BBA - General Subjects (v.1670, #3)

Antioxidant effects of American ginseng berry extract in cardiomyocytes exposed to acute oxidant stress by Zuo-Hui Shao; Jing-Tian Xie; Terry L. Vanden Hoek; Sangeeta Mehendale; Han Aung; Chang-Qing Li; Yimin Qin; Paul T. Schumacker; Lance B. Becker; Chun-Su Yuan (165-171).
It is postulated that antioxidant properties of American ginseng root mediate its cardioprotective actions. The antioxidant capabilities of the American ginseng root have been demonstrated previously, however, the berry of the American ginseng has not yet been evaluated. In this study, we tested the American ginseng berry extract (AGBE) for its antioxidant effects in cell-free chemical systems using H2O2/FeSO4 to generate hydroxyl radicals which were measured by a fluorescent probe, 2′, 7′-dichlorofluorescin diacetate (DCFH/DA). Xanthine/xanthine oxidase was used to generate superoxide anion, which was measured by a fluorescent probe dihydroethidium (DHE). We found that AGBE decreased fluorescence significantly, suggesting that AGBE scavenges oxygen free radicals. We further tested whether AGBE (0.1–1 mg/ml) can protect cardiomyocytes from oxidative injury induced by exogenous or endogenous oxidants. Cells were exposed to either H2O2 or antimycin A (a mitochondrial electron transport chain site III inhibitor that augments mitochondrial oxidant production). The resulting oxidant stress was measured using DCFH/DA and the cell death was assessed using propidium iodide staining. Pretreatment with AGBE (1 mg/ml) significantly attenuated DCF fluorescence by 49% or 85% and reduced cell death by 59% or 63% in cells exposed to H2O2 or antimycin A, respectively. When the effects of extracts from berry and root of American ginseng were compared in cardiomyocytes exposed to antimycin A, we observed that AGBE conferred greater antioxidant protection at the same dose. We conclude that AGBE is a potent antioxidant that protects cardiomyocytes against oxidant-mediated injury and this protection is partly mediated by its free radical scavenging properties.
Keywords: American ginseng; Berry; Root; Cardiomyocyte; Hydroxyl radical; Hydrogen peroxide; Antimycin A; Cell death; Oxidant stress;

Optimization of antitumor efficacy and safety of in vivo cytokine gene therapy using RGD fiber-mutant adenovirus vector for preexisting murine melanoma by Yuka Okada; Naoki Okada; Hiroyuki Mizuguchi; Koichi Takahashi; Takao Hayakawa; Tadanori Mayumi; Nobuyasu Mizuno (172-180).
We previously reported that RGD fiber-mutant adenovirus vector (AdRGD) was a very useful vector system for in vivo cytokine gene therapy for established murine B16BL6 melanoma. However, intratumoral administration of AdRGD expressing tumor necrosis factor α (AdRGD-TNFα) at high dose revealed not only the dramatic reinforcement of anti-tumor effect but also serious adverse effects, such as body weight reduction and sudden death, caused by high-level TNF-α leakage from the tumor into circulation. These results strongly suggested that the determination of ‘limiting dose’, which demonstrated therapeutic effectiveness without adverse effect, against each vector was important for the development of appropriate cytokine gene therapy. In the present study, we investigated the efficacy and the safety of AdRGD expressing interleukin-12 (AdRGD-IL12) in murine melanoma model, and determined its limiting dose. Moreover, we demonstrated that combination therapy using AdRGD-IL12 and AdRGD-TNFα at limiting doses or less could achieve more effective tumor regression without adverse effects. Therefore, we conclude that a combination of multiple AdRGD expressing cytokines having distinct anti-tumor mechanisms can contribute to the establishment of in vivo cytokine gene therapy for melanoma, which possesses both excellent efficacy and high safety.
Keywords: Adenovirus vector; Melanoma; Gene therapy; Fiber-mutant; IL-12; TNF-α;

Tetrahydrobiopterin biosynthesis in white and brown adipose tissues is enhanced following intraperitoneal administration of bacterial lipopolysaccharide by Kentaro Fujiwara; Keiji Mori; Yoko S. Kaneko; Akira Nakashima; Akio Nagasaka; Mitsuyasu Itoh; Akira Ota (181-198).
Tetrahydrobiopterin is an essential cofactor for nitric oxide synthase (NOS). This study was undertaken to examine the effects of intraperitoneally injected lipopolysaccharide on tetrahydrobiopterin biosynthesis in murine white and brown adipose tissues. Tetrahydrobiopterin content, catalytic activity and mRNA expression level of GTP cyclohydrolase I (GCH), rate-controlling enzyme in de novo biosynthesis of tetrahydrobiopterin, in both adipose tissues were up-regulated by 500-μg lipopolysaccharide at 6 h after the injection. On the contrary, treatment of 3T3-L1 adipocytes with lipopolysaccharide alone did not affect GCH mRNA expression level, whereas the combination of lipopolysaccharide, tumor necrosis factor (TNF)-α, and interferon γ induced the increase in expression levels of GCH mRNA and CD14 mRNA. Collectively, our results showed that tetrahydrobiopterin biosynthesis can be augmented by increased GCH activity caused by a synergistic effect of lipopolysaccharide and cytokines in white and brown adipose tissues. These observations support the view that tetrahydrobiopterin biosynthesis in the adipose tissues is a target of inflammatory events triggered by peripheral LPS injection.
Keywords: Adipose tissue; Tetrahydrobiopterin; GTP cyclohydrolase I; Lipopolysaccharide; Inducible nitric oxide synthase;

