Forced expression of miR-143 and -145 in cardiomyocytes induces cardiomyopathy with a reductive redox shift.

BACKGROUND: Animal model studies show that reductive stress is involved in cardiomyopathy and myopathy, but the exact physiological relevance remains unknown. In addition, the microRNAs miR-143 and miR-145 have been shown to be upregulated in cardiac diseases, but the underlying mechanisms associated with these regulators have yet to be explored. METHODS: We developed transgenic mouse lines expressing exogenous miR-143 and miR-145 under the control of the alpha-myosin heavy chain (αMHC) promoter/enhancer. RESULTS: The two transgenic lines showed dilated cardiomyopathy-like characteristics and early lethality with markedly increased expression of miR-143. The expression of hexokinase 2 (HK2), a cardioprotective gene that is a target of miR-143, was strongly suppressed in the transgenic hearts, but the in vitro HK activity and adenosine triphosphate (ATP) content were comparable to those observed in wild-type mice. In addition, transgenic complementation of HK2 expression did not reduce mortality rates. Although HK2 is crucial for the pentose phosphate pathway (PPP) and glycolysis, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was unexpectedly higher in the hearts of transgenic mice. The expression of gamma-glutamylcysteine synthetase heavy subunit (γ-GCSc) and the in vitro activity of glutathione reductase (GR) were also higher, suggesting that the recycling of GSH and its de novo biosynthesis were augmented in transgenic hearts. Furthermore, the expression levels of glucose-6-phosphate dehydrogenase (G6PD, a rate-limiting enzyme for the PPP) and p62/SQSTM1 (a potent inducer of glycolysis and glutathione production) were elevated, while p62/SQSTM1 was upregulated at the mRNA level rather than as a result of autophagy inhibition. Consistent with this observation, nuclear factor erythroid-2 related factor 2 (Nrf2), Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) were activated, all of which are known to induce p62/SQSTM1 expression. CONCLUSIONS: Overexpression of miR-143 and miR-145 leads to a unique dilated cardiomyopathy phenotype with a reductive redox shift despite marked downregulation of HK2 expression. Reductive stress may be involved in a wider range of cardiomyopathies than previously thought.

REGγ ablation impedes dedifferentiation of anaplastic thyroid carcinoma and accentuates radio-therapeutic response by regulating the Smad7-TGF-ß pathway.

Anaplastic thyroid cancer (ATC) is the most aggressive human thyroid malignancy, characterized by dedifferentiation and resistance to radioiodine therapy. The underlying mechanisms regulating ATC dedifferentiation are largely unknown. Here, we show that REGγ, a noncanonical proteasome activator highly expressed in ATC, is an important regulator of differentiation in ATC cells. Ablation of REGγ significantly restored expression of thyroid-specific genes, enhanced iodine uptake, and improved the efficacy of 131I therapy in ATC xenograft models. Mechanistically, REGγ directly binds to the TGF-ß signaling antagonist Smad7 and promotes its degradation, leading to the activation of the TGF-ß signal pathway. With gain- and loss-of-function studies, we demonstrate that Smad7 is an important mediator for the REGγ function in ATC cell dedifferentiation, which is supported by expression profiles in human ATC tissues. It seems that REGγ impinges on repression of thyroid-specific genes and promotion of tumor malignancy in ATC cells by activating the TGF-ß signal pathway via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for allowing radioiodine therapy in anaplastic thyroid cancer patients with poor prognosis.

NADPH oxidase 4 function as a hydrogen peroxide sensor.

Nox4, a member of the NADPH- and oxygen-dependent oxidoreductases that generate reactive oxygen species (ROS), is widely expressed and constitutively active. To understand better its function and regulation, specific mutations in the Nox4 dehydrogenase (DH) domain were examined for effects on Nox4 oxidase activity. Transfection of His6-tagged Nox4 increased the amount of p22phox subunit in HEK293 cells, and a higher level of the heterodimer was observed in the nucleus-enriched fraction (NEF). NEF from Nox4-expressing HEK293 cells exhibited oxygen and H2O2 concentration-dependent NADPH oxidation rate. In Nox4-expressing cells, NEF and its partially purified form, the Nox4(P437H) mutant almost completely lost its oxidase activity, while Nox4(C546S), Nox4(C546L) or/and (C547L) had a significantly decreased rate of ROS production. The NADPH-dependent reduction of cytochrome c or cytochrome b5 by purified Nox4 DH domain was found regulated by the H2O2 concentration, and C546L and C547L mutants showed lower rates of the hemeprotein reduction. These conserved Cys residues in the DH domain respond to the cytosolic H2O2 concentration to regulate Nox4 activity.

