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Kinds of Raw Terms modified by Raw Selected AbstractsSuppression of inflammatory responses by celastrol, a quinone methide triterpenoid isolated from Celastrus regeliiEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2009D. H. Kim Abstract Background, Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, exhibits various biological properties, including chemopreventive, antioxidant and neuroprotective effects. In this study, we showed that celastrol inhibits inflammatory reactions in macrophages and protects mice from skin inflammation. Materials and methods, Anti-inflammatory effects of celastrol (0,1 ,M) were examined in lipopolysaccharide (LPS)-stimulated RAW 264·7 macrophages. To investigate the effects of celastrol (0,50 ,g per mice) in vivo, activation of myeloperoxidase (MPO) and histological assessment were examined in the 12- O -tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear oedema model. Results, Our in vitro experiments showed that celastrol suppressed not only LPS-stimulated generation of nitric oxide and prostaglandin E2, but also expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW264·7 cells. Similarly, celastrol inhibited LPS-induced production of inflammatory cytokines, including tumour necrosis factor-, and interleukin-6. In an animal model, celastrol protected mice from TPA-induced ear oedema, possibly by inhibiting MPO activity and production of inflammatory cytokines. Conclusions, Our data suggest that celastrol inhibits the production of inflammatory mediators and is a potential target for the treatment of various inflammatory diseases. [source] Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: Involvement of ERK1/2, NF-,B and JAK/STAT pathwaysEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Susanta Kar Abstract Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-, in inducing ERK1/2 phosphorylation and NF-,B DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-,B or ERK1/2 pathway blocked the cystatin plus IFN-,-inducible NF-,B activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-,B-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-,-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-,B, including nuclear translocation of NF-,B subunits, I,B phosphorylation and I,B kinase, are all remarkably enhanced in Leishmania -infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general. [source] Bionanotechnology: Enhancement of Aggregation-Induced Emission in Dye-Encapsulating Polymeric Micelles for Bioimaging (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 9 2010Mater. Amphiphilic block copolymers can form polymer micelles for delivering hydrophobic fluorescent probes with aggregation-induced emission properties, as presented by A. K.-Y. Jen et al. on page 1413. By itself, 1,1,2,3,4,5-hexaphenylsilole (HPS) exhibits dramatically enhanced blue-green fluorescent emission efficiencies when encapsulated within the hydrophobic core of a polymeric micelle. When HPS is co-encapsulated with bis(4-(N -(1-naphthyl) phenylamino)-phenyl)fumaronitrile, effective orange-red fluorescence resonance energy transfer can be demonstrated within live RAW 264.7 cells. Illustration provided by Brent Polishak. [source] Enhancement of Aggregation-Induced Emission in Dye-Encapsulating Polymeric Micelles for BioimagingADVANCED FUNCTIONAL MATERIALS, Issue 9 2010Wen-Chung Wu Abstract Three amphiphilic block copolymers are employed to form polymeric micelles and function as nanocarriers to disperse hydrophobic aggregation-induced emission (AIE) dyes, 1,1,2,3,4,5-hexaphenylsilole (HPS) and/or bis(4-(N -(1-naphthyl) phenylamino)-phenyl)fumaronitrile (NPAFN), into aqueous solution for biological studies. Compared to their virtually non-emissive properties in organic solutions, the fluorescence intensity of these AIE dyes has increased significantly due to the spatial confinement that restricts intramolecular rotation of these dyes and their better compatibility in the hydrophobic core of polymeric micelles. The effect of the chemical structure of micelle cores on the photophysical properties of AIE dyes are investigated, and the fluorescence resonance energy transfer (FRET) from the green-emitting donor (HPS) to the red-emitting acceptor (NPAFN) is explored by co-encapsulating this FRET pair in the same micelle core. The highest fluorescence quantum yield (,62%) could be achieved by encapsulating HPS aggregates in the micelles. Efficient energy transfer (>99%) and high amplification of emission (as high as 8 times) from the NPAFN acceptor could also be achieved by spatially confining the HPS/NPAFN FRET pair in the hydrophobic core of polymeric micelles. These micelles could be successfully internalized into the RAW 264.7 cells to demonstrate high-quality fluorescent images and cell viability due to improved quantum yield and reduced cytotoxicity. [source] Bone morphogenetic protein-6 induces the expression of inducible nitric oxide synthase in macrophagesIMMUNOLOGY, Issue 1pt2 2009Seok J. Kwon Summary Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-, (TGF-,) superfamily. In the present study, we investigated the effect of BMPs on the production of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line, RAW 264.7, and in mouse peritoneal macrophages. Among the BMPs, only BMP-6 induced iNOS expression in a time-dependent and dose-dependent