The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes.

BACKGROUND AND PURPOSE: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. EXPERIMENTAL APPROACH: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-kappaB and MAP kinases (MAPKs) in activated HMC-1 cells. KEY RESULTS: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-kappaB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. CONCLUSIONS AND IMPLICATIONS: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-kappaB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells.

Flaxseed lignan attenuates high-fat diet-induced fat accumulation and induces adiponectin expression in mice.

Flaxseed lignan secoisolariciresinol diglucoside (SDG) has been reported to prevent and alleviate lifestyle-related diseases including diabetes and hypercholesterolaemic atherosclerosis. This study assesses the effect of SDG on the development of diet-induced obesity in mice and the effect of the SDG metabolite enterodiol (END) on adipogenesis in 3T3-L1 adipocytes. We compared body weight, visceral fat weight, liver fat content, serum parameters, mRNA levels of lipid metabolism-related enzymes and adiponectin in mice fed either a low-fat diet (5 % TAG), high-fat diet (30 % TAG) or high-fat diet containing 0.5 and 1.0 % (w/w) SDG for 4 weeks. Administration of SDG to mice significantly reduced high-fat diet-induced visceral and liver fat accumulation, hyperlipaemia, hypercholesterolaemia, hyperinsulinaemia and hyperleptinaemia. SDG also suppressed sterol regulatory element binding protein 1c mRNA level in the liver and induced increases in the adiponectin mRNA level in the white adipose tissue and carnitine palmitoyltransferase I mRNA level in the skeletal muscle. Differentiated 3T3-L1 adipocytes were treated with 0, 5, 10 and 20 mumol/l END and then assayed for mRNA expression of adipogenesis-related genes and DNA binding activity of PPARgamma to the PPAR response element consensus sequence. END induced adipogenesis-related gene mRNA expression including adiponectin, leptin, glucose transporter 4 and PPARgamma, and induced PPARgamma DNA binding activity in 3T3-L1 adipocytes. In conclusion, SDG induced adiponectin mRNA expression and showed beneficial effects on lipid metabolism in diet-induced obesity in mice. Flaxseed lignans are suggested to regulate adipogenesis-related gene expressions through an increase in PPARgamma DNA binding activity.

Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells.

BACKGROUND AND PURPOSE: 5alpha,8alpha-Epidioxy-22E-ergosta-6, 22-dien-3beta-ol (ergosterol peroxide) is a major antitumour sterol produced by edible or medicinal mushrooms. However, its molecular mechanism of action has yet to be determined. Here, we examine the anticancer and anti-inflammatory effects of ergosterol peroxide. EXPERIMENTAL APPROACH: After treating RAW264.7 macrophages with LPS and purified ergosterol peroxide or ergosterol, we determined LPS-induced inflammatory cytokines, nuclear DNA binding activity of transcription factors and phosphorylation of MAP kinases (MAPKs). HT29 colorectal adenocarcinoma cells were treated with ergosterol peroxide for 5 days. To investigate the antitumour properties of ergosterol peroxide, we performed DNA microarray and RT-PCR analyses and determined the reactive oxygen species (ROS) in HT29 cells. KEY RESULTS: Ergosterol peroxide suppressed LPS-induced TNF-alpha secretion and IL-1alpha/beta expression in RAW264.7 cells. Ergosterol peroxide and ergosterol suppressed LPS-induced DNA binding activity of NF-kappaB and C/EBPbeta, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. Ergosterol peroxide down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. In addition, ergosterol peroxide showed cytostatic effects on HT29 cells and increased intracellular ROS. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes. CONCLUSION AND IMPLICATION: Our results suggest that ergosterol peroxide and ergosterol suppress LPS-induced inflammatory responses through inhibition of NF-kappaB and C/EBPbeta transcriptional activity, and phosphorylation of MAPKs. Moreover, ergosterol peroxide appears to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells.

Wedelolactone suppresses LPS-induced caspase-11 expression by directly inhibiting the IKK complex.

