Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures

Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold‐free three‐dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19‐day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14‐ and 19‐day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki‐67 immunoreactivity showed an even distribution in two‐dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis‐associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold‐free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell–cell and cell–matrix interactions.     

Detection of DNA of the finch polyomavirus in diseases of various types of birds in the order Passeriformes

Between 2000 and 2004 a disease occurred in an aviary in Germany affecting various bird species belonging to the order Passeriformes including Collared Grosbeaks (Mycerobas affinis), Eurasian Bullfinches (Pyrrhula pyrrhula griseiventris), Brown Bullfinches (Pyrrhula nipalensis), Grey-headed bullfinches (Pyrrhula erythaca) and Yellow-bellied Tits (Periparus venustulus).The major clinical signs included increased mortality of fledglings and young birds, as well as feather disorders and feather loss in adult birds. In addition, adult Eurasian Bullfinches showed in one year a disease course, in which the major symptom was inflammation of the skin beginning on the basis of the beak and spreading over the head occurring a few days before death. Bacteriological and parasitological investigations did not reveal any consistent findings. Using a newly developed polymerase chain reaction protocol, DNA of the recently discovered finch polyomavirus (FPyV) was demonstrated in several affected birds. Because of the consistent detection of FPyV-DNA and the similarity of the symptoms with those observed during infection with the closely related avian polyomavirus in other bird species, an etiological role of FPyV in the observed disease is assumed.     

A historical perspective on measurement of soil and plant water status and plant water status

Summary Surrounded by many sophisticated instruments, data acquisition systems and connecting computers, contemporary soil and plant scientists may not fully appreciate what really was known in earlier times and how the science has developed. A short review and a few examples of the growth of measurements and ideas are presented here.     

Phosphate desorption from the surface of soil mineral particles by a phosphate-solubilizing fungus

High phosphate (Pi) sorption in soils is a serious limiting factor for plant productivity and Pi fertilization efficiency, particularly in highly weathered and volcanic ash soils. In these soils, the sorbed Pi is so strongly held on the surfaces of reactive minerals that it is not available for plant root uptake. The use of phosphate-solubilizing microorganisms (PSM) capable of Pi desorption seems to be a complementary alternative in the management of these soils. The aim of this study was to evaluate the effectiveness of the soil fungus Mortierella sp., a known PSM, to desorb Pi from four soil minerals differing in their Pi sorption capacity. The fungus was effective in desorbing Pi from all tested minerals except from allophane, and its desorption depended on the production of oxalic acid. The effectiveness of the fungus to desorb Pi was ranked as montmorillonite > kaolinite > goethite > allophane. The quantity of desorbed Pi increased by increasing the amount of sorbed Pi. The Pi sorption capacity expressed as P_0.2 value (amount of P required to increase a solution P concentration up to 0.2 mg L^?1) was a good indicator of the effectiveness of Mortierella sp. to desorb Pi from soil minerals.     

Antioxidant capacity of oat (Avena sativa L.) extracts. 1. Inhibition of low-density lipoprotein oxidation and oxygen radical absorbance capacity

Milled oat groat pearlings, trichomes, flour, and bran were extracted with methanol and the fractions tested in vitro for antioxidant capacity against low-density lipoprotein (LDL) oxidation and R-phycoerythrin protein oxidation in the oxygen radical absorbance capacity (ORAC) assay. The oxidative reactions were generated by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or Cu(2+) in the LDL assay and by AAPH or Cu(2+) + H(2)O(2) in the ORAC assay and calibrated against a Trolox standard to calculate Trolox equivalents (1 Trolox equivalent = 1 TE = activity of 1 micromol of Trolox). The antioxidant capacity of the oat fractions was generally consistent with a potency rank of pearlings (2.89-8.58 TE/g) > flour (1.00-3.54 TE/g) > trichome (1.74 TE/g) = bran (1.02-1.62 TE/g) in both LDL and ORAC assays regardless of the free radical generator employed. A portion of the oat antioxidant constituents may be heat labile as the greatest activity was found among non-steam-treated pearlings. The contribution of oat tocols from the fractions accounted for <5% of the measured antioxidant capacity. AAPH-initiated oxidation of LDL was inhibited by the oat fractions in a dose-dependent manner, although complete suppression was not achieved with the highest doses tested. In contrast, Cu(2+)-initiated oxidation of LDL stimulated peroxide formation with low oat concentrations but completely inhibited oxidation with higher doses. Thus, oats possess antioxidant capacity most of which is likely derived from polar phenolic compounds in the aleurone.     