manner in both cell types. Induction of iNOS was inhibited by both cycloheximide and actinomycin D, indicating that the induction of iNOS expression by BMP-6 requires new protein synthesis. Mechanistic studies revealed that the BMP-6-induced iNOS expression requires both Smads and nuclear factor-kappa B (NF-,B) signalling pathways. Furthermore, induction of interleukin-1, (IL-1,) was necessary for iNOS induction by BMP-6. These observations suggest that BMP-6 stimulates macrophages to produce iNOS through IL-1, via Smad and NF-,B signalling pathways and that BMP-6 may be an important regulator of macrophages. [source] Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophagesJOURNAL OF APPLIED TOXICOLOGY, Issue 1 2009Jonghoon Choi Abstract Photoluminescent silicon nanoparticles have a bright and stable fluorescence and are promising candidates for bio-imaging, cell staining and drug delivery. With increasing development of nanotechnology applications for biomedicine, an understanding of the potential toxicity of nanoparticles is needed to assess safety concerns for clinical applications. The objective of this study was to compare biological responses of silicon nanoparticles (SNs, 3 nm diameter) with silicon microparticles (SMs, ,100,3000 nm diameter) in cultured murine macrophages (RAW 264.7) using standard protocols for assessing cytotoxicity/cell viability and inflammatory responses developed for micron-sized particles. SNs and SMs were exposed to macrophages with and without addition of endotoxin lipopolysaccharide (LPS), a positive inducer of tumor necrosis factor-alpha (TNF- ,), interleukin 6 (IL-6), and nitric oxide (NO). Cytotoxicity was assayed using the dye exclusion and MTT assays. Cell supernatants were assayed for production TNF- ,, IL-6 and NO. SNs at concentrations ,20 µg ml,1 exhibited no cytotoxicity or inflammatory responses; however, SNs and SMs >20 and 200 µg ml,1, respectively, increased cytotoxicity compared with controls. SMs induced concentration-related increases in TNF- , and IL-6 production; in contrast, the production of these cytokines was shown to decrease with increasing concentrations of SNs. NO production was not induced by SNs or SMs alone. Fluorescence microscopy demonstrated that SNs were associated with the macrophages, either internalized or attached to cell membranes. In conclusion, evaluating differences in biological responses for nanoparticles compared with microparticles of the same material may help improve tests to assess biological responses of nanoparticles that may be used in biomedical applications. Copyright © 2008 John Wiley & Sons, Ltd. [source] Cholesterol-Sensing Receptors, Liver × Receptor , and ,, Have Novel and Distinct Roles in Osteoclast Differentiation and ActivationJOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2006Kirsten M Robertson Abstract The liver × receptor (,,,) is responsible for regulating cholesterol homeostasis in cells. However, our studies using the LXR,,/,, LXR,,/,, and LXR,,/,,,/, mice show that both LXR, and , are also important for bone turnover, mainly by regulating osteoclast differentiation/activity. Introduction: The liver × receptors (,,,) are primarily responsible for regulating cholesterol homeostasis within cells and the whole body. However, as recent studies show that the role for this receptor is expanding, we studied whether the LXRs could be implicated in bone homeostasis and development. Materials and Methods: pQCT was performed on both male and female LXR,,/,, LXR,,/,, LXR,,/,,,/,, and WT mice at 4 months and 1 year of age. Four-month-old female mice were additionally analyzed with reference to qPCR, immunohistochemistry, histomorphometry, transmission electron microscopy, and serum bone turnover markers. Results: At the mRNA level, LXR, was more highly expressed than LXR, in both whole long bones and differentiating osteoblast-like MC3T3-E1 and osteoclast-like RAW 264.7 cells. Four-month-old female LXR,,/, mice had a significant increase in BMD because of an increase in all cortical parameters. No difference was seen regarding trabecular BMD. Quantitative histomorphometry showed that these mice had significantly more endosteal osteoclasts in the cortical bone; however, these cells appeared less active than normal cells as suggested by a significant reduction in serum levels of cross-linked carboxyterminal telopeptides of type I collagen (CTX) and a reduction in bone TRACP activity. Conversely, the female LXR,,/, mice exhibited no change in BMD, presumably because a significant decline in the number of the trabecular osteoclasts was compensated for by an increase in the expression of the osteoclast markers cathepsin K and TRACP. These mice also had a significant decrease in serum CTX, suggesting decreased bone resorption; however, in addition presented with an increase in the expression of osteoblast associated genes, bone formation markers, and serum leptin levels. Conclusions: Our findings show that both LXRs influence cellular function within the bone, with LXR, having an impact on osteoclast activity, primarily in cortical bone, whereas LXR, modulates trabecular bone turnover. [source] Involvement of the JAK-STAT pathway and SOCS3 in the regulation of adiponectin-generated reactive oxygen species in murine macrophage RAW 264 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010Sumio Akifusa Abstract Adiponectin is a