BioFactors

Published by Wiley
Online ISSN: 1872-8081
Publications
Article
The cells of Corynebacterium (Brevibacterium) ammonia-genes cultivated in a medium supplemented with diquat or benzylviologen accumulate 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate as revealed by 31P-NMR spectroscopy. On heating at 120 degrees C for 30 min the cells still maintain a substantial portion of this compound and acquire new cyclic phosphates characterized by 31P-NMR chemical shifts of +17.3 and +20 p.p.m. The +17.3 p.p.m. component was isolated from the preparation of the purified cyclopyrophosphate kept for some time at pH above 7 and it was shown to be 2-methylbutane-1,2,3,4-tetraol-1,2,- cyclophospho-4-phosphate on the grounds of two-dimensional NMR spectroscopy.
 
Article
1,25(OH)2 Vitamin D3 (1,25(OH)2D3) and adhesion propagate monocyte differentiation. We identified the selenoprotein thioredoxin reductase (TrxR) as a new molecular target for 1,25(OH)2D3 in monocytes during this process. In THP1 monocytic leukemia cells 1,25(OH)2D3 stimulated TrxR mRNA levels 2-4-fold by 4-8 h and enhanced TrxR activity (60%) (as measured by the dithionitrobenzole-assay) after 24 h, which declined below baseline after 96 h. The addition of 100 nM selenite enhanced (approx. 50%) basal and stimulated enzyme activity in THP1 cells. The relative stimulation by 1,25(OH)2D3 was very similar but peak levels were sustained in THP1 cells up to 48 h. Human peripheral blood monocytes (PBM) of different donors showed very low basal TrxR steady state mRNA levels which were markedly enhanced (as analyzed by Northern blotting) after 4 h of adherence to culture dishes. 1,25(OH)2D3 (100 nM) further stimulated TrxR mRNA expression (4 h, 3-fold). TrxR enzyme activity mirrored the mRNA changes. Basal activity was stimulated approx. 25% by adhesion in culture alone and was further stimulated (approximately 15%) by 1,25(OH)2D3 after 4 h. By 24 h similar results were achieved but the effect of 1,25(OH)2D3 could be seen in the presence of 100 nM selenium only. The expression of TrxR and its regulation by 1,25(OH)2D3 and selenite in monocytes might be important for their induction of differentiation and maintenance of function.
 
Article
A highly hydrophobic protein with six transmembrane structure that is coded by the candidate tumor suppressor gene 101F6 located in the human chromosome 3p.21.3 and a possible member of the cytochrome b 561 protein family was expressed, purified, and characterized in its functional form for the first time. The protein was heterologously expressed in methylotrophic yeast Pichia pastoris as a fusion protein containing a C-terminal thrombin-specific sequence and an 8-His residue tag. Purification was achieved by ion exchange chromatography on DEAE-Sepharose and affinity chromatography on Ni-NTA-Sepharose. SDS-PAGE analysis revealed a single protein band with an estimated molecular weight of 26 kDa, while Western blot and MALDI-TOF-MS analysis confirmed the presence of the cytochrome b561 specific sequence in the protein. The 101F6 protein was found to be reducible by ascorbate efficiently and to have two midpoint potentials at +89.5 and +13.1 mV, slightly lower than the corresponding values of +155 and +62 mV, respectively, of bovine adrenal cytochrome b 561, despite a lower conservation of the putative ascorbate binding site sequence in the 101F6 protein. The "modified motif 1" sequence unique in 101F6 protein may be responsible for other molecular functions, such as protein-protein interactions, in the endoplasmic membranes.
 
Article
Interleukin (IL)-11 is a bone marrow fibroblast derived cytokine with a wide spectrum of activities in different biological systems. It has been shown that IL-11 supports the growth of certain types of plasmacytoma and hybridoma cells, enhances antigen-specific antibody responses, synergizes with IL-3 in supporting megakaryocyte colony formation, acts synergistically with IL-3 in shortening the G0 period of early progenitors, induces the synthesis of acute phase proteins, and inhibits lipoprotein lipase activity and adipocyte differentiation. The human IL-11 gene, which is localized at 19q13.3-13.4, consists of five exons and four introns. Initial biochemical characterization has identified a 151 kDa protein as the potential IL-11 binding subunit of the receptor complex. Because of the overlapping biological activities between IL-6 and IL-11, we compared the signal transduction pathways mediated by IL-6 or IL-11 in cell lines responsive to both cytokines. Results from protein tyrosine phosphorylation and immediate response gene expression suggest that there are convergent and divergent points along the signal transduction pathways utilized by IL-6 or IL-11. The IL-6 signal transducer, gp130, appears to be involved in the IL-11 mediated signaling. Other cytokines such as leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor have also been shown to utilize gp130 as a signal transducer. The significance of growth factor sharing common biological activities and signaling pathways will be discussed.
 
Article
Aberrant activation of the canonical Wnt/β-catenin signaling pathway has been reported for numerous tumors of different origins. In most cases, mutations in components of the Wnt signaling pathway or in β-catenin itself were detected which ultimately induce a genetic program that promotes cell proliferation and attenuates apoptosis. Thus, targeting of Wnt/β-catenin signaling is of specific therapeutic interest. Herein, we investigated the plant-derived isoquinoline alkaloid berberine, which has been reported to have anticancer activity, and synthetic 13-arylalkyl derivatives thereof for their effects on Wnt/β-catenin signaling. Berberine did not show major effects on viability of HEK-293 embryonic kidney and HCT116 colon carcinoma cells and was not toxic in concentrations up to 20 µM. Berberine inhibited β-catenin transcriptional activity and attenuated anchorage-independent growth. As a result of berberine treatment, cellular levels of active β-catenin were reduced concomitant with an increase in the expression of E-cadherin. However, in unstimulated cells, the effects on β-catenin levels were low. A screen of synthetic 13-arylalkyl berberine derivatives identified compounds exhibiting activities superior to those of the naturally occurring parent substance with more than 100-fold lower EC50 values for Wnt-repression. Thus, berberine and its synthetic derivatives represent potential therapeutic agents to inhibit Wnt/β-catenin signaling in tumorigenesis. © 2013 BioFactors, 2013.
 
Article
Chronic inflammation can lead to altered extracellular matrix deposition and ultimately fibrosis. Interleukin-13 (IL-13) is a cytokine that was found to promote IgE class switching and inhibit proinflammatory cytokines. However, it is now recognized as an important mediator in allergy and most importantly fibrosis. Indeed, animal studies with genetically deleted mice have demonstrated its critical role in fibrosis and although it shares over lapping functions with IL-4 it is the dominant cytokine in fibrosis. Systemic sclerosis is an autoimmune disease in which there is chronic inflammation and fibrosis. The disease is associated with a Th2 polarization and IL-13 levels are elevated both in the blood and in the skin of patients. This review will examine the role of IL-13 in driving fibrosis with a particular emphasis on systemic sclerosis as a prototypical fibrotic disease. It will highlight recent research into the role of IL-13 and how this cytokine may be targeted in systemic sclerosis. © 2013 BioFactors, 2013.
 
