) forms iron-dependent reactive oxygen species (ROS) which causes DNA damage in vivo, DMA(III) may be responsible for arsenic carcinogenesis in human.
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PMID:Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal, India. 1130 25


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