protein hormone produced by differentiating adipocytes and has been proposed to have anti-diabetic and immunosuppressive properties. We previously reported that the globular form of adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO), followed by caspase-dependent apoptotic cell death in RAW 264 cells. Here, we demonstrate that gAd-induced ROS generation and apoptosis were diminished by suppressor of cytokine signaling 3 (SOCS3). The phosphorylation level of signal transducer and activator of transcription (STAT) 3 detected by Western blotting was highest at 20,min in gAd-treated RAW 264 cells. This phosphorylation was inhibited by AG490, a specific inhibitor of janus-activator kinase (JAK). The gAd-induced ROS and NO were reduced by administration of AG490 and Jak-2-specific siRNA in RAW 264 cells. The gAd stimulation transiently induced SOCS3 mRNA expression and protein production. We examined SOCS3-overexpressing RAW 264 cells to investigate the role of the JAK-STAT pathway in gAd-induced ROS and NO generation. SOCS3 overexpression significantly reduced both ROS and NO generation. Additionally, gAd-induced caspase activation and apoptotic cell death were reduced in SOCS3 transfectants compared with vector control transfectants. These results suggest that the JAK-STAT pathway, which can be suppressed by SOCS3 expression, is involved in gAd-induced ROS and NO generation followed by apoptotic cell death. J. Cell. Biochem. 111: 597,606, 2010. © 2010 Wiley-Liss, Inc. [source] Possible involvement of protein kinase C in the induction of adipose differentiation-related protein by Sterol ester in RAW 264.7 macrophagesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Jin-Shan Chen Abstract The accumulation of lipid droplets in macrophages contributes to the formation of foam cells, an early event in atherosclerosis. It is, therefore, important to elucidate the mechanisms by which lipid droplets accumulate and are utilized. Sterol ester (SE)-laden RAW 264.7 macrophages accumulated lipid droplets in a time-dependent manner up to 16 h, which was enhanced by cotreatment with 0.1 ,M phorbol 12-myristate 13-acetate (PMA). Inhibition of protein kinase C (PKC) activity by cotreatment with 0.3 ,M calphostin C CAL for 16 h resulted in coalescence of small lipid droplets into large ones and increased accumulation of lipid droplets, although to a lesser extent than after PMA cotreatment. Immunostaining for adipose differentiation-related protein (ADRP) revealed a fluorescent rim at the surface of each medium to large lipid droplet. ADRP appearance correlated with lipid droplet accumulation and was regulated by PMA in a time-dependent manner. Induction of ADRP expression by PMA or CAL required SE, since ADRP levels in PMA- or CAL-treated non-SE-laden macrophages were comparable to those in untreated cells. Removal of SE from the incubation medium resulted in the concomitant dissolution of lipid droplets and down-regulation of ADRP. In conclusion, the above results suggest that ADRP may be an important protein in the regulation of lipid droplet metabolism in lipid-laden macrophages and that this regulation may be mediated by PKC activity. J. Cell. Biochem. 83: 187,199, 2001. © 2001 Wiley-Liss, Inc. [source] Regulation of osteoclastogenesis and RANK expression by TGF-,1JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Tao Yan Abstract Transforming growth factor-, (TGF-,) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-, on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-,B ligand (RANK-L) (50 ng/ml), TGF-,1 (0.01,5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-,1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-,1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-,1 resulted in a significant increase in the percentage of RANK+ RAW cells (P,<,0.05), as well as an increase in the fluorescence intensity per cell (P,<,0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-, directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression. J. Cell. Biochem. 83: 320,325, 2001. © 2001 Wiley-Liss, Inc. [source] Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophagesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2001Yen-Chou Chen Abstract Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N -nitro- L -arginine (NLA) or N -nitro- L -arginine methyl ester (L -NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L -arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L -NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L -NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L -arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes. J. Cell. Biochem. 