Article
Expression of cellular adhesion molecules (CAMs) at endothelial surfaces represents a physiological response to vascular damage and mediates the initiation of inflammation and possibly of atherogenesis. The cytokines TNFα and IL-1 are potent inducers of CAMs in endothelial cells. Reactive oxygen species comprising lipid oxidation products have been implicated in the signaling pathways of both TNFα and IL-1 and accordingly could modulate atherogenic events. We, therefore, investigated the potential role of the lipoxygenase product, 13-hydroperoxyoctadecadienoic acid (13-HPODE), which has also been identified in oxidized low density lipoproteins on CAM expression in HUVEC. 13-HPODE induced the expression of ICAM-1 in a concentration dependent manner up to 75 μM. Higher concentrations were toxic. Similar effects were observed with H2O2 and phosphatidylcholine hydroperoxide. VCAM-1 and E-selectin were not induced by 13-HPODE. 13-HPODE administered simultaneously with IL-1 or TNFα induced ICAM-1 additively, suggesting that hydroperoxides and cytokines act on the same signaling pathways. In contrast, pretreatment of cells with 50 μM 13-HPODE for 1 hour rather inhibited subsequent cytokine-induced ICAM-1 and E-selectin expression. Surprisingly, the reduction product of 13-HPODE, 13-hydroxyoctadecadienoic acid (13-HODE) proved to be an even better inducer of ICAM-1 than 13-HPODE. Pretreatment with 13-HODE did not show any inhibitory effect on ICAM-1 expression. Our data show that lipoxygenase products differentially affect CAM expression. 13-HPODE is stimulatory by itself and can positively or negatively affect cytokine signaling depending on time of exposure. 13-HODE induces CAM expression by itself but does not inhibit cytokine signaling. Thus, the interplay of lipoxygenase products with proinflammatory cytokines can not simply be explained by an oxidant-mediated facilitation of cytokine signaling.
 
Article
Purified ATPsynthase of bovine heart mitochondria has been analyzed for its mobility and reactivity of oligomycin-sensitive sulfhydryl regions in presence of the substrate ADP and oligomycin. Labeling of thiol groups at the hydrophobic F_0 region of the ATPsynthase was increased in the enzyme initially treated with SDS, N-ethylmaleimide and dithiothreitol (modified enzyme). After dialysis or gel permeation the ATPsynthase was treated with [14C] alpha lipoic acid at a molar ratio of 35-85/1 (lipoic acid/ATPsynthase) corresponding to 4-8.6 nmol/mg protein. Under these conditions, ATPase activity of the native enzyme was significantly decreased. After preincubation with ADP, PAGE of the native, [14C] labeled enzyme revealed an increase of radioactivity at a region of 25 kDa deduced to Cys 197 of subunit b. In the modified enzyme the increase in radioactivity was found at 10 kDa. In this context, the sequence Lys-Cys-Ile around Cys 197 of subunit b suggests excessive reactivity of this thiol, as well as ready reversibility by -SH-S-S- interchange. Therefore, previously observed reaction by thiol reagents and antioxidants from outside the mitochondrion can be interpreted with Cys 197 of F0 b. It accounts for sulfhydryl unmasked by binding of ADP at F1.
 
Article
Retinol and its metabolite retinoic acid play a critical role in immunity, reproduction, and development. Retinoids are known to influence renal development, and show beneficial effects in experimental models of renal disease. ß-Carotene (provitamin A) is cleaved to retinal by ß-carotene 15,15′- monooxygenase (BCM), which is an essential enzyme for retinoid biosynthesis. However, the metabolism of retinol and ß-carotene in renal diseases such as nephrosis remains unclear. We studied BCM gene expression and retinol status in rats with nephrotic syndrome induced by puromycin aminonucleoside (PAN). BCM gene expression in the liver and intestines of PAN-treated rats was decreased compared with that in controls, while the expression in the kidney of PAN-treated animals was increased. Plasma retinol and retinol-binding protein levels were decreased in PAN-treated rats, but hepatic retinol level did not differ between PAN-treated and control rats. Up-regulation of BCM gene expression in the kidneys of rats with nephrotic syndrome may result in increased conversion of ß-carotene to retinal, so this change might supply more retinoic acid to the damaged glomeruli. Changes in the metabolism of retinol and ß-carotene might have an important role in protection against the development of nephrosis.
 
Article
The vitamin A status has been studied in type 2 diabetes and it is known that plasma retinol levels of patients with type 2 diabetes are elevated. However, the details of vitamin A metabolism in type 2 diabetes are unclear. beta-Carotene exhibits biological activity as provitamin A and beta-carotene 15,15'-monooxygenase (BCM) cleaves beta-carotene to form retinal. We studied BCM gene expression in type 2 diabetic Goto-Kakizaki (GK) rats. BCM gene expression was analyzed in the liver, intestine, and testis of 8- and 13-week-old GK rats and Wistar rats (control). The plasma and liver retinol levels were measured, and plasma retinol-binding protein (RBP) was detected. BCM gene expression in the liver, intestine and testis of GK rats was increased compared with that in controls. Plasma retinol levels and RBP levels were increased in GK rats, but hepatic retinol levels did not differ between GK rats and controls. BCM gene expression in the liver and intestine might affect retinol levels in type 2 diabetes. Conversion of beta-carotene to retinal might be accelerated in the presence of insulin resistance status, so that plasma retinol levels are increased in type 2 diabetes.
 
Article
The precise mechanisms of antioxidant-mediated longevity are poorly understood. We show that an antioxidant treatment can extend the lifespan of Caenorhabditis elegans (C. elegans) through the nuclear translocation of the forkhead box O transcription factor (FoxO) homolog DAF-16. This pathway was found to involve 3-phosphoinositide-dependent kinase-1 (PDK-1) and serum- and glucocorticoid-regulated kinase-1 (SGK-1), distinct from the known oxidative stress-mediated mechanism in which FoxO3a translocation is regulated by c-Jun N-terminal kinase (JNK) and mammalian sterile 20-like kinase-1 (MST-1). The differences in the mechanisms of FoxO activation by antioxidants and oxidants result in differences in FoxO phosphorylation and target gene expression. Based on these results, we found that a combination of early antioxidant treatment and late oxidant treatment is most effective for lifespan extension in C. elegans. © 2013 BioFactors, 2013.
 