A method for studying insoluble immune complexes by Boris N Khlebtsov; Gennadii L Burygin; Larisa Yu Matora; Sergei Yu Shchyogolev; Nikolai G Khlebtsov (199-207).
Two variants of a method for determining the average composition of insoluble immune complex particles (IICP) are described. The first variant is based on measuring the specific turbidity (the turbidity per unit mass concentration of the dispersed substance) and the average size of IICP determined from dynamic light scattering (DLS). In the second variant, the slope of the logarithmic turbidity spectrum (wavelength exponent) is used instead of DLS particle size. Both variants allow the average biopolymer volume fraction to be determined in terms of the average refractive index of IICP. The method is exemplified by two experimental antigen+antibody systems: (i) lipopolysaccharide–protein complex (LPPC) of Azospirillum brasilense Sp245+rabbit anti-LPPC; and (ii) human IgG (hIgG)+sheep anti-hIgG. We have found that IICP can be modeled by incompact porous particles that contain about 30% of biopolymer substance and 70% of buffer.
Keywords: Antigen–antibody interaction; Insoluble immune complex; Dynamic light scattering; Turbidimetry; Particle composition;

Sensitive immunoassays for human and rat GMFB and GMFG, tissue distribution and age-related changes by Masaaki Inagaki; Mineyoshi Aoyama; Kazuya Sobue; Naoki Yamamoto; Tetsuro Morishima; Akihiko Moriyama; Hirotada Katsuya; Kiyofumi Asai (208-216).
We developed sensitive and specific two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) using specific antibodies raised in rabbits. These assay systems enabled us to identify GMFB and GMFG (GMFs) in both human and rat samples and they were used to investigate the tissue distribution and serum concentrations of human and rat GMFs. In the case of rat, relatively high levels of GMFB were found in the central nervous system, except for the spinal cord, and in thymus and colon. Higher levels of GMFG were found in the thymus, spleen and colon. The distribution of GMFs in human was similar to that in rat. In the rat, the maximum serum concentration of GMFG was at 4 weeks of age. The decrease in its level was rapid for the first 30 days of life in both sexes. On the other hand, the concentration of GMFB in serum did not change significantly with age. Similarly, in human, the concentration of GMFG in serum was highest in the 21–30-year-old group and began to decrease rapidly in the 30-year-old group. In contrast, the concentration of GMFB did not change significantly during this period. No significant sex differences in the serum levels of GMFs were observed in human and rat. The present EIA systems are sufficiently sensitive for studying GMFs in human and rat organs.
Keywords: Glia maturation factor beta; Glia maturation factor gamma; Enzyme immunoassay; Tissue distribution; Age-related change;

Determination of binding constant of transcription factor myc–max/max–max and E-box DNA: the effect of inhibitors on the binding by Seyeon Park; Sunah Chung; Kyung-Mee Kim; Kyung-Chae Jung; Chihoon Park; Eun-Ryeong Hahm; Chul-Hak Yang (217-228).
The truncated myc and max proteins, only containing basic regions and helix–loop–helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc–max (or max–max)–DNA complex formation. The association kinetic constants (k 1 and k −1) of truncated max–max or myc–max dimer and DNA were determined as k 1=(1.7±0.6)×105 M−1 s−1, k −1=(3.4±1.2)×10−2 s−1 for max–max and DNA or k 1=(2.1±0.7)×105 M−1 s−1, k −1=(3.2±1.4)×10−2 s−1 for myc–max and DNA. The equilibrium binding constant (K 1) was determined using these kinetic parameters [K XXD=(7.8±2.6)×106 M−1 for max–max and DNA or K XYD=(6.9±2.2)×106 M−1 for myc–max and DNA]. The binding constants of myc–max or max–max dimer formation were K XX=(2.6±0.9)×105 M−1 or K XY=(1.3±0.4)×104 M−1, respectively. When truncated proteins were used, the max–max dimer formation was easier than the myc–max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc–max−DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max–max dimer and the inhibitor was 9 μM, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc–max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over-expressed human stomach cancer cell line SNU16.
Keywords: myc–max; Binding constant; Inhibitor; SNU16 cell; Target gene;

Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function by Gerald Rimbach; Peter D Weinberg; Sonia de Pascual-Teresa; Maria Garcia Alonso; Ben A Ewins; Rufus Turner; Anne Marie Minihane; Nigel Botting; Brian Fairley; Seiichi Matsugo; Yuzo Uchida; Aedin Cassidy (229-237).
Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological properties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4′-sulfate and genistein-4′-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4′-sulfate was less potent than genistein, and genistein-4′-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis.
Keywords: Genistein; Isoflavone; Sulfation; Cell adhesion; Platelet aggregation; Inflammation;