82: 537,548, 2001. © 2001 Wiley-Liss, Inc. [source] DETERMINATION OF pH CHANGE KINETICS DURING DIFFERENT STAGES OF KASHAR CHEESE MANUFACTURING FROM RAW AND PASTEURIZED MILK WITH ADDITION OF THERMOPHILIC, MESOPHILIC AND MIXED THERMOPHILIC CULTUREJOURNAL OF FOOD PROCESS ENGINEERING, Issue 4 2008AHMET FERIT ATASOY ABSTRACT The pH change kinetics during Kashar cheese production from raw and pasteurized milk with addition of thermophilic, mesophilic and mixed thermophilic-mesophilic lactic acid bacteria were evaluated. The kinetics of pH change were determined during milk ripening, cooking/holding and pressing/fermentation phases of Kashar cheese. The pH decreased logarithmically, nonlinearly, with time in the milk ripening period, and reduced linearly with time in the cooking/holding and pressing/fermentation stages. Pasteurization of milk retarded the rate of change in pH during the three periods. The highest rate of pH change was determined in the addition of thermophilic culture, followed by mixed thermophilic-mesophilic and then mesophilic ones during milk ripening. The pH change characteristics of cheese made with thermophilic starter were similar to the cheese made with mixed thermophilic-mesophilic culture, but different from mesophilic lactic acid bacteria during cooking/holding and pressing/fermentation stages. PRACTICAL APPLICATIONS One of the important factors in the control of cheese quality is the extent of acid production in the vat. Acid development at a desired rate is important during cheese making. The progress of acidification is monitored by pH change in the industrial Kashar cheese production. Three main stages have been recognized with respect to pH change: milk ripening, cooking/holding and pressing/fermentation. This study evaluated and compared the pH change kinetics during various stages of Kashar cheese making using raw, pasteurized milk with the addition of thermophilic, mesophilic and mixed thermophilic culture. This work may help in the comparison of raw and pasteurized milk, and in the selection of appropriate starter culture for Kashar cheese production. [source] RHEOLOGY AND TEXTURE OF COMMERCIAL QUESO FRESCO CHEESES MADE FROM RAW AND PASTEURIZED MILKJOURNAL OF FOOD QUALITY, Issue 2010MICHAEL H. TUNICK ABSTRACT Queso Frescos made in Mexico from raw milk (RM) were compared with cheeses made in Mexico and the U.S.A. from pasteurized milk (PM) to determine textural and rheological differences. RM cheese, considered the ideal Queso Fresco, contained more moisture than PM cheeses, displayed higher cohesiveness and shear strain and exhibited lower hardness and shear stress. The U.S.-made cheeses were harder and more brittle and crumbly than the Mexican cheeses. The shear stress decreased as fat content increased in all samples, and the shear strain decreased as the pH increased in the Mexican cheeses. An understanding of the differences between the RM and PM versions should allow cheese makers to adjust manufacturing procedures so that PM Queso Fresco cheeses will meet consumer expectations. PRACTICAL APPLICATIONS Hispanic cheeses such as Queso Fresco are gaining in popularity in the U.S.A. The milk for making Queso Fresco in the U.S.A. must be pasteurized, and the resulting cheese is perceived by some consumers to be inferior in flavor and texture to the traditional variety made from raw milk. Queso Fresco samples made in Mexico from both kinds of milk and made in the U.S.A. from pasteurized milk were analyzed to see if there were differences in texture, structure, and the ability to twist and break pieces of the cheese. The cheeses made in the U.S.A. contained less moisture and were too hard, crumbly, and brittle when compared to the raw milk cheeses. The differences observed should enable U.S. companies to make Queso Fresco with properties close to those of the raw milk variety. [source] BAKING PERFORMANCE AND CONSUMER ACCEPTABILITY OF RAW AND EXTRUDED COWPEA FLOUR BREADSJOURNAL OF FOOD QUALITY, Issue 5 2004K.H. MCWATTERS ABSTRACT Cowpea flour was used to partially replace wheat flour in yeast bread, using automatic household-type bread machines for mixing, proofing and baking. Loaves containing 15 or 30% extruded cowpea flour weighed more (683.4 g) than loaves from other treatments (641.1,652.6 g). The 100% wheat had the highest loaf volume (2.58 L) and the 30% extruded cowpea the lowest (1.64 L). Cowpea flour breads contained more protein (13.9,15.4%) than the 100% wheat (4.1% fat, 12.5% protein). Bread made with 15% extruded cowpea flour was not different (P < 0.05) from the all-wheat control in sensory quality and acceptability. Hedonic ratings for the control and 15% extruded cowpea flour ranged from 6.6 (like slightly) to 7.4 (like moderately) for all sensory attributes. The least liked samples contained either 30% raw or 30% extruded cowpea flour, receiving ratings for all attributes ranging from 4.8 (disliked slightly) to 6.2 (liked slightly). Overall, 15% extruded cowpea flour demonstrated successful bread making performance without compromising sensory quality. [source] Lycopene Inhibits LPS-Induced Proinflammatory Mediator Inducible Nitric Oxide Synthase in Mouse Macrophage CellsJOURNAL OF FOOD SCIENCE, Issue 1 2007Mohamed M. Rafi ABSTRACT:, Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 ,M) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene. [source] (-)-Epigallocatechin gallate induces apoptosis, via caspase activation, in osteoclasts differentiated from RAW 264.7 cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2007J.