Article
It has been well-established that type-2 immunity, characterized by eosinophilia, goblet cell hyperplasia, mucus production, and B cell class switching to IgE, is highly dependent on the production of the type-2 cytokines, interleukin (IL)-4, IL-5, IL-9, and IL-13, by T helper 2 (Th2) cells. However, it is less clear how the type-2 cytokine effector response is induced and in addition what innate cell type produces the initiating factor. Recent reports highlight IL-25 as a type-2 inducing factor, with IL-25 administration resulting in severe gut and lung type-2 pathologies. The expression of IL-25 is also necessary for initiation of a robust type-2 response both at the genesis of the response, as with helminth infection, and during the response, as has been shown in experimental allergic asthma. It is also apparent that, as well as directly controlling type-2 immunity via IL-4, IL-5, and IL-13, IL-25 may also interact with other cytokines and their receptors, such as IL-17A and the IL-17RA receptor. Here, we review the role of IL-25 as an important factor in controlling the initiation and severity of the type-2 response, and as an alternative therapeutic target to the type-2 cytokine family, for the treatment of allergic asthma. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.
 
Article
The present study evaluated protein oxidation, alteration in hydroxysteroid dehydrogenases (3β- and 17β HSD) in testes and serum hormonal profiles of dietary zinc deficient Wistar rats. Pre-pubertal rats were divided into three groups: zinc control (ZC), pairfed (PF), and zinc deficient (ZD) and fed 100 ppm (ZC and PF groups) and 1.0 ppm (ZD group) zinc diet for 2- and 4-weeks. The testes from zinc deficient groups exhibited significant increase in total protein (2 weeks) and protein carbonyl (2- and 4-weeks) concentration as well as 3β- and 17β-hydroxysteroid dehydrogenase activities (4 weeks), whereas a significant decrease was recorded in total protein (testes 4 weeks; serum 2- and 4-weeks), total zinc (testes and serum 2- and 4-weeks), 3β- and 17β-hydroxysteroid dehydrogenase activities (testes 2 weeks), and serum hormonal profiles (FSH and testosterone 2- and 4-weeks). However, LH was below the detectable limits. These results reflect that zinc deficiency during pre-pubertal period affected total protein and zinc status, elevates protein oxidation, and causes dysregulation of the hydroxysteroid dehydrogenases. Low level of zinc attenuated the gonadal physiology which indicates that the metabolic regulation of testes is mediated by combined effects of a specific response (caused by decreased zinc concentration) and a nonspecific response (inhibition of gonadotrophin secretion). All these contribute to testicular dysfunction.
 
Article
Homocysteine, cytokines (IL-18, IL-6, IL-8) are involved in vascular inflammation and coronary artery disease. Homocysteine influences endothelial IL-6 and IL-8 cytokine expression and release, however, an association between homocysteine and IL-18 has not been previously investigated in endothelial/smooth muscle cells and or in coronary artery disease. We report in 9 coronary artery bypass surgery (CABG) patients a positive correlation r = 0.86 between homocysteine and IL-18 plasma levels (p < 0.05). Plasma IL-18 levels are significantly higher in those patients with elevated homocysteine compared to those with normal levels (p < 0.02; 153 +/- 19 pg/ml versus 116 +/- 14 pg/ml respectively). Our in vitro cell culture studies suggest that the source of IL-18 in CABG patients with elevated homocysteine is not from vascular smooth muscle or endothelial cells.
 
Article
Decomposition of lipid hydroperoxides (LOOH) is known to generate toxic products capable to induce tissue injury. We have recently confirmed that decomposition of LOOH into peroxyl radicals is a potential source of singlet oxygen ((1)O(2) in biological system. Using (18)O-labeled linoleic acid hydroperoxide (LA(18)O(18)OH) in the presence of Ce(4+) or Fe(2+), we observed the formation of (18)O-labeled (1)O(2) ((18)[(1)O(2)]) by chemical trapping of (1)O(2) with 9,10-diphenylanthracene (DPA) and detecting the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS). (18)O-Labeled alcohol and ketone were also detected providing further evidence for the generation of (1)O(2) by the Russell mechanism. Similarly the reaction of LA(18)O(18)OH with peroxynitrite also generated (18)[(1)O(2)].In conclusion, these results indicates that the use of (18)O-labeled LOOH associated with HPLC-MS/MS can be an useful tool to clarify mechanistic features involved in the reaction of LOOH in biological media.
 
Article
Induction of xenobiotics metabolizing enzymes is related to the formation and chemoprevention of cancer. Since cytochrome P450s (CYPs) including CYP1A subfamily metabolize certain pro-carcinogens to their ultimate forms, down-regulation of CYP1As by food factors leads to the prevention of cancer. Mushroom polysaccharides, especially beta-glucans such as lentinan from Lentinus edodes, possess the anti-tumor and immunomodulating activities through the cytokine production from immunocytes. Recent our studies have demonstrated that lentinan suppresses hepatic CYP1As expression in the both constitutive and inducible levels through the production of tumor necrosis factor-alpha and an increase in the DNA-binding activity of nuclear factor-kappaB. This paper discusses on the effective lentinan dosage and route of administration for suppression of CYP1As.
 
Article
High resolution 1H NMR spectroscopy has been employed to investigate the detection and quantification of the illicit "date-rape" drug gamma-hydroxybutyrate (GHB) in both human saliva and a commonly-consumed low-alcohol beer product. Data acquired revealed that this multicomponent analytical technique provided unequivocal evidence for the detection of this agent by this technique in both of these matrices, i.e., all three of its resonances [those ascribable to the alpha-CH2 (t, delta=2.25 ppm), beta-CH2 (tt, delta=1.81 ppm) and gamma-CH2 (t, delta=3.61 ppm) group protons] were present in spectra acquired on human saliva, and two of these (the alpha- and beta-CH2 group signals) in the beverage product examined, the latter observation attributable to overlap of the gamma-CH2 1H resonance with those of carbohydrates. Since good linear calibration relationships between the intensities of each of the NMR-visible signals and added GHB concentration (the former normalised to that of an external 3-trimethylsilyl [2,2,3,3-2H4]- propionate standard present in a coaxial NMR tube insert) were observed, this illicit drug is also readily quantifiable in such multicomponent samples. Our data demonstrate the advantages offered by this technique when applied to the analysis of illicit drugs in multicomponent sample matrices such as human biofluids and beverage products.
 
