吉田 ルシア幸子

We aimed to find candidate molecules possibly involved in the anti-inflammatory activity of shikonin (active compound of "Shikon") by analyzing its effects on gene expression of lipopolysaccharide (LPS)-treated THP-1 macrophages. Polysome-associated mRNAs (those expected to be under translation: translatome) from cells treated with LPS alone (LPS: 5 A mu g/mL), shikonin alone (S: 100 nM), or LPS plus shikonin (LPS&S) for 3 h were analyzed by DNA microarray followed by detection of enriched pathways/gene ontologies using the tools of the STRING database. Candidate genes in enriched pathways in the comparison of LPS&S cells vs. LPS cells were analyzed by reverse-transcription quantitative real-time PCR (RT-qPCR; 1, 2, and 3 h). DNA microarray showed shikonin significantly influences gene expression. Gene expression changes between LPS&S cells and LPS cells were compared to detect relevant proteins and/or mRNAs underlying its anti-inflammatory effects: shikonin downregulated pathways which were upregulated in LPS cells, for example, 'innate immune response'. Within changed pathways, three genes were selected for RT-qPCR analyses as key candidates influencing inflammatory responses: CYBA (component of the superoxide-generating Nox2 enzyme), GSK3B (controller of cell responses after toll-like receptor stimulation), and EIF4E (a key factor of the eukaryotic translation initiation factor 4F complex that regulates abundance of other proteins involved in immune functions). All three mRNAs were decreased at 2 h, and CYBA continued low at 3 h relative to LPS cells. Given that shikonin decreased the expression of CYBA gene of Nox2, in addition to the direct inhibition of the Nox2 activity that we have previously shown, it is suggested that one of its anti-inflammatory mechanisms could be attenuation of oxidative stress.

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4 alpha) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4 alpha in myofibrils, the effects of TPM4 alpha-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, alpha-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4 alpha-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, alpha-actinin and obvious myofibril striations were undetectable. It is possible that TPM4 alpha-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4 alpha-suppression significantly affects actin filament, and that TPM4 alpha plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD(2)' value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 mu M. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC(50)s, 4-7 mu M), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.

Tumor necrosis factor (TNF)-alpha, which potentiates reactive oxygen species (ROS) generation, is crucial for the development of coronary arteritis and aneurysm in Kawasaki disease. We hypothesized that vascular NADPH oxidase (Nox) enzymes participate in the TNF-alpha-triggered endothelial damage through elevating ROS generation. Thus, we herein examine the expression of Nox enzymes in human coronary artery endothelial cells (HCAEC) and the effects of TNF-alpha on Nox-mediated ROS generation. We show that HCAEC in culture spontaneously generate H(2)O(2) at basal level (0.53 nmol/min/mg protein). In searching for Nox components responsible for the H(2)O(2) generation, two distinct isoforms of Nox4 are found expressed in HCAEC: the prototype Nox4A and the shorter Nox4B, respectively in the postnuctear supernatant and the nuclear fractions. Other expressed Nox family components are: as mRNAs, Nox4C, Nox4D, Nox1, p51(nox), and Racs; as mRNAs and proteins, Nox2, p22(phox), p47(phox), and p67(phox). The H(2)O(2)-generating activity increases up to three-fold upon inclusion of TNF-alpha in culture, concomitantly with augmented expressions of Nox4A, p22(phox), p47(phox) and p67(phox) proteins. Together, these results suggest that Nox2 and Nox4A enzymes are induced by TNF-alpha endowing HCAEC with enhanced ROS-generating activity, which may play a rote in the initial endothelial dysfunction through oxidative stress. (c) 2008 Elsevier B.V. All rights reserved.

Objective: Shikonin, a component of the herbal medicine "Shikon", is known to suppress inflammatory reactions, but its molecular targets are not identified. This study examines the effect of shikonin on human basophil degranulation response and aims to identify its targets. Materials: Human basophils in isolated leukocytes from healthy volunteers' peripheral bloods recombinant human Syk and Lyn tyrosine kinases. Methods: Histamine release from basophils stimulated with anti-IgE antibody was analyzed fluorimetrically. Syk and Lyn kinase activities were tested in vitro with recombinant proteins and analyzed by off-chip mobility shift assay. Results: Shikonin dose-dependently inhibited the histamine release from basophils induced by anti-IgE antibody (IC(50) = 2.6 +/- 1.0 mu M; mean +/- SEM). A search for the target(s) of shikonin in the signal cascade of IgE-mediated activation showed that it strongly inhibits Syk (IC(50) = 7.8 mu M, in the recombinant kinase assay), which plays a pivotal role in the degranulation response. A less significant inhibition was found for Lyn, which phosphorylates Fc epsilon RI-beta gamma, subunits and also Syk. Conclusions: These results indicate that the inhibition of Syk-dependent phosphorylation events might underlie the blocked histamine release from human basophils, thus contributing to the anti-inflammatory effects of shikonin.

We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.

Fungal gliotoxin (GT) is a potent inhibitor of the O-2(-)-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47(phox), a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C betaII with p47(phox) on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O-2(-)-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b(558) is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b(558) and cytosolic components (native cytosol or recombinant p67(phox), p47(phox), and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 muM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67(phox), p47(phox), and Rac2 with flavocytochrome b(558) is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b(558)'s ability to reconstitute O-2(-) generation, supporting the conclusion at the cellular level. The flavocytochrome b(558) heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b(558), which is accessible only before oxidase activation.

Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated 02 generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47(phox) phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67(phox), p47(phox), and p40(phox), which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47(phox) phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47(phox) phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) betaII rather than an effect of GT on PKC betaII activity, suggesting a failure of PKC betaII to associate with the substrate, p47(phox), on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.

Nox1, a homologue of gp91(phox) subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O-2(-)) generation in guinea pig gastric mucosal cells (GMC), but involvement of regulatory components (p67(phox), p47(phox), and Rac) which are essential in phagocytes remains unknown. Here, we aimed to figure out how Nox1 of GMC achieves an active oxidase status. GMC in primary culture show low O-2(-) generation but acquire a 9-fold higher activity when cultured with Helicobacter pylori lipopolysaccharide (LPS), in correlation with a far increased Nox1 expression. Investigation into the O-2(-)-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O-2(-) per se; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O-2(-) generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91(phox), i.e., to produce O-2(-) appreciably in response to myristate stimulation; 4) Nox1 prefers NADPH to NADH under the in vitro assay with neutrophil cytosol plus myristate (K-m = 10.4 mum); 5) substitution of neutrophil cytosol by a set of recombinant cytosolic components (rp67(phox), rp47(phox), Rac2) is, however, ineffective and still requires GMC cytosol. Thus, Nox1 probably requires an additional cytosolic factor(s). In contrast, GMC cytosol enables cytochrome b(558) to generate plenty of O-2(-), on condition that rp47(phox) is added. This result suggests that GMC cytosol contains a component with p67(phox)-ability, and also Rac, but lacks p47(phox). These data indicate that GMC Nox1 requires at least a p67(phox) counterpart and Rac to acquire NADPH oxidase activity.

Gliotoxin from Aspergillus, bearing a SS bond in its structure, prevented the onset of O-2(-) generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA), Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O-2(-) generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC50) of 5.3, and 3.5 mu M for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC50 of 0.009 mu M. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation. (C) 2000 Academic Press.

Defective NADPH oxidase components prevent superoxide (O-2(radical anion)) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91(phox) subunit of cytochrome b(558) and usually lack gp91(phox) protein completely (X91(0)). gp91(phox) is considered to be a flavocytochrome that contains binding sites for NADPH, FAD, as well as heme. We here report a rare X-linked CGD patient whose neutrophils entirely failed to produce O-2(radical anion), but presented a diminished expression of gp91(phox) containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67(phox) and p47(phox) was normal. However, the FAD content in his neutrophil membranes was as low as that of X91(0) patients, suggesting complete depletion of FAD in his gp91(phox). This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91(phox) in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FAD-rich dehydrogenase fraction isolated from normal neutrophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91(phox) and p22(phox) subunits) and two cytosolic factors (p47(phox) and p67(phox)), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40(phox), as a molecule that associates with p67(phox) in human neutrophils. Although it has been found that this p40(phox) protein is defective in patients with chronic granulomatous disease (CGD) who lack p67(phox), evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40(phox) as well as against the COOH-terminal polypeptide of p67(phox) to examine the mode of interaction between p40(phox) and p67(phox) in a complex. The antibody against the COOH terminus of p67(phox) was able to coimmunoprecipitate g40(phox) in conjunction with p67(phox) itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40(phox) completely dissociated the p67(phox) molecule from the p40(phox)-p67(phox) complex unit without any detectable coimmunoprecipitation of p67(phox), despite their tight association, whereas that against the NH2 terminus of p40(phox) had absolutely no dissociation effect. Similar results were found regarding their effects on the O-2(-)-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40(phox). However, this dissociation did not affect the translocation of the cytosolic components including p47(phox) to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40(phox)-p67(phox) complex. These results Provide the first demonstration that p40(phox) is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67(phox).

Cytochrome b(558) purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O-2-radical-anion generating activity. ESR spectra of cytochrome b(558) either from resting or stimulated neutrophils showed a low-spin hemoprotein with g(1,2,3) of 3.2, 2.1, and 1.3 (estimated), At physiological pH, the oxidized cytochrome b(558) is in a purely low-spin state, On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin cytochrome b(558) was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in cytochrome b(558) was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b(558) was varied to form different ratios of the low-spin to high-spin states of the heme, and its O-2-radical-anion generating activity was examined in cell-free systems, O-2-radical-anion farming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b(558) is essential to generate O-2-radical-anion and the heme retains the low-spin state through the redox cycle.

A high incidence of hepatocellular carcinoma (HCC) was observed in mice fed a choline-deficient diet containing 0.1% ethionine (CDE) for 19 months. HCC was present in 85% of CDE mice and in 22% of choline-deficient (CD) mice not receiving ethionine. This strong hepatocarcinogenicity of the CDE diet was concomitant with a severe decrease in plasma and liver alpha-tocopherol (Toc) to 60 and 35%, respectively, of those contained in choline-supplemented (CS) control mice. We previously found that this dietary-induced HCC was preceded at 4-week feeding by a depletion of Toc and a remarkable increase of phosphatidylcholine hydroperoxide (PCOOH) in the livers of CDE mice.1 When HCC was prominent in CDE mice, PCOOH was still elevated. Mouse glutathione S-transferase (GST) M II isozyme, which is related to rat GST-P form, a positive marker for rat hepatic preneoplastic and neoplastic lesions, revealed an inverse histochemical pattern as that seen in rats (i.e., the HCC lesions tended to decreased staining). The aforementioned results taken together indicate that decreases in Toc and enhanced PC peroxidation are important events in CDE-induced mice liver tumors.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2--generating activity in the absence of exogenous FAD. However, the system showed high O2--generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2--generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2--generating activity in the reconstituted system. The O2--forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2--generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2--forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.