[Measuring anxiety with behavior analysis software and comparing human observations with EthoVision XT in the elevated plus maze paradigm.] / Not available.

INTRODUCTION: The reproduction of anxiety in laboratory animals is a renewing problem whenever a new drug is to be tested for its anxiolytic effect. Some gold-standard tests, such as the elevated plus maze test, are always considered to be used as reference. However, many controversial results for different anxiolytics were reported in elevated plus maze test. The analysis methods used by different labs could be the source of variability of the results. Human observations were the most commonly used since the 90's, when behavior analysis software appeared. In each lab, specific procedures for reducing bias in ethopharmacological experiments were implemented, but the performance of human observers was rarely compared to software assisted analysis. METHODS: Four analysts and 24 trials were involved, each analyst having to do all the analyses during which they had to register eight parameters. All trials were also analyzed with the EthoVision XT (Noldus IT, Netherlands). RESULTS: Several crucial parameters of the elevated plus maze test were significantly different between the analysts (p<0.05). The results registered by human observers were summarized and compared to the results of automatic analysis, which showed significant difference in the case of closed arm entry and total distance (p<0.001). CONCLUSIONS: In this study, it was shown that despite all precautionary measures taken to reduce the variability and bias among observers the results were clearly different from those registered by behavior analysis software. As a conclusion, it can be stated that the behavior analysis methods need some kind of standardization in order to be comparable between labs, preferably the use of the same software and/or settings.

Different action of IBMX, isoproterenol and rutin on orthovanadate-induced nitric oxide release in mouse macrophage cells.

The effects of cAMP-elevating compounds IBMX (3-isobutyl-1-methylxanthine) and isoproterenol, and that of rutin (an effective superoxide scavenger) were studied on orthovanadate--(a putative protein-phosphotyrosine phosphatase inhibitor) induced nitric oxide (NO) production in J774A.1 mouse macrophage cells. As we previously reported (Koncz and Horváth, 2000), rutin and sodium orthovanadate act synergistically to induce production of high amount of NO in J774A.1 cells. IBMX, an agent that can elevate cAMP level in the cells, can reduce the production of both the LPS- and rutin + orthovanadate-induced NO in macrophages. In contrast, isoproterenol, a non-selective beta-adrenergic receptor agonist, that reduced the LPS-induced NO production in macrophage cells, was unable to reduce the rutin + orthovanadate-induced NO production without negatively affecting cell viability. Moreover, isoproterenol dramatically enhanced the orthovanadate-induced NO synthesis in J774A.1 cells. Our previous study clarified that rutin and orthovanadate, in a specific concentration ratio of both, were able to produce hydrogen peroxide (H2O2). Using 2',7'-dichlorofluorescein-diacetate as a marker for H2O2, isoproterenol alone induced its oxidation but the rutin plus orthovanadate-induced H2O2 production was reduced by isoproterenol. These observations have revealed that, in some cases, H2O2 and superoxide (O2-) scavengers can act in a reverse mode on macrophage cells depending on the presence or absence of orthovanadate.

Synergistic action of rutin and orthovanadate on nitric oxide release from mouse macrophage cells.

The cooperative action of sodium orthovanadate (a putative protein-phosphotyrosine phosphatase inhibitor) and rutin (an effective superoxide scavenger) on the nitric oxide (NO) production of J774A.1 mouse macrophage cells has been investigated. Orthovanadate alone caused a mild but significant increase in NO production of the cells at its highest concentration used (500 microM). Orthovanadate and rutin together caused a significant increase in the nitrite level of the supematants of the J774A.1 cells after a 24-hour incubation period, in a concentration dependent manner. The optimal doses for orthovanadate and rutin were 50 microM and 100 microM, respectively. This cooperative action of rutin and orthovanadate was totally inhibitable by catalase, reduced glutathion, N-acetylcystein, cycloheximide, pyrrolidine dithiocarbamate (a putative NF-kappaB inhibitor), genistein and tyrphostin-AG126 (two protein tyrosine-kinase inhibitors). Superoxide dismutase had no inhibitory effect. Orthovanadate and rutin (only together) could induce the oxidation of 2',7'-dichlorofluorescein-diacetate, a marker of hydrogen peroxide. This effect was inhibitable by reduced glutathion, a hydrogen peroxide specific scavenger. These findings suggest, that orthovanadate can induce the production of NO by J774A.1 macrophages not only by inhibition of protein tyrosine-phosphatases, but, using it with rutin, by increasing the level of hydrogen peroxide in the cells.

Ipriflavone metabolite-III inhibits LPS-induced nitric oxide release from RAW-264.7 cells.

Ipriflavone [CAS No.: 35212-22-7] is a novel drug used in the treatment of osteoporosis successfully. However, its mechanism of action has not been fully clarified yet. We investigated the effects of ipriflavone and its metabolites (I, II, III, V, VI, VII) on lipopolysaccharide (LPS)-induced nitric oxide (NO) release from RAW-264.7 mouse macrophage cells. Our data show that the LPS-induced NO release from RAW-264.7 cells was significantly inhibited by ipriflavone metabolite-III [7-isopropoxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one], [CAS No.: 97846-18-9] in a dose (3 x 10(-8)-10(-5) M)-dependent manner. Ipriflavone itself and its other metabolites had much lower inhibitory effect on the LPS-induced NO release from RAW-264.7 cells. The IC50-value of ipriflavone metabolite-III (1.0 x 10(-6) M) was between the IC50-values of the two reference compounds dexamethasone (4.0 x 10(-8) M) and NG-Nitro-L-arginine (L-NNA, 7.5 x 10(-5) M). Our finding suggests that some of the beneficial effects of ipriflavone in the treatment of osteoporosis might be mediated by its metabolite-III.

Differential sensitivity to Arg side chain modification of IL-1 beta binding to type I and type II receptors.

The central role of interleukin-1 (IL-1) in several disease processes, including fever and inflammation, makes the characterization of ligand-receptor interaction of prime importance. The role of arginine (Arg) side chains of hr-IL-1 beta in receptor recognition was studied by the modification of Arg residues with the specific reagent 1,2-cyclohexanedione. It was found that chemical modification of Arg residues decreased the binding potential of IL-1 beta to type I receptor dramatically (by 230-fold) while the affinity to type II receptor was reduced only moderately (by 10-fold), with an insignificant reduction of the dissociation rate. These studies suggest that intact Arg side chains of IL-1 beta may be necessary for high affinity binding to type I IL-1 receptor, but have less importance for the interaction of IL-1 beta with type II IL-1 receptor. This observation may be useful in the study of type II IL-1 receptor-mediated biological responses and design of receptor-subtype specific ligands as well.

The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin.

A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109. The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen-binding site. Studies on various fragments of fibronectin have also implicated the two type-II units of this molecule in collagen-binding. In the present work we have found that type-II domains of human fibronectin, expressed in Escherichia coli as beta-galactosidase fusion proteins, bind specifically to immobilized collagen.