Article
Bone resorption is known to accelerate during the onset of several disorders, including osteoporosis (OP) and rheumatoid arthritis (RA). Some epidemiological surveys have suggested that a high intake of vegetables and fruits has an inverse relation to such disease incidence, though the number of active constituents elucidated thus far is limited. In the present study, we examined the efficacy of various food phytochemicals using two animal models. First, female ddY mice were ovariectomized (OVX) or sham-operated (sham), after which five different compounds (phenethyl isothiocyanate, zerumbone, auraptene, 1'-acetoxychavicol acetate, and nobiletin) were administered separately to OVX mice with a mini-osmotic pump at doses of 0.25 or 0.5 mg/day for 4 weeks, with 17beta-estradiol (E_{2}, 0.03 microg/day) used as a positive control. Nobiletin, in contrast to the other tested phytochemicals, significantly (P<0.05) suppressed the reduction of whole bone mineral density by 61%, which was comparable to or higher than the efficacy of E_{2}. Next, nobiletin given as an i.p. administration at 20 mg/kg of body weight, but not 2 mg/kg, to male DBA/1J mice every 2 days for 12 days led to a marked decrease in type II collagen-induced arthritis by 45% (P < 0.05). Furthermore, the flavonoid (4-50 microM) attenuated receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells, as detected by tartarate-resistant acid phosphatase activity and microscopic observations. Of note, nobiletin also suppressed RANKL-activated extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2, and p38 mitogen-activated protein kinase activities, and thereby regulated the promoter activation of nuclear factor kappaB (NFkappaB) and activator protein-1, key transcription factors for differentiation. Together, our results suggest that nobiletin is a promising phytochemical for the prevention or treatment of osteoclastogenesis-related disorders, including OP and RA, with reasonable action mechanisms.
 
Article
1 alpha, 25(OH)2 vitamin D3 (1,25(OH)2D3) is a potent hormone, stimulating bone cell growth and differentiation. In order to detect novel targets for 1,25(OH)2D3 action, we applied differential display PCR (ddPCR) to human fetal osteoblasts (FOB cells). By ddPCR analysis, we identified the selenoprotein thioredoxin reductase (TRR) as a 1,25(OH)2D3-responsive gene. In FOB cells, the response of TRR mRNA steady state levels to 1,25(OH)2D3 was fast and transient. Maximal stimulation was observed after one hour of 1,25(OH)2D3 treatment, thereafter TRR steady state mRNA levels declined to control levels. This transient response of TRR mRNA was not reflected at the TRR enzyme activity level upon treatment with 1,25(OH)2D3 for up to 48 h. Sodium selenite added to differentiated FOB cells increased TRR enzyme activity 2.6-fold, whereas no selenite effect on TRR mRNA steady state levels was measurable. Our data might provide a link between the induction of a differentiation program by 1,25(OH)2D3 and the expression of the selenium responsive TRR system in human osteoblasts.
 
Article
Endothelial cells respond to hypoxia by decreased degradation of hypoxia-inducible factor 1alpha (HIF-1alpha), accumulation of which leads to increased transcription of numerous proteins involved in cell growth and survival. Ascorbic acid prevents HIF-1alpha stabilization in many cell types, but the physiologic relevance of such effects is uncertain. Given their relevance for angiogenesis, endothelial cells in culture were used to evaluate the effects of ascorbate on HIF-1alpha expression induced by hypoxia and the hypoxia mimic cobalt. Although EA.hy926 cells in culture under oxygenated conditions did not contain ascorbate, HIF-1alpha expression was very low, showing that the vitamin is not necessary to suppress HIF-1alpha. On the other hand, hypoxia- or cobalt-induced HIF-1alpha expression/stabilization was almost completely suppressed by what are likely physiologic intracellular ascorbate concentrations. Increased HIF-1alpha expression was not associated with significant changes in expression of the SVCT2, the major transporter for ascorbate in these cells. Cobalt at concentrations sufficient to stabilize HIF-1alpha both oxidized intracellular ascorbate and induced an oxidant stress in the cells that was prevented by ascorbate. Whereas the interaction of ascorbate and cobalt is complex, the presence of physiologic low millimolar concentrations of ascorbate in endothelial cells effectively decreases HIF-1alpha expression and protects against cobalt-induced oxidant stress.
 
Article
Hyaluronan (HA) fragments produced by degradation of native highly polymerized HA during inflammation may exacerbate proinflammatory responses in different pathologies. In contrast, the nucleoside adenosine (ADO) interacting with cell surface adenosine receptors A(2A) R, A(2B) R, A(1,) and A(3) , acts as endogenous modulator of the inflammation. The engagement of high-affinity A(2A) R by ADO activates a pathway leading to increased cAMP production. Elevated levels of cAMP associate with the activation of protein kinase A (PKA) able to inhibit NF-kB, hence exerting anti-inflammatory activity. In this study the effect of ADO treatment in normal murine chondrocytes stimulated with interleukin-1beta (IL-1beta) was investigated. mRNA and related protein levels were measured for enzymes, receptors and pro-inflammatory cytokines TNF-alpha, IL-6 and Il-18. IL-1beta stimulation significantly up-regulated HA levels, its fragmentation, cAMP, PKA, cytokine levels, and activated NF-kB. ADO treatment increased cAMP and PKA levels, while reduced NF-kB activation and cytokine levels. HA inhibition by specific synthetic HA blocking peptide (Pep-1) reduced IL-1beta action but not ADO activity. While A(2A) R inhibition by specific small interference RNA (siRNA) increased inflammation and decreased cAMP and PKA levels. This study suggests that HA is partially responsible for the up-regulation of proinflammatory cytokines in chondrocytes and that endogenous/exogenous ADO may reduce inflammation via PKA. © 2012 International Union of Biochemistry and Molecular Biology, Inc.
 
Article
Transient activation of fibroblasts or fibroblast-like cells to proliferate and to produce elevated quantities of extracellular matrix is essential to fibrosis. This activation is regulated by several cytokines produced by various inflammation-associated cells. Among these, transforming growth factor beta1 (TGFβ1) is considered of major importance. Many studies have shown that lipid peroxidation play a key role in the initiation and progression of fibrosis in different organs. In fact, 4-hydroxy-2,3-nonenal (HNE), the major aldehydic product of lipid peroxidation, is able to induce TGFβ1 expression and synthesis, and activation of activator protein-1 (AP-1) transcription factor. In this study, using the murine macrophage line J774-A1, we show that these effects are strictly related to the chemical structure of HNE, since neither 2-nonenal nor nonanal are biologically active to the same extent. Moreover, we demonstrate that HNE can indeed contribute to the onset of fibrosis by stimulating AP-1 binding to DNA and consequently inducing TGFβ1 expression, since thiol-group reagents, such as N-ethylmaleimide and 4-(chloro-mercuri)-benzenesulfonic acid, that down-modulate HNE entrance and localisation inside the cell, prevent both phenomena. The possibility to control fibrogenic cytokine levels by means of antioxidant or dietetic treatments opens new potential pharmacological and nutritional horizons in the treatment of many chronic diseases characterised by excessive fibrosis.
 