Molecular hydrogen modulates gene expression via histone modification and induces the mitochondrial unfolded protein response.

Molecular hydrogen (H2) is a biologically active gas that is used medically to ameliorate various systemic pathological conditions. H2 also regulates gene expression involved in intracellular signaling and metabolic pathways. However, it is unclear whether H2 affects gene expression directly or through indirect effects as a consequence of health improvement. Therefore, we attempted to identify genes that exhibit similar changes in expression in response to H2 by employing DNA microarrays and gene set enrichment analysis to analyze RNA from liver and lung of rats and mice with or without dietary stress. We found that H2 activated the expression of sets of genes regulated by histone H3K27 methylation status. H2 also modified the expression of many genes regulated by a wide variety of signaling pathways. RT-qPCR showed that H2 up-regulated expression of Kcnc3, a H3K27-regulated gene, in organs such as liver, lung, kidney and brain. Furthermore, using immunohistochemistry and immunoblot analysis, we observed changes in H3K27 methylation status in the liver of mice and rats administered H2. Moreover, we showed that H2 simultaneously induced the H3K27 demethylase, Jmjd3, and mitochondrial unfolded protein response (mtUPR)-related genes. Recently, alteration of mitochondrial function was shown to cause induction of H3K27 demethylase or chromatin restructuring, followed by mtUPR activation through the alteration of H3K27 or H3K9 methylation states. Taken together, our study suggests that H2 can induce beneficial effects through mtUPR activation via epigenetic histone modification and by modification of gene expression.

Characterization of gene expression profiling of mouse tissues obtained during the postmortem interval.

Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval. We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3h after death, and 3) mice that were stored at 4°C for 18h after death, as this storage resembles the human autopsy process in Japan. The RNA quality of the brain and the liver was maintained up to 18h during the postmortem interval. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained due to high RNA integrity number (RIN) values, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. Taken together, our findings suggest that under typical autopsy conditions, gene expression profiles that reflect disease pathology can be examined by understanding comprehensive recognition of postmortem fluctuation of gene expression.

Prevention of allergic rhinitis by ginger and the molecular basis of immunosuppression by 6-gingerol through T cell inactivation.

The incidence of allergies has recently been increasing worldwide. Immunoglobulin E (IgE)-mediated hypersensitivity is central to the pathogenesis of asthma, hay fever and other allergic diseases. Ginger (Zingiber officinale Roscoe) and its extracts have been valued for their medical properties including antinausea, antiinflammation, antipyresis and analgesia properties. In this study, we investigated the antiallergic effects of ginger and 6-gingerol, a major compound of ginger, using a mouse allergy model and primary/cell line culture system. In mice with ovalbumin (OVA)-induced allergic rhinitis, oral administration of 2% ginger diet reduced the severity of sneezing and nasal rubbing by nasal sensitization of OVA and suppressed infiltration of mast cells in nasal mucosa and secretion of OVA-specific IgE in serum. 6-Gingerol inhibited the expression of not only Th2 cytokines but also Th1 cytokines in OVA-sensitized spleen cells. Accordingly, 6-gingerol suppressed in vitro differentiation of both Th1 cells and Th2 cells from naïve T cells. In addition, 6-gingerol suppressed both superantigen staphylococcal enterotoxin B (SEB)- and anti-CD3-induced T cell proliferation. 6-Gingerol also abrogated PMA plus ionomycin- and SEB-induced IL-2 production in T cells, suggesting that 6-gingerol affected T cell receptor-mediated signal transduction rather than the antigen-presentation process. Indeed, 6-gingerol inhibited the phosphorylation of MAP kinases, calcium release and nuclear localization of c-fos and NF-κB by PMA and ionomycin stimulation. Thus, our results demonstrate that 6-gingerol suppresses cytokine production for T cell activation and proliferation, thereby not causing B cell and mast cell activation and resulting in prevention or alleviation of allergic rhinitis symptoms.

Simultaneous oral and inhalational intake of molecular hydrogen additively suppresses signaling pathways in rodents.