-H. Yun Background and Objective:, Alveolar bone resorption is a characteristic feature of periodontal diseases and involves removal of both the mineral and the organic constituents of the bone matrix, a process mainly carried out by multinucleated osteoclast cells. (-)-Epigallocatechin gallate, the main constituent of green tea polyphenols, has been reported to induce the apoptotic cell death of osteoclasts and to modulate caspase activation in various tumor cells. In the present study, we investigated the inhibitory effect of (-)-epigallocatechin gallate on osteoclast survival and examined if (-)-epigallocatechin gallate mediates osteoclast apoptosis via caspase activation. Material and Methods:, The effect of (-)-epigallocatechin gallate on osteoclast survival was examined by tartrate-resistant acid phosphatase (TRAP) staining in osteoclasts differentiated from RAW 264.7 cells. In addition, we evaluated the apoptosis of osteoclasts by (-)-epigallocatechin gallate using a DNA-fragmentation assay. Involvement of caspase in (-)-epigallocatechin gallate-mediated osteoclast apoptosis was evaluated by treatment with a general caspase inhibitor, Z-VAD-FMK. Moreover, the effect of (-)-epigallocatechin gallate on the activation of caspase-3 was assessed by a colorimetric activity assay and western blotting. Results:, (-)-Epigallocatechin gallate significantly inhibited, in a dose-dependent manner, the survival of osteoclasts differentiated from RAW 264.7 cells and induced the apoptosis of osteoclasts. Treatment with (-)-epigallocatechin gallate resulted in DNA fragmentation and induced the activation of caspase-3 in RAW 264.7 cell-derived osteoclasts. Additional treatment with Z-VAD-FMK suppressed these effects of (-)-epigallocatechin gallate. Conclusion:, From these findings, we could suggest that (-)-epigallocatechin gallate might prevent alveolar bone resorption by inhibiting osteoclast survival through the caspase-mediated apoptosis. [source] Bis-(3-hydroxyphenyl) diselenide inhibits LPS-stimulated iNOS and COX-2 expression in RAW 264.7 macrophage cells through the NF- kB inactivationJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2009Kyung-Min Shin Abstract Objectives Previously, we reported that diaryl diselenide compounds have strong inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophages. In this study, we investigated the molecular mechanisms underlying NO suppression and prostaglandin E2 (PGE2) production by diaryl diselenide compounds, bis-(2-hydroxyphenyl) diselenide (DSE-A), bis-(3-hydroxyphenyl) diselenide (DSE-B), bis-(4-hydroxyphenyl) diselenide (DSE-C), dipyridyl diselenide (DSE-D) and diphenyl diselenide (DSE-E). Methods The effect of these compounds on NO suppression and PGE2 production was investigated in RAW 264.7 macrophages. Key findings Our data indicate that of the above, DSE-B most potently inhibits NO and PGE2 production, and that it also significantly reduces the releases of tumour necrosis factor (TNF)-,, interleukin(IL)-1, and IL-6. Consistent with these observations, DSE-B also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), and the mRNA levels of iNOS, COX-2, TNF-,, IL-1, and IL-6. Furthermore, DSE-B inhibited LPS-induced nuclear factor-,B (NF-,B) activation, which was associated with the prevention of the inhibitor ,B-, (I,B-,) degradation and a subsequent reduction in nuclear p65 protein levels. Conclusions Taken together, our data suggest that the anti-inflammatory properties of DSE-B are due to reduction in the expression of iNOS, COX-2, TNF-,, IL-1, and IL-6 through the down-regulation of NF-,B binding activity. [source] Eutigoside C inhibits the production of inflammatory mediators (NO, PGE2, IL-6) by down-regulating NF-,B and MAP kinase activity in LPS-stimulated RAW 264.7 cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2008Hye-Ja Lee Eutigoside C, a compound isolated from the leaves of Eurya emarginata, is thought to be an active anti-inflammatory compound which operates through an unknown mechanism. In the present study we investigated the molecular mechanisms of eutigoside C activity in lipopolysacchardide (LPS)-stimulated murine macrophage RAW 264.7 cells. Treatment with eutigoside C inhibited LPS-stimulated production of nitric oxide (NO), prostaglandin E2 (PGE2) and interleukin-6 (IL-6). To further elucidate the mechanism of this inhibitory effect of eutigoside C, we studied LPS-induced nuclear factor (NF)-,B activation and mitogen-activated protein (MAP) kinase phosphorylation. Eutigoside C suppressed NF-,B DNA binding activity, interfering with nuclear translocation of NF-,B. Eutigoside C suppressed the phosphorylation of three MAP kinases (ERK1/2, JNK and p38). These results suggest that eutigoside C inhibits the production of inflammatory mediators (NO, PGE2 and interleukin-6) by suppressing the activation and translocation of NF-,B and the phosphorylation of MAP kinases (ERK1/2, JNK and p38) in LPS-stimulated murine macrophage RAW 264.7 cells. [source] Anti-inflammatory activity of the synthetic C-C biflavonoidsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2006Haeil Park To find anti-inflammatory agents based on plant constituents, the effects of six synthetic C-C biflavonoids connecting with different positions of C-C bond between flavone monomers (a: 4,-4,, b: 4,-3,, c: 4,-6, d: 3,-6, e: 6-6, f: 4,-3) were examined on PGE2 and nitric oxide (NO) production from lipopolysaccharide (LPS)-treated macrophages, RAW 264.7. Among the compounds tested, the biflavonoids d, e, and f showed a considerable inhibition of cyclooxygenase-2 (COX-2)-mediated PGE2 production at concentrations up to 50 ,M, while the derivative c exerted cytotoxic effects on RAW cells. Especially, the biflavonoid e possessed the most potent inhibitory activity of PGE2 production with an IC50 of 3.7 ,M, compared with an IC50 of 8.2,20.7 ,M by ginkgetin (natural biflavonoid). Western blot and reverse transcriptase-polymerase chain reaction analyses have shown that the inhibition of PGE2 production by these