Article
2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) identified as a new bacterial oxidative stress substance (Ostrovsky D. et al. (1993) Biochem. J., 295, 901-902) was shown to accumulate in Corynebacterium (Brevibacterium) ammoniagenes cells aerobically cultivated in peptone-yeast extract-glucose broth on heating for 1 hour at 45 degrees C. The enzyme(s) responsible for MEC biosynthesis is evidently oxidized for activation and is completely loosing its activity on anaerobic incubation at this temperature in an hour. Salt stress or drying did not provoke the MEC biosynthesis.
 
Article
The concept that the location of an AAA-ATPase associated with the plant plasma membrane may be indicative of a functional relationship to growth or cell enlargement by analogy with roles in physical membrane displacements as proposed for AAA-ATPases associated with internal membranes was tested. A plant growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans was utilized in a completely cell-free system. The rate of enlargement was accelerated by the synthetic plant growth factor 2,4-dichlorophenoxyacetic acid (2,4-D) in a log dose-dependent manner and was increased approximately 2-fold with the addition of 1 microM 2,4-D plus 100 microM ATP compared to 100 microM ATP alone, 1 microM 2,4-D alone or no additions. The cell-free enlargement was inhibited by AAA-ATPase-specific antisera and by CoCl2, an inhibitor specific for AAA-ATPases. The responsible ATP site appears to be on the inside of the cell, since right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination.
 
Article
Interleukin-21 (IL-21), a cytokine produced by various subsets of activated CD4+ T cells, regulates multiple innate and adaptive immune responses. Indeed, IL-21 controls the proliferation and function of CD4+ and CD8+ T lymphocytes, drives the differentiation of B cells into memory cells and Ig-secreting plasma cells, enhances the activity of natural killer cells and negatively regulates the differentiation and activity of regulatory T cells. Moroever, IL-21 can stimulate nonimmune cells to synthesize various inflammatory molecules. Excessive production of IL-21 has been described in many human chronic inflammatory disorders and there is evidence that blockade of IL-21 helps attenuate detrimental responses in mouse models of immune-mediated diseases. In this article we briefly review data supporting the pathogenic role of IL-21 in immune-inflammatory pathologies and discuss the benefits and risks of IL-21 neutralization in patients with such diseases. © 2013 BioFactors, 2013.
 
Article
A human glutathione peroxidase cDNA has been used as a probe to hybridize to DNAs isolated from human - rodent somatic cell hybrids that have segregated human chromosomes. A 609 bp probe which contains the entire coding region hybridizes to human chromosomes 3, 21 and Xp. Fragments of the cDNA coding sequence and of the 3' untranslated region were also used as probes. These fragments hybridized to each of the three chromosomes with the same efficiency, suggesting similarity between the loci, whereas an intronic probe detected only the gene on chromosome 3. The general organization of each gene was determined from the hybridization data. The data suggest that the locus on chromosome 3 is a functional gene containing a single intron and a pattern of restriction sites identical to those found in the cDNA coding sequence. The data also suggest that the sequences on chromosomes X and 21 have equal conservation of the 3' untranslated and coding sequences but do not contain introns, providing evidence that the latter two sequences are processed pseudogenes. A simple two allele polymorphism in PvuII digests was detected at the locus on chromosome 21.
 
Article
Little is known about the direct effect of broccoli sprouts on human health. So we investigated the effect of broccoli sprouts on the induction of various biochemical oxidative stress markers. Twelve healthy subjects (6 males and 6 females) consumed fresh broccoli sprouts (100 g/day) for 1 week for a phase 1 study. Before and after the treatment, biochemical examination was conducted and natural killer cell activity, plasma amino acids, plasma PCOOH (phosphatidylcholine hydroperoxide), the serum coenzyme Q(10), urinary 8-isoprostane, and urinary 8-OHdG (8-hydroxydeoxyguanosine) were measured. With treatment, total cholesterol and LDL cholesterol decreased, and HDL cholesterol increased significantly. Plasma cystine decreased significantly. All subjects showed reduced PCOOH, 8-isoprostane and 8-OHdG, and increased CoQ(10)H(2)/CoQ(10) ratio. Only one week intake of broccoli sprouts improved cholesterol metabolism and decreased oxidative stress markers.
 
Article
Human amyloid beta peptides Abeta1-40 and Abeta1-42 exhibit NADH oxidase activity with regular oscillations at intervals of ca 6 min. In the presence of copper, the oscillations in Abeta1-40 and Abeta1-42 become more pronounced and now assume a period length of 24 min. In the presence of copper, the oscillations are similar to those observed with NADH oxidase activities of cell surface ECTO-NOX proteins in general including a period length of 24 min. Solutions of copper sulphate in the presence of all the reagents except for the peptides did not exhibit the oscillatory behavior. NOX proteins have been reported previously to have properties of prions and to form amyloid rods of indeterminant length similar to those formed by the 39-43 residue amyloid beta proteins (Abeta). In this report, we demonstrate a second similarity between ECTO-NOX proteins and amyloid beta, that of an oscillating NADH oxidase activity with a period length of 24 min when assayed in the presence of copper.
 
Article
Iron is one of the trace elements playing a key role in the normal brain metabolism. An excess of free iron on the other hand is catalyzing the iron-mediated oxygen radical production. Such a condition might be a harmful event leading perhaps to serious tissue damage and degeneration. Therefore, during evolution a complex iron sequestering apparatus developed, minimizing the amount of redox-reactive free iron. However, this system might be severely disturbed under pathophysiological conditions including hypoxia or anoxia. Since little is known about the non-transferrin-mediated iron metabolism of the brain during anoxia/reoxygenation, we tested the ability of the microglial cell line RAW 264.7 to take up iron independently of transferrin under various oxygen concentrations. Microglial cells are thought to be the major player in the maintenance of the extracellular homeostasis in the brain. Therefore, we investigated the iron metabolism of microglial cells employing radiolabeled ferric chloride. We tested the uptake of iron under normoxic, anoxic and postanoxic conditions. Furthermore, the amount of ferritin was measured by immunoblotting. We were able to show that iron enters the microglial cell line in the absence of extracellular transferrin under normoxic, anoxic and postanoxic conditions. Interestingly, the amount of ferritin is decreasing in the early reoxygenation phase. Therefore, we concluded that microglia is able to contribute to the brain iron homeostasis under anoxic and postanoxic conditions.
 