Molecular hydrogen (H2) is an agent with potential applications in oxidative stress-related and/or inflammatory disorders. H2 is usually administered by inhaling H2-containing air (HCA) or by oral intake of H2-rich water (HRW). Despite mounting evidence, the molecular mechanism underlying the therapeutic effects and the optimal method of H2 administration remain unclear. Here, we investigated whether H2 affects signaling pathways and gene expression in a dosage- or dose regimen-dependent manner. We first examined the H2 concentrations in blood and organs after its administration and found that oral intake of HRW rapidly but transiently increased H2 concentrations in the liver and atrial blood, while H2 concentrations in arterial blood and the kidney were one-tenth of those in the liver and atrial blood. In contrast, inhalation of HCA increased H2 equally in both atrial and arterial blood. We next examined whether H2 alters gene expression in normal mouse livers using DNA microarray analysis after administration of HCA and HRW. Ingenuity Pathway Analysis revealed that H2 suppressed the expression of nuclear factor-kappa B (NF-κB)-regulated genes. Western blot analysis showed that H2 attenuated ERK, p38 MAPK, and NF-κB signaling in mouse livers. Finally, we evaluated whether the changes in gene expression were influenced by the route of H2 administration and found that the combination of both HRW and HCA had the most potent effects on signaling pathways and gene expression in systemic organs, suggesting that H2 may act not only through a dose-dependent mechanism but also through a complex molecular network.

Forced expression of miR-143 represses ERK5/c-Myc and p68/p72 signaling in concert with miR-145 in gut tumors of Apc(Min) mice.

Recently, miR-143 and miR-145 have been shown to belong to a subset of microRNAs whose expression is controlled by a complex of a tumor suppressor p53 and DEAD-box RNA helicase subunits p68/p72. While accumulating studies have acknowledged that both miRNAs function as tumor suppressors and are similarly regulated, evidence of their coordinated action against tumorigenesis has been poorly presented. Herein, we establish transgenic mice that express miR-143 under the control of the CAG regulatory unit. When crossbred with Apc(Min/+) mice, the development of tumors in the small intestines is significantly attenuated. In the transgenic small intestine tumors, the endogenous miR-145 is also enhanced and the expression of c-Myc and p68/p72, both of which have been reported to be pivotal for gut tumor development, is suppressed, corresponding to the downregulation of ERK5. We demonstrate that the combination of miR-143 and miR-145 inhibits the expression of c-Myc in human colon cancer cells, whereas miR-145 retards that of p72. Moreover, we show the possibilities that miR-145 modulates p72 expression through its 3' untranslated region and that c-Myc downregulation is involved in both p68 suppression and miR-145 induction. These findings suggest that forced expression of miR-143, probably interacting with endogenous miR-145, inhibits ERK5/c-Myc and p68/p72/ß-catenin signaling and hampers small intestine tumor development in Apc(Min/+) mice. This unique cascade, in turn, may prevent overproduction of a subset of tumor suppressive miRNAs by repressing their own modulators, p68/p72.

3ß-Hydroxysterol-Delta24 reductase plays an important role in long bone growth by protecting chondrocytes from reactive oxygen species.

Desmosterolosis is an autosomal recessive disease caused by mutations in the 3ß-hydroxysterol-Delta24 reductase (DHCR24) gene, with severe developmental anomalies including short limbs. We utilized DHCR24 knockout (KO) mice to study the underlying bone pathology. Because the KO mice died within a few hours after birth, we cultured metatarsal bones from newborn mice. The growth of bones from KO mice was significantly retarded after 1 week of culture. Absence of proliferating chondrocytes in the growth plate and abnormal hypertrophy of prehypertrophic chondrocytes were observed in the bones from KO mice. Hypertrophic differentiation was evidenced by higher expression of Indian hedgehog, alkaline phosphatase, and matrix metalloproteinase 13. Since elevated levels of reactive oxygen species (ROS) during chondrogenesis are known to inhibit proliferation and to initiate chondrocyte hypertrophy in the growth plate, and since DHCR24 acts as a potent ROS scavenger, we hypothesized that the abnormal chondrocyte proliferation and differentiation in KO mice were due to decreased ROS scavenging activity. Treatment with an antioxidant, N-acetyl cysteine, could correct the abnormalities observed in the bones from KO mice. Treatment of bones from wild-type mice with U18666A, a chemical inhibitor of DHCR24, resulted in short broad bones with a disrupted proliferating zone. Treatment of ATDC cells with hydrogen peroxide (H(2)O(2)) induced hypertrophic changes as evidenced by the expression of the marker genes specific for hypertrophic chondrocyte differentiation. H(2)O(2)-induced hypertrophic change was prevented by adenoviral delivery of DHCR24. Induction of chondrocyte differentiation in ATDC cells by insulin was associated with increased ROS production that was markedly enhanced by treatment of ATDC5 cells with DHCR24 siRNA. This is the first demonstration that DHCR24 plays an important role in long bone growth by protecting chondrocytes from ROS.

Mimosine-induced apoptosis in C6 glioma cells requires the release of mitochondria-derived reactive oxygen species and p38, JNK activation.

