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.
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.
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.
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.
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.
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.
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. ß‐Carotene exhibits biological activity as provitamin A and ß‐carotene 15,15′‐monooxygenase (BCM) cleaves ß‐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 ß‐carotene to retinal might be accelerated in the presence of insulin resistance status, so that plasma retinol levels are increased in type 2 diabetes.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