Caspase-11 is a key regulator of proinflammatory cytokine IL-1beta maturation and pathological apoptosis. Caspase-11 is not expressed in most tissues under normal condition, but highly inducible upon pathological stimulation such as in the presence of lipopolysaccharide (LPS). Here, we describe the identification and characterization of wedelolactone, a natural compound that inhibits LPS-induced caspase-11 expression in cultured cells by inhibiting NF-kappaB-mediated transcription. We demonstrate that wedelolactone is an inhibitor of IKK, a kinase critical for activation of NF-kappaB by mediating phosphorylation and degradation of IkappaBalpha.

"A system biology" approach to bioinformatics and functional genomics in complex human diseases: arthritis.

Human and other annotated genome sequences have facilitated generation of vast amounts of correlative data, from human/animal genetics, normal and disease-affected tissues from complex diseases such as arthritis using gene/protein chips and SNP analysis. These data sets include genes/proteins whose functions are partially known at the cellular level or may be completely unknown (e.g. ESTs). Thus, genomic research has transformed molecular biology from "data poor" to "data rich" science, allowing further division into subpopulations of subcellular fractions, which are often given an "-omic" suffix. These disciplines have to converge at a systemic level to examine the structure and dynamics of cellular and organismal function. The challenge of characterizing ESTs linked to complex diseases is like interpreting sharp images on a blurred background and therefore requires a multidimensional screen for functional genomics ("functionomics") in tissues, mice and zebra fish model, which intertwines various approaches and readouts to study development and homeostasis of a system. In summary, the post-genomic era of functionomics will facilitate to narrow the bridge between correlative data and causative data by quaint hypothesis-driven research using a system approach integrating "intercoms" of interacting and interdependent disciplines forming a unified whole as described in this review for Arthritis.

Analysis of increased leukocyte ratio induced by tumor necrosis factor-alpha using several inhibitors in the undisturbed microcirculation of the rat mesentery.

Increased leukocyte ratio induced by intraperitoneal injection of TNF-alpha was analyzed in undisturbed rat mesentery venules, a long-term experimental model. TNF-alpha induced a biphasic increase in PMNL ratio in the mesentery venules; in particular, there was a large increase in PMNL ratio 3 h after injection of TNF-alpha. Anti-P and anti-L-selectin compounds, fucoidin and dextran sulfate, inhibited the PMNL ratio by about 40%. Dexamethasone completely inhibited the increased ratio. These results suggest that all selectins may be involved in the increased ratio, i.e. rolling and adhesion reaction.

Effects of brushing with a dentifrice for sensitive teeth on tubule occlusion and abrasion of dentin.

By using a dentifrice or toothpaste for sensitive teeth, the brushing-induced effects on dentinal tubule occlusion and abrasion of human sound dentin were investigated with a scanning electron microscope and a scanning laser microscope. The dentifrice contained diatomaceous earth and silica as abrasives and strontium chloride hexahydrate as an active ingredient. Thirty dentin pieces of human premolar teeth with an average of 20% occluded dentinal tubules were attached to resin plates and exposed to the oral cavities of five adult subjects for 2, 4, and 8 weeks. Brushing with and without dentifrice was performed 1 min per day, respectively. Brushing with the dentifrice gradually decreased the mean average of occluded tubules from about 91 to 77% during 2 to 8 weeks, although there were no significant differences among the individual values. However, the mean abrasive loss of the dentin surfaces brushed with dentifrice significantly increased from about 52 to 143 microm in depth. The brushed surfaces of the dentin showed a rough topography with numerous toothbrush scratches but no organic pellicle was found. On the other hand, brushing without dentifrice caused about 99% of the dentinal tubules to occlude in 2 and 4 weeks and 100% in 8 weeks. The brushed dentin surfaces at 8 weeks were entirely covered with organic pellicle containing fine mineral granules derived from saliva, and the abrasive loss was about 1.4 microm in mean depth. Such results indicate that brushing with abrasive dentifrices for sensitive teeth remarkably erodes dentin, and suggest that the brushing should cause the dentinal tubules to open again for a certain period of time.

Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells.

We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.

Revision of failed metal-backed patellar component of Miller/Galante-I total knee prosthesis.