Article
Oxidative stresses are involved in the process of chronic inflammatory diseases. The objective of this study was to evaluate and compare the anti-inflammatory activity of major dietary antioxidants. Murine RAW264.7 macrophages (4 x 10(6) cells) were incubated in DMEM containing 10% FBS supplemented with 5 to 20 microM of alpha-tocopherol, beta-carotene or quercetin for 24 hrs, and then treated with LPS for 16 hrs. The medium was collected to measure TNF-alpha, IL-6, PGE2 and nitrite. Expressions of cyclooxygenase-2(COX-2) and inducible nitric oxide synthase(iNOS) were determined using the harvested cells. Results indicate that TNF-alpha and IL-6 accumulations were significantly reduced by 5 to 20 microM quercetin treatment, and 20 microM of alpha-tocopherol treatment. Nitrite release was significantly reduced by 5 microM quercetin treatment. However, PGE2 accumulation was not affected by any of the antioxidants used. Expressions of COX-2 and iNOS were effectively reduced by 5 microM quercetin treatment. These findings indicate that dietary antioxidants possess significant anti-inflammatory activities, and quercetin is the most potent antioxidant.
 
Article
Current intakes of very long chain omega-3 fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are low in most individuals living in Western countries. A good natural source of these fatty acids is seafood, especially oily fish. Fish oil capsules contain these fatty acids too. Very long chain omega-3 fatty acids are readily incorporated from capsules into transport, functional, and storage pools. This incorporation is dose-dependent and follows a kinetic pattern that is characteristic for each pool. At sufficient levels of incorporation, EPA and DHA influence the physical nature of cell membranes and membrane protein-mediated responses, eicosanoid generation, cell signaling and gene expression in many different cell types. Through these mechanisms, EPA and DHA influence cell and tissue physiology, and the way cells and tissues respond to external signals. In most cases, the effects seen are compatible with improvements in disease biomarker profiles or in health-related outcomes. As a result, very long chain omega-3 fatty acids play a role in achieving optimal health and in protection against disease. Long chain omega-3 fatty acids protect against cardiovascular morbidity and mortality, and might be beneficial in rheumatoid arthritis, inflammatory bowel diseases, childhood learning, and behavior, and adult psychiatric and neurodegenerative illnesses. DHA has an important structural role in the eye and brain, and its supply early in life is known to be of vital importance. On the basis of the recognized health improvements brought about by long chain omega-3 fatty acids, recommendations have been made to increase their intake.
 
Article
Adenosine is known to produce biphasic effects in the renal tissues via adenosine receptors. However, the presence of more than one subtype of adenosine receptor on a type of kidney cell or tissue has not been conclusively demonstrated. To address this issue, we investigated the presence of A1 and A2 adenosine receptors in baby hamster kidney (BHK) cells by use of radioligand binding and the reverse transcription-polymerase chain reaction. Ligand binding studies with (3H)-DPCPX revealed a single class of binding site with a K(D) of 9.2 +/- 2.0 nM, a Bmax of 1.7 +/- 0.2 pmol/mg protein and a pharmacological profile characteristic of A1 adenosine receptor on the BHK cell membrane. As the presence of A2 adenosine receptors could not be conclusively determined by ligand binding studies, the more sensitive method of RT-PCR was employed. The presence of A1 and A2B adenosine receptors was detected by RT-PCR with specific primers and the subsequent sequencing of the resultant amplification product. The sequences obtained were 75-90% homologous to the respective adenosine receptor mRNA of rat, mouse and human.
 
Article
Resveratrol (Res) and its two natural analogs that are also related to Res metabolism, piceatannol (Pic) and 3,5,4'-trans-trimethoxystilbene (TMS), were compared in their ability to suppress lipopolysaccharide (LPS)-induced production of proinflammatory tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and to induce anti-inflammatory heme oxygenase-1 (HO-1) expression in RAW264.7 macrophages. At non-cytotoxic concentrations, they differentially suppressed LPS-induced production of TNF-α and IL-1β; the relative potency for suppression of TNF-α and IL-1β production was Pic > Res > TMS. Res and Pic differentially induced HO-1 expression; Pic, which possesses four hydroxyl groups, was more active in inducing HO-1 expression than Res that contains three hydroxyl groups. TMS, which has none of hydroxyl groups, failed to induce HO-1 expression. These findings suggest that the hydroxyl groups of Res analogs are important for suppression of TNF-α and IL-1β production and HO-1 expression. Interestingly, protoporphyrin-IX, a competitive inhibitor of HO-1 activity, partly attenuated the inhibitory effects of Res and Pic (but not TMS) on TNF-α and IL-1β production, suggesting that suppression of TNF-α and IL-1β production correlates at least in part with HO-1 expression. Overall, the ability of Res analogs to induce HO-1 expression may provide one of possible mechanisms of their anti-inflammatory action. © 2013 BioFactors, 2013.
 
Article
(Z)-3,5,4'-Trimethoxystilbene is a natural polyphenol present in five different plants, Virola cuspidata, Virola elongata, Centipeda minima, Schoenus nigricans and Rheum undulatum. This molecule was prepared in a three-step sequence in good overall yield. The isomerisation from the (E)- to (Z)-isomer is performed using UV irradiation. Biological investigations were conducted on a human colon cancer cell line (Caco-2) with anti-mitotic activities. Growth was completely arrested at an added 0.4 microM level of (Z)-3,5,4'-trimethoxystilbene. This agent is 100-fold more active than resveratrol or (E)-3,5,4'-trihydroxystilbene, and the mechanism of this process involves an inhibition of tubulin polymerisation in a dose dependent manner.
 
Article
Molybdopterin guanine dinucleotide was studied by 31P-NMR in the free, iodoacetamide derivatized form [di(carboxamidomethyl)molybdopterin] and in the native state in the dimethyl sulfoxide reductase from Rhodobacter sphaeroides. The spectra confirm the presence of a pyrophosphate moiety in the cofactor molecule. Comparison of the spectrum of the free pterin with that of the protein-bound cofactor reveals a substantial upfield shift of the 31P resonances in the enzyme-bound form with respect to the free form. This shift is attributed to differences in the bond and torsional angles of the phosphates. The spectrum of the protein suggests significant coupling between the two phosphorus nuclei with coupling constants of approximately 200 Hz. Comparison of the 31P-NMR spectra of molybdopterin guanine dinucleotide and flavin adenine dinucleotide suggests that the two cofactors have similar conformations in both their free and protein-bound forms.
 