synthetic derivatives was mediated at least in part by COX-2 inhibition, but not by COX-2 down-regulation. Meanwhile, these synthetic biflavonoids did not considerably inhibit inducible nitric oxide synthase-mediated NO production at concentrations up to 50 ,M. When intraperitoneally administered, the biflavonoid e showed a significant anti-inflammatory activity (22.2% inhibition) against rat carrageenan-induced paw oedema at 5 mg kg,1. The biflavonoid e may be used as a synthetic lead for developing new anti-inflammatory agents. [source] Induction of apoptosis of RAW 264.7 cells by the cytostatic macrolide apicularen AJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2003JangJa Hong ABSTRACT In RAW 264.7 cells, a mouse leukaemic monocyte cell line, apicularen A decreased cell growth and survival as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in a concentration-dependent manner at 10,1000 nM. Apicularen B, an N -acetyl-glucosamine glycoside of apicularen A, was 10,100-fold less effective than apicularen A. Apicularen A induced a DNA ladder, an increase in the percentage of sub-G1 cells and annexin V-binding cells, and promoted the activation of caspase as revealed by the cleavage of poly(ADP-ribose) polymerase, indicating that apicularen A induced apoptosis in RAW 264.7 cells. In addition, apicularen A phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The p44/42 MAPK inhibitor PD98059 rescued the cells from apicularen-induced decrease in cell growth and survival as determined by the MTT assay, while the p38 MAPK inhibitor SB203580 augmented the effect of apicularen A. This suggested the activation of p44/42 MAPK to be pro-apoptotic and the activation of p38 MAPK anti-apoptotic in apicularen A-treated RAW 264.7 cells. [source] Participation of various kinases in staurosporine-induced apoptosis of RAW 264.7 cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2002Kouya Yamaki Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage-like cell line, as determined by DNA fragmentation, the increase of annexin V-stained cells, and the cleavage of poly(ADP- ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub-G1 cells revealed that the DNA fragmentation occurred in a time- and concentration-dependent manner at 0.021,2.1 ,m of staurosporine. Staurosporine induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal-regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002 potentiated the staurosporine-induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H-89 potentiated the staurosporine-induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro-31,8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine-induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and P13K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation. [source] HDL2 of Heavy Alcohol Drinkers Enhances Cholesterol Efflux From Raw Macrophages via Phospholipid-Rich HDL2b ParticlesALCOHOLISM, Issue 6 2008Sanna M. Mäkelä Background:, Alcohol consumption is associated with increased serum high density lipoprotein (HDL) cholesterol levels and a decreased risk for the development of atherosclerosis. However, the effects of heavy alcohol intake on reverse cholesterol transport, one of the key anti-atherogenic processes related to HDL, are poorly known. Methods:, The ability of total HDL as well as HDL2 and HDL3 subclasses to promote cholesterol efflux from 3H-cholesterol-labeled RAW 264.7 macrophages was studied among 6 heavy alcohol drinkers and 6 controls. Distribution of HDL subclasses was analyzed by 4 to 30% native gradient gels. Serum phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) activities were analyzed among several other biochemical measures. Results:, Cholesterol efflux to HDL2 of heavy drinkers was 22% (p = 0.025) higher relative to controls. The increase in HDL2 phospholipids, with a concomitant 2-fold (p = 0.055) increase in large HDL2b particles, was associated with enhanced cholesterol efflux to HDL2. Interestingly, the cholesterol efflux to HDL3 did not differ between the 2 study groups. These findings may be partially explained by a decreased CETP activity (,26%, p = 0.037) and an increased PLTP activity (39%, p = 0.045) in heavy drinkers. Conclusions:, The increased cholesterol efflux potential of HDL2 is most likely an anti-atherogenic feature linked to heavy alcohol consumption. The cholesterol efflux and HDL phospholipids also associated strongly within the whole study group (rs = 0.910, p , 0.01) suggesting a common pathway of enhanced cholesterol efflux via enlarged phospholipid-rich HDL particles. [source] Differences in Children's Recess Physical Activity: Recess Activity of the Week InterventionJOURNAL OF SCHOOL HEALTH, Issue 9 2010Megan Babkes Stellino EdD BACKGROUND: The increased prevalence in recent childhood obesity rates raises concern about youth health and the role that lack of physical activity plays in this trend. A focus on how children today choose to spend their discretionary time is one approach that may yield ideas for how to reduce childhood obesity. The purpose of the present study was to examine whether 3 separate recess activities of the week (RAWs) would make a difference in children's discretionary time physical activity levels. METHODS: Children (N = 65: 30 boys, 35 girls; 32 first and second graders; 33 third and fourth graders; 45 healthy body mass index [BMI], 20 overweight BMI) at 