Structural observations during androgenic microspore culture of the 4c1 genotype of Zea mays L.

Summary The capacity of the maize genotype 4c1 to regenerate microcalli and embryos from cultured microspores has been examined by comparing various cold pretreatments and culture media, using microspores and pollen at different stages of development. Viability of cultured cells was tested with FDA and their development was traced with light and fluorescence microscopy using DAPI as a nuclear dye.It was found that a pre-incubation of dissected flowers floating in a liquid nutrient medium at 8°C during 10–14 days was most successful for the induction of cell division. Among the developmental stages tested only the microspores appeared to regenerate. Subculture at 25°C in the same liquid medium, supplemented with 0.1 mg/l TIBA, gave highest rates of microspore division, i.e. up to 70% at 4 to 6 days of culture.All pathways described earlier for maize androgenic embryogenesis were observed within the 4c1 genotype. Symmetric divisions occurred in cultured microspores but most frequently asymmetric divisions lead to the formation of microcalli within 12 days of culture. In at least 60% of all dividing microspores cells were derived from the generative nucleus. Microcalli further developed either into loose or compact calli. Compact calli formed embryo-like structures.Abbreviations DAPI 4,6-diamidino-2-phenylindole - Dicamba 3,6-dichloro-2-methoxy benzoic acid - 2,4D 2,4 dichlorophenoxyacetic acid - FDA fluorescein diacetate - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid - YP medium Yu-Pei basal salt medium     

Determination of Atrazine Residues in Electro-Ultrafiltration Soil Extracts by a Sensitive Enzyme Immunoassay

A sensitive enzyme immunoassay (EIA) based on polyclonal antibodies from sheep was used to screen for atrazine in electro-ultrafiltration (EUF) soil extracts without clean-up. Matrix effects were circumvented by diluting the aqueous EUF extracts. The EUF proved to be a convenient method for the extraction of atrazine residues in soil. The efficiency of EUF appeared to be equivalent to that of organic extraction methods except on weathered residues, which generally resulted in lower yields. Both the combined gas chromatography/automated Soxhlet (GC/Soxtec) and the immunochemical technique EIA/EUF yielded similar data for the 26 soil samples identified as positive (> 0.02 mg/kg) during the first screening of 479 EUF extracts by the EIA.     

Genotoxicity of melanoidin fractions derived from a standard glucose/glycine model

Genotoxic compounds can act at various levels in the cell (causing gene, chromosome, or genome mutations), necessitating the use of a range of genotoxicity assays designed to detect these different types of mutations. The production of melanoidins during the processing and cooking of foods is associated with changes in their nutritional character, and the discovery of mutagenic substances in pyrolyzed protein and amino acids has raised concern about the safety of these foods. The aim of this work was to test melanoidin fractions in three different in vitro assays (Ames test, Vitotox test, and micronucleus test). These melanoidin fractions were produced from the condensation of glucose with glycine and their separation was conducted by dialysis. The crude reaction mixture (before dialysis) and both the LMW and HMW fractions obtained by dialysis showed no genotoxicity in these assays, despite being tested at concentrations much higher than those naturally found in food products. The LMW fraction, however, showed toxicity at these high concentrations. The volatile fraction produced in this reaction showed genotoxicity only in the Vitotox test, at high concentrations.     

Bioassay-directed Fractionation of Organic Extracts of Marine Surface Sediments from the North and Baltic Sea - Part II: Results of the biotest battery and development of a biotest index (8 pp)

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Part I: Determination and identification of organic pollutants Part II: Results of the biotest battery and development of a biotest index

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Preamble. This series of two papers presents the results of an interdisciplinary research project (ISIS) dealing with bioassay-directed fractionation of marine sediment extracts. Part I presents the extraction and fractionation procedure as well as the results of chemical analysis, including non-target analysis of sediments. Part II describes the results of the biotest battery in relation to chemicals possibly causing parts of the observed effects. A biotest index is used to compare the toxicities of the samples.

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AUTHORS / AFFILIATIONS Ninja Reineke (3), Werner Wosniok (4), Dirk Danischewski (1), Heinrich Hühnerfuss (3), Angelika Kinder (5), Arne Sierts-Herrmann (5), Norbert Theobald (2), Hans-Heinrich Vahl (6), Michael Vobach (1), Johannes Westendorf (6) and Hans Steinhart (5).

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(1) Federal Research Centre for Fisheries, Institute for Fishery Ecology, Palmaille 9, 22767 Hamburg, Germany (2) Federal Maritime and Hydrographic Agency, Bernhard-Nochtstr. 78, 20359 Hamburg, Germany (3) University of Hamburg, Institute for Organic Chemistry, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany (4) University of Bremen, Institute of Statistics, Bibliothekstr. 1, 28334 Bremen, Germany (5) University of Hamburg, Institute for Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany (6) University of Hamburg, University Hospital Hamburg-Eppendorf, Department for Toxicology, Vogt-Kölln-Str. 30, 22527 Hamburg, Germany (7) Eurofins Wiertz-Eggert-Jörissen, Stenzelring 14b, 21107 Hamburg, Germany

Goal, Scope and Background

The ecological relevance of contaminants in mixtures is difficult to assess, because of possible interactions and due to lacking toxicity data for many substances present in environmental samples. Marine sediment extracts, which contain a mixture of environmental contaminants in low concentrations, were the object of this study. The extracts were investigated with a set of different biotests in order to identify the compound or the substance class responsible for the toxicity. For this goal, a combination of biotests, biotest-directed fractionation and chemical analysis has been applied. Further on, a strategy for the development of a biotest index to describe the toxicity of the fractions without a prior ranking of the test results is proposed. This article (Part II) focuses on the biological results of the approach.