Article
Yerba mate tea (YMT) has a chemopreventive role in a variety of inflammatory diseases. The objective was to determine the capability of YMT and mate saponins to prevent azoxymethane (AOM)-induced colonic inflammation in rats. YMT (2% dry leaves, w/v, as a source of drinking fluid) (n = 15) and mate saponins (0.01% in the diet, at a concentration present in one cup of YMT) (n = 15) were given ad libitum to rats 2 weeks prior to AOM-injection until the end of the study; while control rats (n = 15) received a basal diet and drinking water. After 8-weeks of study, total colonic mucosa was scraped (n = 3 rats/group) and the remaining colons (n =12 rats/group) were cut into three equal sections and aberrant crypt foci (ACF) were analyzed. YMT reduced ACF formation from 113 (control group) to 89 (P < 0.05). YMT and mate saponins reduced the expression of proinflammatory molecules COX-2 and iNOS with concomitant reduction in p-p65 (P < 0.05). Immunohistochemical analysis of the formalin-fixed middle colons showed that YMT and mate saponins reduced the expression of p-p65(ser311) by 45.7% and 43.1%, respectively, in comparison to the control (P < 0.05). In addition, the expression of molecules upstream of NF-κB such as p-IκB-α and p-GSK-3β(Y216) was downregulated by YMT 24.7% and 24.4%, respectively (P < 0.05). Results suggest the mechanism involved in the chemopreventive effect of YMT and mate saponin consumption in AOM induced-colonic inflammation in rats is through inhibition of NF-κB. © 2013 BioFactors, 2013.
 
Article
We used in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and delta G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six-months of treatment with CoQ (150 mg/day), all brain MRS-measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.
 
Article
Nitric oxide (NO.) can lead to damaging or protective actions in the central nervous system. Here we consider the chemistry of the NO group and its redox-related species that can lead to these exactly opposite ends. In the neurodestructive mode, NO. reacts with superoxide anion (02.-) to form peroxynitrite (ONOO-), which leads to neuronal injury. In contrast, the reaction of the NO group with cysteine sulfhydryls on the NMDA receptor leads to a decrease in receptor/channel activity. avoidance of excessive Ca2+ entry, and thus neuroprotection. Site-directed mutagenesis of recombinant NMDA receptor subunits has recently increased our knowledge of such redox modulation by NO. Transfer of the NO group to cysteine sulfhydryls on the NMDA receptor or other proteins, known as S-nitrosylation, is becoming recognized as a ubiquitous regulatory reaction, skin to phosphorylation, and represents a form of redox modulation in diverse tissues including the brain.
 
Article
The NDR/LATS family of kinases is a subgroup of the AGC group of protein kinases and is conserved from lower eukaryotes to humans. Like other AGC kinases, NDR/LATS kinases require phosphorylation of conserved Ser/Thr residues for activation. On the one hand, binding of the coactivator MOB to NDR/LATS allows autophosphorylation. On the other hand, MST kinases directly phosphorylate NDR/LATS kinases. In addition to our understanding of the molecular activation mechanisms, recent studies have shown that LATS kinases play a central role in Hippo/SWH (Salvador/Warts/Hippo) tumor suppressor pathways, which coordinate cell proliferation and apoptosis by regulating proto-oncogenes, such as YAP and TAZ. In this review, we summarize current knowledge of Merlin/MST/SAV/MOB/LATS/NDR/YAP/TAZ networks (also termed mammalian Hippo signaling) and their roles in mammalian cellular transformation.
 
Article
Factor 390 (F390), an adenylylated or guanylylated derivative of the methanogen coenzyme factor 420 (F420), was previously detected in Methanobacterium thermoautotrophicum cells exposed to air. Of six other methanogenic species that have now been tested, only Methanobacterium formicicum was found to produce F390 upon oxygen exposure. Aerobic conditions led to an immediate cessation of methanogenesis, whereas only 51% of cellular F420 was slowly converted to F390 over 4 h in Mb.formicicum at 37 degrees C. F390 formation is reversible. When oxidized cells were re-introduced into anoxic medium, F390 reverted to F420 prior to recovery of methanogenesis. Anaerobic cultures of Mb.formicicum were subjected to alternative stresses such as exposure to heavy metals, methanogenesis inhibitors and eubacterial alarmone-producing chemicals; however, only oxygen was found to induce F390 formation.
 
Article
In this study, the CYP3A inducer pregnenolone-16alpha-carbonitrile (PCN) and the CYP3A inhibitor ketoconazole (KCZ) were used to investigate whether the metabolism of alpha-tocopherol to its metabolite, alpha-carboxyethyl hydroxychroman (alpha-CEHC), is CYP3A-dependent in rats. In experiment 1, two groups of Wistar rats were fed for 3 wk with either a basal diet (containing 50~ppm of alpha-tocopherol) or the same diet containing 10-fold more alpha-tocopherol. In the last 3 days, each group was divided into 2 subgroups which were given a single i.p. injection of either PCN at 75 mg/kg/d (P50 & P500 groups) or DMSO (D50 & D500 groups). The liver TBARS concentration was highest in the P50 group. Two-way ANOVA analysis showed that alpha-tocopherol levels in the plasma and liver were both significantly decreased by PCN (p < 0.0001), as were alpha-CEHC levels in the urine (p = 0.0004). In experiment 2, alpha-tocopherol levels in the liver were increased and alpha-CEHC excretion in the urine decreased in the Wistar rats fed with KCZ containing diet. In experiment 3, Wistar rats administered with dexamethasone (DEX) significantly decreased alpha-tocopherol levels in the plasma and liver and alpha-CEHC levels in the urine. These data showed CYP3A is not a major contributor of the metabolism of alpha-tocopherol to alpha-CEHC. Nevertheless, vitamin E status was markedly reduced by CYP3A inducers due to increased lipid peroxidation and this would increase the consumption of alpha-tocopherol in the liver.
 
Article
Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.
 
Article
The intracellular level of taurine is maintained both by the taurine transporter (TAUT) and by endogenous synthesis from Met and Cys. We investigated in the present study the regulation of TAUT and of cysteine dioxygenase (CDO), one of the major taurine biosynthetic enzymes, in 3T3-L1 adipocytes. The TAUT activity, expression of TAUT and CDO mRNA were up-regulated by hypertonicity. In contrast, the TAUT activity, expression of TAUT and CDO mRNA were down-regulated by taurine-rich conditions. Furthermore, it was indicated that the up-regulation of TAUT activity resulted from the increased number of expressed TAUT, and not by the change in affinity of TAUT. On the other hand, the taurine-induced down-regulation of TAUT activity resulted not only from a decrease in the number of expressed TAUT but also from a decrease in their affinity. These results suggest that murine TAUT and CDO were cooperatively regulated in response to hypertonicity and taurine-rich conditions.
 