1 Midwest elementary school wore pedometers for each 15-minute morning recess period for 4 weeks. Following 1 no RAW (#1), a new RAW was introduced each subsequent week: #2, circuit course; #3, obstacle course; and #4, Frisbees. RESULTS: Repeated measures factorial analysis of variance results revealed that children were significantly more active during the no RAW and circuit course week than the Frisbee week. Males were significantly more physically active than females during the obstacle course week. Older children were significantly more active during the Frisbee week than younger children. Healthy BMI children were significantly more physically active during the circuit course week than children in the overweight/obese BMI category. CONCLUSIONS: Results imply that it is important for schools to consider demographic factors in the creation of recess opportunities to increase physical activity. [source] Effect of pistachio oil on gene expression of IFN-induced protein with tetratricopeptide repeats 2: A biomarker of inflammatory responseMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue S1 2010Jun Zhang Abstract When incorporated into the diet, pistachios have a beneficial effect on lipid and lipoprotein profiles. However, little is known about potential anti-inflammatory properties. This study was conducted to determine whether pistachio oil and an organic extract from pistachio oil extract (PE) regulated expression of inflammation-related genes. A mouse macrophage cell line (RAW 264.7 cells) was treated with pistachio oil and gene expression microarray analyses were performed. Pistachio oil significantly affected genes involved in immune response, defense response to bacteria, and gene silencing, of which INF-induced protein with tetratricopeptide repeats 2 (Ifit-2) was the most dramatically reduced. PE reduced the LPS-induced Ifit-2 by 78% and the bioactive molecules contained in PE, linoleic acid, and ,-sitosterol recapitulated this inhibition. Promoter analysis identified two adjacent IFN-stimulated response elements, which lie between ,110 and ,85bp of the 5,-flanking region of the Ifit-2 promoter, as being responsive to LPS activation and inhibition by PE. Our results indicate that pistachio oil and bioactive molecules present therein decrease Ifit-2 expressions, and due to the sensitivity of this effect, this gene is a potential biomarker for monitoring diet-induced changes in inflammation. [source] Protein hydrolysates from ,-conglycinin enriched soybean genotypes inhibit lipid accumulation and inflammation in vitroMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 8 2009Cristina Martinez-Villaluenga Abstract Obesity is a worldwide health concern and a well recognized predictor of premature mortality associated with a state of chronic inflammation. The objective was to evaluate the effect of soy protein hydrolysates (SPH) produced from different soybean genotypes by alcalase (SAH) or simulated gastrointestinal digestion (SGIH) on lipid accumulation in 3T3-L1 adipocytes. The anti-inflammatory effect of SPH produced by alcalase on LPS-induced macrophage RAW 264.7 cell line was also investigated. SAH (100 ,M) derived from soybean enriched in ,-conglycinin (BC) (up to 47% total protein) decreased lipid accumulation (33,37% inhibition) through downregulation of gene expression of lipoprotein lipase (LPL) and fatty acid synthase (FAS). SGIH (100 ,M) inhibited lipid accumulation to a lesser extent (8,14% inhibition) through inhibition of LPL gene expression. SAH (5 ,M) decreased the production of nitric oxide (NO) (18,35%) and prostaglandin E2 (PGE2) (47,71%) and the expression of inducible nitric oxide synthase (iNOS) (31,53%) and cycloxygenase-2 (COX-2) (30,52%). This is the first investigation showing that soy hydrolysates inhibit LPS-induced iNOS/NO and COX-2/PGE2 pathways in macrophages. Soybeans enriched in BCs can provide hydrolysates that limit fat accumulation in fat cells and inflammatory pathways in vitro and therefore warrant further studies as a healthful food. [source] 6-Shogaol suppressed lipopolysaccharide-induced up-expression of iNOS and COX-2 in murine macrophagesMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 12 2008Min-Hsiung Pan Abstract Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, antinausea, anti-inflammatory, and anticarcinogenic properties. In this study, we investigated the inhibitory effects of 6-shogaol and a related compound, 6-gingerol, on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS. Western blotting and reverse transcription-PCR analyses demonstrated that 6-shogaol significantly blocked protein and mRNA expression of inducible NOS (iNOS) and COX-2 in LPS-induced macrophages. The in vivo anti-inflammatory activity was evaluated by a topical 12- O -tetradecanoylphorbol 13-acetate (TPA) application to mouse skin. When applied topically onto the shaven backs of mice prior to TPA, 6-shogaol markedly inhibited the expression of iNOS and COX-2 proteins. Treatment with 6-shogaol resulted in the reduction of LPS-induced nuclear translocation of nuclear factor-,B (NF,B) subunit and the dependent transcriptional activity of NF,B by blocking phosphorylation of inhibitor ,B (I,B), and p65 and subsequent degradation of I,B,. Transient transfection experiments using NF,B reporter constructs indicated that 6-shogaol inhibits the transcriptional activity of NF,B in LPS-stimulated