Occurrence of Polycyclic Musks in Sewage Sludge and their Behaviour in Soils and Plants. Part 1: Behaviour of Polycyclic Musks in Sewage Sludge of Different Treatment Plants in Summer and Winter (5 pp)

Background, Aim and Scope   Part 1: Behaviour of Polycyclic Musks in Sewage Sludge of Different Treatment Plants in Summer and Winter Part 2: Investigation of Polycyclic Musks in Soils and Plants -  Preamble. In Part 1 of the study, screening tests were performed to investigate the occurrence of PCMs in sewage sludges. For a preliminary risk assessment, further information is needed about their behaviour in the terrestrial environment. Hence, Part 2 examined the adsorption of PCMs to soil, their dissipation and leaching in soil and their uptake by plants. Background, Aim and Scope   Polycyclic Musks (PCMs) enter the environment via the waste water system. Because of their persistence, they can accumulate in different matrices like sewage sludge or biota. By the use of sewage sludge as a fertilizer, PCMs are transferred to agricultural soils. Therefore, in Part 1 of the study, screening tests were performed to investigate the occurrence of PCMs in sewage sludge. For a preliminary risk assessment, further information is needed about their behaviour in the terrestrial environment. Hence, Part 2 of the study examined the adsorption of PCMs to soil, their dissipation and leaching in soil, and their uptake by plants. Materials and Methods: In the screening study, samples of activated sewage sludge were taken both in summer and in winter at 21 treatment plants. In order to get an overview of the contamination situation, sampling covered different types of treatment plants (in rural, urban, industrial areas). Analytical methods for the determination of HHCB, AHTN, ADBI, ATTN, AHDI and ATII in the sludge samples were developed and applied. Results: The analytical screening of PCMs showed their presence in activated and dried sewage sludge samples. HHCB and AHTN represented about 95% of the PCMs investigated. Their concentrations in the activated sludge samples varied between 2.9 and 10.4 mg/kg dry mass (dm) and 1.1 to 4.2 mg/kg dm, respectively. Although different types of sewage treatment plants were investigated, similar PCM levels were found, showing the widespread input of these compounds into domestic waste water. Discussion: PCM concentrations in activated sludge varied widely. The variation drops substantially when concentrations are related to the varying dry mass. In dehydrated sludge, PCM concentrations were up to 24 mg/kg dm for HHCB and up to 6.9 mg/kg dm for AHTN. These high values are comparable to those obtained in other investigations analysing PCMs. If the degradation of organic mass during anaerobic decomposition is included in the evaluation, the figures obtained are comparable to those for activated sludge. Elimination in sewage sludge was higher in summer than in winter. Therefore, the contamination of the sludges in winter reached higher levels compared to the summer. Conclusions: The results show that PCMs are widespread contaminants in sewage sludge. Recommendations and Perspectives: PCM should be considered in a risk assessment as potential contaminants of sewage sludge destined for agricultural use. Due to the high PCM levels in sewage sludge, further investigations into the degradation and elimination behaviour in sewage sludge have to be carried out, including that involving PCM metabolites such as lactone derivatives.     

Proton toxicity interferes with the screening of common bean (Phaseolus vulgaris L.) genotypes for aluminium resistance in nutrient solution

Common bean (Phaseolus vulgaris L.) proved to be very sensitive of low pH (4.3), with large genotypic differences in proton sensitivity. Therefore, proton toxicity did not allow the screening of common bean genotypes for aluminium (Al) resistance using the established protocol for maize (0.5 mM CaCl₂, 8 μM H₃BO₃, pH 4.3). Increasing the pH to 4.5, the Ca²⁺ concentration to 5 mM, and addition of 0.5 mM KCl fully prevented proton toxicity in 28 tested genotypes and allowed to identify differences in Al resistance using the inhibition of root elongation by 20 μM Al supply for 36 h as parameter of Al injury. As in maize, Al treatment induced callose formation in root apices of common bean. Aluminium‐induced callose formation well reflected the effect of Ca supply on Al sensitivity as revealed by root‐growth inhibition. Aluminum‐induced callose formation in root apices of 28 bean genotypes differing in Al resistance after 36 h Al treatment was positively correlated to Al‐induced inhibition of root elongation and Al contents in the root apices. However, the relationship was less close than previously reported for maize. Also, after 12 h Al treatment, callose formation and Al contents in root apices did not reflect differences in Al resistance between two contrasting genotypes, indicating a different mode of the expression of Al toxicity and regulation of Al resistance in common bean than in maize.     

Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes—Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.     

Concept and feasibility study for the integrated evaluation of environmental monitoring data in forests

In the 1970s unexpected forest damages, called “new type of forest damage” or “forest decline”, were observed in Germany and other European countries. The Federal Republic of Germany and the German Federal States implemented a forest monitoring system in the early 1980s, in order to monitor and assess the forest condition. Due to the growing public awareness of possible adverse effects of air pollution on forests, in 1985 the ICP Forests was launched under the convention on long-range transboundary air pollution (CLRTAP) of the United Nations Economic Commission for Europe (UN-ECE). The German experience in forest monitoring was a base for the implementation of the European monitoring system. In 2001 the interdisciplinary case study “concept and feasibility study for the integrated evaluation of environmental monitoring data in forests”, funded by the German Federal Ministry of Education and Research, concentrated on in-depths evaluations of the German data of forest monitoring. The objectives of the study were: (a) a reliable assessment of the vitality and functioning of forest ecosystems, (b) the identification and quantification of factors influencing forest vitality, and (c) the clarification of cause-effect-relationships leading to leaf/needle loss. For these purposes additional data from external sources were acquired: climate and deposition, for selected level I plots tree growth data, as well as data on groundwater quality. The results show that in particular time series analysis (crown condition, tree growth, and tree ring analysis), in combination with climate and deposition are valuable and informative, as well as integrated evaluation of soil, tree nutrition and crown condition data. Methods to combine information from the extensive and the intensive monitoring, and to transfer process information to the large scale should be elaborated in future.