Article
Adipocyte dysfunction is strongly associated with the development of insulin resistance and diabetes, and regulation of adipogenesis is important in prevention of diabetes. We prepared a 100% methanol fraction of methanolic extract from unripe kiwi fruit (Actinidia deliciosa), designated KMF (kiwi fruit methanol fraction). When applied to 3T3-L1 preadipocyte cells, KMF promoted adipocyte differentiation, increased glycerol-3-phosphate dehydrogenase (GPDH) activity, and increased triglyceride (TG) content. KMF markedly increased mRNA expression of peroxisome proliferator-activated receptor gamma (PPARgamma)-the master adipogenic transcription factor-and its target genes. Moreover, KMF increased mRNA expression and protein secretion of adiponectin, whereas mRNA expression and secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were decreased. Compared with troglitazone, KMF decreased the production of reactive oxygen species (ROS) and nuclear factor-kappaB (NFkappaB) activation. Glucose uptake was stimulated by KMF in differentiated 3T3-L1 adipocytes. Taken together, these results indicate that KMF may exert beneficial effects against diabetes via its ability to regulate adipocyte differentiation and function.
 
Article
Polyphenolic compounds were examined for their effects on suppressing adipocyte differentiation in 3T3-L1 cells. Most polyphenolic compounds inhibited adipocyte development from 3T3-L1 cells to some extent. Among them, rutin was the most effective in suppressing adipocyte differentiation in a dosage dependant manner. Activity of glycerol-3-phosphate dehydrogenase (GPDH), which has a central position in lipogenesis in adipose cells, was also decreased by rutin addition at the induction stage. RT-PCR results demonstrated that mRNA expression of adipogenic transcription factors such as peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha) in 3T3-L1 cells were remarkably down regulated by rutin treatment. For further investigation on anti-adipogenic activities of rutin, it was orally administered (25 and 50 mg/kg b.w/daily) with high-fat diet (64.4% of total calories as fat) to C57BL/6 mice. Body weight gains were less in high-fat diet + rutin fed groups (HFR) than high-fat diet alone fed group (HF) after 4 weeks. Total cholesterol contents in blood were significantly lower in HFR groups. When mRNA expressions of PPARgamma and C/EBPalpha in hepatocytes were compared between the control HF and HFR groups, their expressions in hepatocytes of HFR groups were significantly suppressed. These results indicate that rutin inhibits adipogenic development in pre-adipocytes and hepatocytes by down regulating expressions of key adipogenic transcription factors.
 
Article
To understand the mechanisms of the anti-obesity effects of dietary caffeine, the effects of caffeine and its metabolites on adipocyte differentiation and insulin-stimulated glucose uptake in murine 3T3-L1 adipocytes were investigated. Caffeine did not inhibit the differentiation of 3T3-L1 pre-adipocytes to mature adipocytes, but it did suppress the intracellular lipid accumulation after complete differentiation in a dose-dependent manner (0.125-1.0 mM). This effect was also observed in 1,3,7-trimethyluric acid- 3,7-dimethyluric acid- and 5-acetylamino-6-formylamino-3-methyluracil-treated cells. Caffeine also inhibited insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes in a dose-dependent manner. Treatment with theophylline, paraxanthine, 1-methylxanthine (MX), 3-MX, or 7-MX also inhibited glucose uptake in differentiated adipocytes. These results suggest that the anti-obesity activity of dietary caffeine is due to the additive and/or synergistic inhibitory effects of caffeine and its metabolites on intracellular lipid accumulation and that caffeine does not affect adipocyte differentiation.
 
Article
Withaferin A (WA), a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera (also called ashwagandha) has been reported to have anti-tumor, anti-angiogenesis, and pro-apoptotic activity. We investigated the effects of WA on viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. Pre- and post-confluent preadipocytes and mature adipocytes were treated with WA (1-25 microM) up to 24 hrs. Viability and apoptosis were measured by CellTiter-Blue Cell Viability Assay and single strand DNA ELISA Assay, respectively. WA decreased viability and induced apoptosis in all stages of cells. Induction of apoptosis by WA in mature adipocytes was mediated by increased ERK1/2 phosphorylation and altered Bax and Bcl2 protein expression. The effect of WA on adipogenesis was examined by AdipoRed Assay after treating with WA (0.1-1 microM) during the differentiation period. WA decreased lipid accumulation in a dose-dependent manner and decreased the expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte fatty acid binding protein. The effects on apoptosis and lipid accumulation were also confirmed with Hoechst staining and Oil Red O staining, respectively. These results show that WA acts on adipocytes to reduce cell viability and adipogenesis and also induce apoptosis.
 
Article
This study examined the effects of fargesin, a neolignan isolated from Magnolia plants, on obesity and insulin resistance and the possible mechanisms involved in these effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Fargesin promoted the glucose uptake in 3T3-L1 adipocytes. In HFD-induced obese mice, fargesin decreased the body weight gain, white adipose tissue (WAT), and plasma triglyceride, non-esterified fatty acid and glucose levels, and improved the glucose tolerance. Fargesin increased glucose transporter 4 (GLUT4) protein expression and phosphorylation of Akt, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in both 3T3-L1 adipocytes and WAT of HFD-induced obese mice. Fargesin also decreased the mRNA expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1 (CPT-1), uncoupling protein-2 (UCP-2) and leptin in WAT. Taken together, the present findings suggest that fargesin improves dyslipidemia and hyperglycemia by activating Akt and AMPK in WAT.
 
Article
Genistein (G), an isoflavone, and guggulsterone (GS), the active substance in guggulipid, have been reported to possess therapeutic effects for obesity. In the present study, we investigated the effects of combinations of G plus GS on apoptosis and adipogenesis in 3T3-L1 cells. In mature adipocytes, G and GS individually caused apoptosis, but combination of G plus GS significantly increased apoptosis, more than either compound alone. Furthermore, G plus GS caused a greater increase in procaspase-3 cleavage, Bax expression, cytochrome c release, and proteolytic cleavage of PARP than either compound alone. In maturing preadipocytes G and GS each suppressed lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. These results suggest that combination of genistein and guggulsterone may exert anti-obesity effects by inhibiting adipogenesis and inducing apoptosis in adipocytes.
 
Top-cited authors
Young-Sup Lee
  • Kyungpook National University
Adeeb Shehzad
  • National University of Sciences and Technology
Stephan Schiekofer
  • Bezirksklinikum Regensburg / Regensburg University
Bharat Aggarwal
  • University of Texas MD Anderson Cancer Center
Gorkem Kismali
  • Ankara University