Of 38 Miller/Galante-I total knee prostheses implanted between 1987 and 1990, 20 (53%) were revised. Failed metal-backed patella (15 knees; 40%) was the most frequent reason for revision. In most patients, the components were found well fixed to bone. The purpose of this study was to describe the revision technique we used for failed metal-backed patella. Total revision of all components would be ideal. However, removal of all components results in large bone loss, large blood loss, and a longer operation time. Because of the patients' relatively old age (78.7 years), less demanding activities of daily life (ADL), and the lower body weight of the Japanese patient, we chose a less invasive approach. The revision technique had the following features: (1) the grooved femoral component was not replaced, (2) the failed patellar component was replaced by an all-polyethylene patella, (3) synovectomy was performed, and (4) realignment of patellar tracking was done by lateral release and medial imbrication of the quadriceps mechanism. The mean follow-up after revision was 2 years, 4 months, and good short-term results were obtained. This less invasive approach would be beneficial for elderly patients whose ADL are less demanding.

Abrasion of human enamel by brushing with a commercial dentifrice containing hydroxyapatite crystals in vitro.

Automatic toothbrushing with a commercial dentifrice containing hydroxyapatite (HAP) crystals was performed on the tangential polished surfaces of sound human enamel, mainly consisting of biological apatite similar to HAP, for 10 min in vitro. The X-ray diffraction peaks of HAP, brushite (DCPD), and monetite (DCP) crystals were detected from the dentifrice. After brushing, the enamel surfaces were observed with a scanning electron and a confocal scanning laser microscope. The brushing caused larger abrasive loss and more remarkable roughness of the enamel surfaces following the broad traces of brush bristles and the exposure of prism structures than brushing with a dentifrice containing only DCPD, which we previously reported. We claim that the fine granular-shaped HAP crystals of the dentifrice indicated as an active ingredient for preventing enamel caries possess stronger abrasivity of sound enamel than the DCPD and DCP as abrasives on account of their Mohs hardness values rather than sizes and shapes. The HAP crystals of dentifrices may not occlude the small defects of early caries enamel, but erode them more strongly as an abrasive than the other abrasives.

Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.

The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.

Proanthocyanidins from barley bran potentiate retinoic acid-induced granulocytic and sodium butyrate-induced monocytic differentiation of HL60 cells.

Polyphenol extract from barley bran (BPE) induced nitro blue tetrazolium (NBT) reducing activity and alpha-naphthyl butyrate esterase activity in HL60 human myeloid leukemia cells. Because BPE induced the biochemical markers of HL60 cell differentiation, we investigated the effects of proanthocyanidins isolated from BPE on the HL60 cell differentiation of HL60 cells. Prodelphinidin B-3, T1, T2, and T3 induced 26-40% NBT-positive cells and 22-32% alpha-naphthyl butyrate esterase-positive cells. Proanthocyanidins potentiated retinoic acid (all-trans-retinoic acid)-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells.

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

BACKGROUND: Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast-specific biological functions remain elusive because only a limited number of osteoclast-specific genes have been identified. To circumvent this, we isolated a large number of osteoclast-specific genes by preparing a subtracted cDNA library of high quality. RESULTS: We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast-specific cDNA library of high complexity by subtracting it with biotin-labelled mRNA, derived from rabbit spleen through the biotin-avidin method. The resulting library included a high proportion of full-length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast-specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple-tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast-specific genes. CONCLUSION: From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up- or down-regulated genes in general.

Expression and localization of matrix metalloproteinase-12 in the aorta of cholesterol-fed rabbits: relationship to lesion development.

Degradation of extracellular matrix (ECM) proteins in the aorta is a critical step for the development of atherosclerosis. Expression of matrix metalloproteinase (MMP)-12 (macrophage elastase), an elastin-degrading proteinase in the MMP family, was investigated in the thoracic aorta of rabbits fed a 1% cholesterol-containing diet for 16 weeks. In the atherosclerotic lesions, MMP-12 was produced abundantly at both the mRNA and protein levels, whereas no expression was observed in the normal rabbit aortas. The principal source of MMP-12 was macrophage foam cells (MFCs) that had infiltrated the atherosclerotic intima; this was demonstrated in both in vitro culture studies of MFCs purified from atherosclerotic lesions and immunohistochemical studies of aortic lesions. Additional biochemical studies using recombinant rabbit MMP-12 revealed that MMP-12 digested elastin, type IV collagen, and fibronectin and also activated MMP-2 and MMP-3. Expression of MMP-12 by human macrophage cell lines was increased by stimulation with acetylated low-density lipoprotein, implying augmentation of MMP-12 production during foam cell formation. Increased expression of MMP-12 in atherosclerotic lesions, concomitant with foam cell generation, which triggers the acceleration of ECM breakdown, is likely to be a critical step in the initiation and progression of the atherosclerotic cascade.

Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells.

Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption.

Structure-activity relationships of flavonoids and the induction of granulocytic- or monocytic-differentiation in HL60 human myeloid leukemia cells.

The flavones apigenin and luteolin strongly inhibited the growth of HL60 cells and induced morphological differentiation into granulocytes. The flavonol quercetin inhibited the cell growth and induced a differentiation marker, i.e., NBT reducing ability. However quercetin-treated cells were not morphologically differentiated into granulocytes. The chalcone phloretin weakly induced NBT reducing ability and a marker of monocytic differentiation alpha-naphthyl butyrate esterase activity in the cells. Quercetin and phloretin appeared to induce the differentiation of HL60 cells into monocytes. The proportion of alpha-naphthyl butyrate esterase-positive cells induced by genistein was less than that of the NBT-positive cells. Some of the nuclei in genistein-treated HL60 cells morphologically changed. Genistein must have induced both granulocytic and monocytic differentiation of HL60 cells. The flavonols galangin and kaempferol, which had fewer hydroxyl group(s) in the B-ring than quercetin, and the flavanone naringenin inhibited the growth but did not induce the differentiation of HL60 cells.

Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein.

Royal jelly was fractionated by ion-exchange chromatography and a protein (DIII protein) that had growth stimulating activity to the U-937 human myeloid cell line was obtained. The molecular weight of the DIII protein was 58 kDa on SDS-PAGE. The growth stimulating activity of the DIII protein was shown to be relatively heat and pH stable.

25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis.

Renal 25-hydroxyvitamin D3 1alpha-hydroxylase [1alpha(OH)ase] catalyzes metabolic activation of 25-hydroxyvitamin D3 into 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], an active form of vitamin D, and is inhibited by 1alpha,25(OH)2D3. 1alpha(OH)ase, which was cloned from the kidney of mice lacking the vitamin D receptor (VDR-/- mice), is a member of the P450 family of enzymes (P450VD1alpha). Expression of 1alpha(OH)ase was suppressed by 1alpha, 25(OH)2D3 in VDR+/+ and VDR+/- mice but not in VDR-/- mice. These results indicate that the negative feedback regulation of active vitamin D synthesis is mediated by 1alpha(OH)ase through liganded VDR.

Competitive ELISA of bovine lactoferrin with bispecific monoclonal antibodies.

A mouse hybrid-hybridoma, HH1-4-3, secreting IgG1 class bispecific antibodies to bovine lactoferrin (bLF) and horseradish peroxidase (HRPO), was established previously. The competitive enzyme-linked immunosorbent assay (ELISA) of bLF using the HH1-4-3 culture supernatant was not sensitive enough to measure bLF concentration in biological fluids. To improve the sensitivity of the competitive ELISA, we fractionated the bispecific antibodies by antigen affinity column chromatography. A column immobilized with bLF adsorbed 60% of the antibodies of the HH1-4-3 supernatant, and the amount of antibodies adsorbed on a column immobilized with HRPO was less than 5%. The competitive ELISA of bLF using the affinity purified bispecific antibodies through an HRPO-immobilized column chromatography showed a good standard curve at bLF concentrations of 10 ng/ml to 100 micrograms/ml.

Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport.

Phloretin, a naturally occurring dihydrochalcone, is known to inhibit tumor cell growth in vitro and in vivo. To clarify the anti-tumor effects of phloretin, its apoptosis-inducing effects in B16 melanoma 4A5 cells were examined. Phloretin induced the internucleosomal DNA fragmentation typical of apoptosis in B16 melanoma cells. The addition of extracellular glucose remarkably inhibited the phloretin-induced apoptosis in the cells. When apoptosis was strongly induced in the B16 cells by phloretin, protein kinase C activity was inhibited in the cells. Our results suggest that phloretin induced apoptosis in B16 melanoma 4A5 cells mainly through the inhibition of glucose transmembrane transport. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in the cells.