Growth-inhibitory effects of mimosine, a plant amino acid, on rat C6 glioma cells were analyzed. Mimosine markedly inhibited proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent manner. Mimosine-mediated apoptosis was accompanied by promoting reactive oxygen species (ROS) generation in mitochondria, and by decreased mitochondrial membrane potential (Δψ), and release of cytochrome c from mitochondria, followed by caspase 3 activation. Furthermore, mimosine increased the phosphorylation level of c-Jun-N-terminal protein kinase and p38, which was the downstream effect of ROS accumulation. Mimosine was confirmed to show profound effects on apoptosis of C6 glioma cells by ROS-regulated mitochondria pathway, and these results bear on the hypothesized potential for mimosine as promising agents in the treatment of malignant gliomas.

Assessment of factors influencing FDG uptake in non-small cell lung cancer on PET/CT by investigating histological differences in expression of glucose transporters 1 and 3 and tumour size.

PURPOSE: The objective of this study was to evaluate the major factors influencing on FDG uptake in non-small cell lung cancer (NSCLC) by investigating histological difference in the expression of glucose transporters 1 and 3 (Glut-1 and Glut-3) and tumour size. METHODS: This study enrolled 32 patients including 9 with squamous cell carcinoma (SCC) and 23 with adenocarcinoma (AC). The AC cases comprised 16 AC with mixed subtypes (AC-mixed) and 7 localized AC in situ (localized bronchioloalveolar carcinoma). Partial volume effect corrected maximum standardized uptake values (cSUVmax) and tumour size were obtained using FDG PET/CT. Glut-1 and Glut-3 expression were evaluated using five-point grading scales. RESULTS: Overexpression of Gluts was observed at high rates (88% for Glut-1 and 97% for Glut-3). They were mutually correlated. cSUVmax showed better correlation with size than with Gluts overexpression. AC and SCC showed a high positive expression rate for both Glut-1 and Glut-3, although the degree of overexpression was significantly higher in SCC than AC. In addition, localized AC in situ revealed a considerably higher positive expression rate and similar degrees of overexpression for both Glut-1 and Glut-3 compared with AC-mixed. By contrast, localized AC in situ alone was significantly smaller in both cSUVmax and size than either SCC or AC-mixed. No significant difference was found in cSUVmax or size between SCC and AC-mixed. CONCLUSIONS: The FDG uptake of NSCLC might be dependent on size rather than on overexpression of Glut-1 or Glut-3. Low FDG uptake in localized AC in situ might result from its small size rather than Glut overexpression.

REGgamma modulates p53 activity by regulating its cellular localization.

The proteasome activator REGγ mediates a shortcut for the destruction of intact mammalian proteins. The biological roles of REGγ and the underlying mechanisms are not fully understood. Here we provide evidence that REGγ regulates cellular distribution of p53 by facilitating its multiple monoubiquitylation and subsequent nuclear export and degradation. We also show that inhibition of p53 tetramerization by REGγ might further enhance cytoplasmic relocation of p53 and reduce active p53 in the nucleus. Furthermore, multiple monoubiquitylation of p53 enhances its physical interaction with HDM2 and probably facilitates subsequent polyubiquitylation of p53, suggesting that monoubiquitylation can act as a signal for p53 degradation. Depletion of REGγ sensitizes cells to stress-induced apoptosis, validating its crucial role in the control of apoptosis, probably through regulation of p53 function. Using a mouse xenograft model, we show that REGγ knockdown results in a significant reduction of tumor growth, suggesting an important role for REGγ in tumor development. Our study therefore demonstrates that REGγ-mediated inactivation of p53 is one of the mechanisms involved in cancer progression.

Requirement of DHCR24 for postnatal development of epidermis and hair follicles in mice.

Desmosterolosis is an autosomal recessive disorder with severe developmental anomalies due to mutations in the DHCR24 gene, encoding an enzyme to convert desmosterol to cholesterol. We reported that DHCR24 [knockout (KO)] mice were born with wrinkleless taut skin and with impaired development of epidermis. In this study, we investigated the postnatal development of epidermis and hair follicle in the skin of KO mice grafted to the nude mice. Skin graft was required since the KO mice die within few hours after birth. Forty days after the skin graft, epidermis from the KO mice revealed the characteristic phenotype observed at birth. Furthermore, the number of hair follicles in the skin graft from KO mice to the nude mice was significantly less and development was delayed than that from control. These findings implicate that DHCR24 plays important roles for normal development of epidermis and hair follicle even in postnatal life.

Iron-dependent oxidative inactivation with affinity cleavage of pyruvate kinase.

Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg2+, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg2+ implies the identical binding sites of Fe2+ and Mg2+, but the cleavage sites were discriminated from the cofactor Mg2+-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe2+ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg2+ concentrations are responsible for the control of cellular respiration and glycolysis.

Activation of NADPH oxidase 1 in tumour colon epithelial cells.

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.

Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities.

We developed a simple algorithm, i-Score (inhibitory-Score), to predict active siRNAs by applying a linear regression model to 2431 siRNAs. Our algorithm is exclusively comprised of nucleotide (nt) preferences at each position, and no other parameters are taken into account. Using a validation dataset comprised of 419 siRNAs, we found that the prediction accuracy of i-Score is as good as those of s-Biopredsi, ThermoComposition21 and DSIR, which employ a neural network model or more parameters in a linear regression model. Reynolds and Katoh also predict active siRNAs efficiently, but the numbers of siRNAs predicted to be active are less than one-eighth of that of i-Score. We additionally found that exclusion of thermostable siRNAs, whose whole stacking energy (DeltaG) is less than -34.6 kcal/mol, improves the prediction accuracy in i-Score, s-Biopredsi, ThermoComposition21 and DSIR. We also developed a universal target vector, pSELL, with which we can assay an siRNA activity of any sequence in either the sense or antisense direction. We assayed 86 siRNAs in HEK293 cells using pSELL, and validated applicability of i-Score and the whole DeltaG value in designing siRNAs.

Prooxidant action of rosmarinic acid: transition metal-dependent generation of reactive oxygen species.

Rosmarinic acid and its constituent caffeic acid produced reactive oxygen species in the presence of transition metals. Complex of rosmarinic acid or caffeic acid with iron inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the rosmarinic acid/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Direct spectrophotometric determination of hydrogen peroxide and superoxide anion confirmed the rosmarinic acid/iron-dependent production of reactive oxygen species. Treatment of DNA from plasmid pBR322 and calf thymus with rosmarinic acid plus copper caused strand scission and formed 8-hydroxy-2'-deoxyguanosine in DNA. Rosmarinic acid and caffeic acid showed a potent activity that reduces transition metals. These results suggest that transition metals reduced by rosmarinic acid can form superoxide radical by one electron reduction of oxygen molecule: superoxide radical in turn converts to hydrogen peroxide and hydroxyl radical causing the formation of DNA base adduct. Cytotoxicity of rosmarinic acid may be related to the prooxidant action resulting from metal-reducing activity.

Inhibitory action of eugenol compounds on the production of nitric oxide in RAW264.7 macrophages.

Effects of eugenol compounds on the production of nitric oxide (NO) in RAW264.7 macrophages were analyzed in relation to the anti-inflammatory action of these compounds. Eugenol and isoeugenol inhibited lipopolysaccharide (LPS)-dependent production of NO, which was due to the inhibition of protein synthesis of inducible nitric oxide synthase (iNOS). Isoeugenol showed the most effective inhibitory effect and eugenol was less effective. LPS-dependent expression of cyclooxygenase-2 (COX-2) protein was also inhibited markedly by isoeugenol, and less effectively by eugenol. Anti-inflammatory action of eugenol compounds may be explained by the inhibition of NO production and COX-2 expression, the pro-inflammatory mediators.

Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages.

Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Ikappa-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Ikappa-Balpha was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Ikappa-Balpha.

Characterization of the differential expression of uncoupling protein 2 and ROS production in differentiated mouse macrophage-cells (Mm1) and the progenitor cells (M1).

The expression status of mitochondrial uncoupling protein 2 (UCP2) was investigated in undifferentiated mouse myeloid leukemia (M1) and its differentiated macrophage-like cells (Mm1). Mm1 cells have a high ability of phagocytosis along with significantly high levels of reactive oxygen species (ROS) production, UCP2 protein and manganese superoxide dismutase (Mn-SOD), in contrast to undifferentiated leukemia cells (M1). Mm1 cells expressed 10-fold more UCP2 protein compared with undifferentiated M1 cells, although the UCP2 mRNA levels in both cell types were similar. The higher expression of UCP2 in the Mm1 cells suggests a regulatory role of UCP2 in the ROS production. Furthermore, the transfection of UCP2-GFP-expression vector in Mm1 cells dissipated the mitochondrial membrane potential and reduced ROS production, which was shown by their direct visualization using MitoTracker Red CM-H2Xros. The macrophage gp91phox protein, a membrane catalytic component of the NADPH oxidase complex, was at a similar level in both of UCP2-GFP expressed and non-expressed Mm1 cells. These results suggest that the UCP2 protein of the undifferentiated cell is regulated at a quite low level and the higher UCP2 protein of the differentiated macrophages involves with the regulation of ROS production.