Assessment of antioxidant activity by using different in vitro methods

In this study, six common tests for measuring antioxidant activity were evaluated by comparing four antioxidants and applying them to beverages (tea and juices): Trolox equivalent antioxidant capacity assay (TEAC I-III assay), Total radical-trapping antioxidant parameter assay (TRAP assay), 2,2-diphenyl- l -picrylhydrazyl assay (DPPH assay), N , N -dimethyl- p -phenylendiamine assay (DMPD assay), Photochemiluminescence assay (PCL assay) and Ferric reducing ability of plasma assay (FRAP assay). The antioxidants included gallic acid representing the group of polyphenols, uric acid as the main antioxidant in human plasma, ascorbic acid as a vitamin widely spread in fruits and Trolox ® as water soluble vitamin E analogue. The six methods presented can be divided into two groups depending on the oxidising reagent. Five methods use organic radical producers (TEAC I-III, TRAP, DPPH, DMPD, PCL) and one method works with metal ions for oxidation (FRAP). Another difference between these tests is the reaction procedure. Three assays use the delay in oxidation and determine the lag phase as parameter for the antioxidant activity (TEAC I, TRAP, PCL). They determine the delay of radical generation as well as the ability to scavenge the radical. In contrast, the assays TEAC II and III, DPPH, DMPD and FRAP analyse the ability to reduce the radical cation (TEAC II and III, DPPH, DMPD) or the ferric ion (FRAP). The three tests acting by radical reduction use preformed radicals and determine the decrease in absorbance while the FRAP assay measures the formed ferrous ions by increased absorbance. Gallic acid was the strongest antioxidant in all tests with exception of the DMPD assay. In contrast, uric acid and ascorbic acid showed low activity in some assays. Most of the assays determine the antioxidant activity in the micromolar range needing minutes to hours. Only one assay (PCL) is able to analyse the antioxidant activity in the nanomolar range. Black currant juice showed highest antioxidant activity in all tests compared to tea, apple juice and tomato juice. Despite these differences, results of these in vitro assays give an idea of the protective efficacy of secondary plant products. It is strongly recommended to use at least two methods due to the differences between the test systems investigated.     

Increased Parasitization of Aphids on Trap Plants Alongside Vials Releasing Synthetic Aphid Sex Pheromone and Effective Range of the Pheromone

Aphid-infested cereal trap plants were used to detect the effects of aphid sex pheromone components on aphid parasitoid activity in arable field margins. The presence of aphid sex pheromones significantly increased parasitization levels by aphid parasitoids on the plants. By placing plants alongside and at varying distances away from a pheromone-releasing vial, the technique was used to measure the distance over which a point source of aphid sex pheromone could increase parasitization levels. Pheromone-releasing vials significantly increased parasitization by the generalist parasitoid Praon volucre on plants adjacent to vials and on plants placed 20 cm away. When the distance between pheromone-releasing vials and aphid-infested plants was increased to 1 m, parasitization by P. volucre was increased only on the plant adjacent to the vial, whereas parasitization by the specialist parasitoid Aphidius rhopalosiphi was also increased on plants placed 1 m away. This indicates a possible difference between the parasitoids in their foraging behaviour in response to semiochemical cues during host selection. When the experiment was repeated with some trap plants placed 3 m away from the pheromone-releasing vial, parasitization was again concentrated on plants directly alongside the vial, but only P. volucre appeared to be active in the field at the time of this experiment, so the effect on A. rhopalosiphi could not be assessed. The results are encouraging for the prospects of using aphid sex pheromones to manipulate parasitoids in order to improve aphid population control.     

Arthropods in tropical oaks: differences in their spatial distributions within tree crowns

The arthropod activity in the upper and the lower zone of nine emergent oak trees (genus Quercus) of a submontane forest in Sabah, Borneo, Malaysia, was investigated by using flight interception traps, yellow colour traps, and arboreal pitfall traps. Number of arthropods did not differ significantly between the upper and the lower region of the tree crowns. Nevertheless, the results reveal significant differences in the abundances of several arthropod groups between the two zones. In the lower part of the oak crowns Homoptera and ants (mostly alates) were sampled in higher numbers, whereas the upper zone was dominated by Diptera and Hymenoptera (ants excluded). Beetles and most other groups showed no significant differences in abundance between the lower and upper zone of the oak trees. Thus, the arthropods of the investigated community are not evenly distributed. The arthropod community in tree crowns, which often has been treated as a homogenous unit, is in fact divided into subunits formed by differently composed arthropod assemblages.     

Gene Trap Mice Reveal an Essential Function of Dual Specificity Phosphatase Dusp16/MKP-7 in Perinatal Survival and Regulation of Toll-like Receptor (TLR)-induced Cytokine Production

MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16.     

L’image de l’ovaloïde dans les gravures rupestres de la Tunisie

Rock art engravings discovered in the central (Jebel Ousselat) and centro-southern Tunisia (Gafsa region), show ovaloids of various types and morphologies associated with wild and domestic animals mainly ornate rams. We examine here these cases of associations and propose some interpretations about.     

Space partitioning among the wolf spiderPardosa amentata species group in Hokkaido,Japan

Population Ecology - There are 4 species belonging to the wolf spiderPardosa amentata species group in Hokkaido. The 4 species have separately occurred in Hokkaido in general, but a few species...     

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria¹, fungi² and parasites³. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent⁴. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase⁵. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility⁶.We describe methods to isolate Neutrophil Granulocytes from peripheral human blood⁷ and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.     

A population study of the japanese wood mouse,Apodemus speciosus (Mammalia: Muridae), with reference to its social behavior

The role of social behavior on the population regulation of the Japanese wood mouse, Apodemus speciosus, was studied in the Ashu Experimental Forest of Kyoto University by monthly trapping and direct observation on the social behavior at the artificial feeding site. The census was carried out from May to October in 1974 and 1975, and the direct observation in June, August and October, 1975. In the study area, the Japanese wood mouse has two breeding seasons in a year: in spring and in autumn. Minimum survival rate was high in the non-breeding season and low in the breeding season. It was negatively correlated to the number of sexually active adults in both sexes. Home ranges of females tended to be mutually exclusive, and female residents prevented the settlement of female immigrants. On the other hand, home ranges of males overlapped largely and males had a straight social hierarchy. Subordinate males tended to disappear more often than dominant males did, but male residents did not hinder the settlement of male immigrants. Males may regulate their number by driving out the subordinate males, while females by spacing behavior.     

The population characteristics of the East African red legged diplopodMetiche tanganyciense Kraus (Diplopoda: Spirobolidae)

A marked variation exists in the numbers and distribution of East African red legged millipede Metiche tanganycienseKraus on the surface and in the lower layer (15 cm) of the soils. Stadia 5 cm and lesser in length are found in the lower layer and those 5 cm and above on the surface soil. Adults are common in varying numbers in both the habitats. It is suggested that after becoming surface active during March–August, and mating, adults migrate to lower layer during September to oviposit. Eggs and early developmental stages are passed here, and when they are 5 cm in length, the stadia move to the surfface soil to complete their development. In keeping with this behaviour, spatial distribution of the adults may change from non-aggregation on surface soil to aggregation in the lower layer. The simulataneous presence of adults and developing stadia in the two habitats suggests and overlapping of generations.