Methods

The toxicological potential of organic extracts of sediments from the North Sea and the Baltic Sea was analyzed in a bioassay-directed fractionation procedure with a set of biotests: luciferase reporter gene assays on hormone receptor and Ah receptor, arabinose resistance test, fish embryo test (Danio rerio), comet assay, acetylcholinesterase inhibition test, heat-shock protein 70 induction, oxidative stress and luminescence inhibition test (Vibrio fischeri). The test results provided the basis for the calculation of a biotest index by factor analysis to compare the toxicity of the samples and fractions.

Results and Discussion

Results of 11 biotests on different fractionation levels of the samples were described and discussed with regard to the occurrence of contaminants and their toxic potentials. Polychlorinated biphenyls, polycyclic aromatic hydrocarbons, quinones, brominated indoles and brominated phenols were in the focus of interest. A biotest index was constructed to compare the toxic responses in the samples and to group the biotest results.

Conclusion

The procedure presented in this study is well suited for bioassay-directed fractionation of marine sediment extracts. However, in relatively low contaminated samples, high enrichment factors and sufficient fractionation is necessary to allow identification of low concentrations of contaminants which is required to link effects and possible causes. In the present case, the relation between substances and effects was difficult to uncover due to relatively low concentrations of pollutants compared to the biogenic matrix and to the remaining complexity of the fractions. The results, with respect to the brominated phenols and indoles in the samples, highlight the successful use of bioassay directed fractionation in the case of high concentrations and high toxicity.

Recommendation and Outlook

In general, it has been shown that a marine risk assessment requires focusing on the input of diffuse sources and taking into account the fact of mixture toxicity. Effects resulting from biogenic substances will make the assessment of the influence of anthropogenic substances even more difficult.     

A Method for Determining Phosphate Diffusion Coefficients by Bulk Diffusion in Soil

A method is described for the determination of phosphate diffusion coefficients by bulk diffusion in soil using the concentration distance method. Two soil blocks only differing in phosphate concentration are brought into contact. After a diffusion period of two weeks the soil blocks are separated, frozen in liquid nitrogen and sliced into layers about 0.02 cm thick by means of a refrigerated microtome. The soil samples are extracted with 4 N HCl, a procedure which fully recovers the added amount of fertilizer P and thus includes the total amount of P that diffuses from one soil block to the other. A concentration distance profile for P and a calculation of the P diffusion coefficient is presented.     

Phosphorylation networks regulating JNK activity in diverse genetic backgrounds

Cellular signaling networks have evolved to enable swift and accurate responses, even in the face of genetic or environmental perturbation. Thus, genetic screens may not identify all the genes that regulate different biological processes. Moreover, although classical screening approaches have succeeded in providing parts lists of the essential components of signaling networks, they typically do not provide much insight into the hierarchical and functional relations that exist among these components. We describe a high-throughput screen in which we used RNA interference to systematically inhibit two genes simultaneously in 17,724 combinations to identify regulators of Drosophila JUN NH(2)-terminal kinase (JNK). Using both genetic and phosphoproteomics data, we then implemented an integrative network algorithm to construct a JNK phosphorylation network, which provides structural and mechanistic insights into the systems architecture of JNK signaling.     

Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi

Basal bodies isolated from Chlamydomonas reinhardi will serve as initiation centers for the assembly of chick brain microtubule protein subunits (tubulin) into microtubules. The rate of microtubule assembly is tubulin-concentration dependent; this assembly occurs onto both distal and proximal ends of the basal body mnicrotubules, with distal assembly greatly favored. In vitro assembly of brain tubulin also occurs onto the mid-lateral aspects of the basal bodies, presumably onto the fiber connecting the two basal bodies.     

The surface of Mars: there view from the viking 1 lander

The first photographs ever returned from the surface of Mars were obtained by two facsimile cameras aboard the Viking 1 lander, including black-and-white and color, 0.12 degrees and 0.04 degrees resolution, and monoscopic and stereoscopic images. The surface, on the western slopes of Chtyse Planitia, is a boulder-strewn deeply reddish desert, with distant eminences-some of which may be the rims of impact craters-surmounted by a pink sky. Both impact and aeolian processes are evident. After dissipation of a small dust cloud stirred by the landing maneuvers, no subsequent signs of movement were detected on the landscape, and nothing has been observed that is indicative of macroscopic biology at this time and place.     

Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.     

Quantitative morphological signatures define local signaling networks regulating cell morphology

Although classical genetic and biochemical approaches have identified hundreds of proteins that function in the dynamic remodeling of cell shape in response to upstream signals, there is currently little systems-level understanding of the organization and composition of signaling networks that regulate cell morphology. We have developed quantitative morphological profiling methods to systematically investigate the role of individual genes in the regulation of cell morphology in a fast, robust, and cost-efficient manner. We analyzed a compendium of quantitative morphological signatures and described the existence of local signaling networks that act to regulate cell protrusion, adhesion, and tension.