mouse macrophages. We found that 6-shogaol also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2, but not p38 mitogen-activated protein kinase (MAPK). Taken together, these results show that 6-shogaol downregulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NF,B by interfering with the activation PI3K/Akt/I,B kinases IKK and MAPK. [source] Expression of receptor activator of nuclear factor-,B ligand by B cells in response to oral bacteriaMOLECULAR ORAL MICROBIOLOGY, Issue 3 2009X. Han Introduction:, We investigated receptor activator of nuclear factor-,B ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Methods:, Expression of messenger RNA transcripts (tumor necrosis factor-,, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription,polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. Results:, The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans -immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans -immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. Conclusion:, This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease. [source] Pre-junctional ,2 -adrenoceptors modulation of the nitrergic transmission in the pig urinary bladder neck,NEUROUROLOGY AND URODYNAMICS, Issue 4 2007Medardo Hernández Abstract Aims To investigate the nitric oxide (NO)-mediated nerve relaxation and its possible modulation by pre-junctional ,2 -adrenoceptors in the pig urinary bladder neck. Methods Urothelium-denuded bladder neck strips were dissected, and mounted in isolated organ baths containing a physiological saline solution (PSS) at 37°C and continuously gassed with 5% CO2 and 95% O2, for isometric force recording. The relaxations to transmural nerve stimulation (electrical field stimulation [EFS]) or exogenously applied NO were carried out on strips pre-contracted with 1 µM phenylephrine (PhE) and treated with guanethidine (10 µM) and atropine (0.1 µM), to block noradrenergic neurotransmission and muscarinic receptors, respectively. Results EFS (0.2,1 Hz, 1 msec duration, 20 sec trains, current output adjusted to 75 mA) evoked frequency-dependent relaxations which were abolished by the neuronal voltage-activated Na+ channel blocker tetrodotoxin (TTX, 1 µM). These responses were potently reduced by the nitric oxide synthase (NOS) inhibitor NG -nitro- L -arginine (L-NOARG, 30 µM) and further reversed by the NO synthesis substrate L -arginine (L-ARG, 3 mM). The ,2 -adrenoceptor agonist BHT-920 (2 µM) reduced the electrically evoked relaxations, its effectiveness being higher on the responses induced by low frequency stimulation. BHT-920-elicited reductions were fully reversed by the ,2 -adrenoceptor antagonist rauwolscine (RAW, 1 µM). Exogenous NO (1 µM,1 mM) induced concentration-dependent relaxations which were not modified by BHT-920, thus eliminating a possible post-junctional modulation. Conclusions These results indicate that NO is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission in the pig urinary bladder neck, the release of NO from intramural nerves being modulated by pre-junctional ,2 -adrenoceptor stimulation. Neurourol. Urodynam. 26:578,583, 2007. © 2007 Wiley-Liss, Inc. [source] Suppressive effect of inducible nitric oxide synthase (iNOS) expression by the methanol extract of Actinodaphne lancifoliaPHYTOTHERAPY RESEARCH, Issue 10 2004Youngleem Kim Abstract Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has played a crucial role in various pathophysiological processes including in,ammation and carcinogenesis. Therefore, the inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-in,ammatory and cancer chemopreventive agents. In our continuous search for iNOS inhibitors from natural products we have evaluated indigenous Korean plant extracts using an assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells. As a result, the methanolic stem extract of Actinodaphne lancifolia showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 2.5 µg/ml). Additional study demonstrated that the extract of Actinodaphne lancifolia signi,cantly suppressed the iNOS protein and gene expression in a dose-dependent manner. These results suggest that Actinodaphne lancifolia could be a potential candidate for developing an iNOS inhibitor from natural products. Further elucidation of active principles for development of new cancer chemopreventive and/or anti-in,ammatory agents could be warranted. Copyright © 2004 John Wiley & Sons, Ltd. [source] Butanolides from Machilus thunbergii and their inhibitory activity on nitric oxide synthesis in activated macrophagesPHYTOTHERAPY RESEARCH, Issue 4 2003Nam Yi Kim Abstract In activated macrophages the inducible form of nitric oxide synthase (iNOS) generates high amounts of the toxic mediator, nitric oxide (NO), that contributes to the circulatory failure associated with septic shock. Three butanolides were isolated from Machilus thunbergii as active principles which inhibit the production of NO in lipopolysaccharide-activated RAW 264.7 cells, and their structures were identi,ed as litsenolide A2 (1), B1 (2) and B2 (3). They showed dose-dependent inhibition of NO syntheses and the IC50s were 3.36, 3.70 and 6.19 µm, respectively. These new inhibitors of iNOS may have potential in the treatment of endotoxaemia and in,ammation accompanied by the overproduction of NO. Copyright © 2003 John Wiley & Sons, Ltd. 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