Estimating the ecological status and change of riparian zones in Andalusia assessed by multi-temporal AVHHR datasets

Following the European Commission's Water Framework Directive all surface waters in EU's Member States must reach a good status by 2015. The evaluation of this status will be partly based on ecological criteria, such as the hydro-morphological quality criteria which also evaluate the structure and condition of the riparian zone. Riparian zones with undisturbed or nearly undisturbed condition are given high-ecological status. The agri-environmental measures in the EU promote an extensive use of land to protect the farmed environment and its biodiversity. Recent studies in Andalusia and elsewhere suggest that extensification leads to riparian zones with higher ecological status compared to intensively used areas. We suggest that extensification and thus better ecological status of the riparian zone can be partly approximated by the amount of vegetation permanently present on the area. For this the so-called permanent vegetation fraction was derived from a multi-temporal advanced very high-resolution radiometer (AVHRR) dataset and was used (1) to classify the ecological status of the riparian zone into two classes, favourable and unfavourable, and (2) to assess the effect of agricultural practices on these areas. The classification was validated by field observations in the Guadalquivir river basin while detailed information on farming practices helped to assess the effect of agriculture on the riparian zone. The assessment was carried out in olive land cover because erosion control in olive cultivations is the most widely implemented measure in Andalusia. Results suggest that the remotely sensed permanent vegetation fraction is a good indicator of the favourable and unfavourable ecological status of the riparian zone. Furthermore, extensification of agricultural practices expressed in terms of increasing permanent vegetation cover was shown to have positive effect on the riparian zone as opposed to areas where no measures were implemented.     

Heterocyclic analogues of squamocin as inhibitors of mitochondrial complex I. On the role of the terminal lactone of annonaceous acetogenins

Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.     

Identification of polysaccharide hydrolases involved in autolytic degradation of zea cell walls

Cell walls of Zea mays (cv L.G.11) seedlings labeled with ¹⁴C were treated with α-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 m LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previously found to occur in cell walls during auxin induced extension.     

A New In Vitro Model to Study Cellular Responses after Thermomechanical Damage in Monolayer Cultures

Although electrosurgical instruments are widely used in surgery to cut tissue layers or to achieve hemostasis by coagulation (electrocautery), only little information is available concerning the inflammatory or immune response towards the debris generated. Given the elevated local temperatures required for successful electrocautery, the remaining debris is likely to contain a plethora of compounds entirely novel to the intracorporal setting. A very common in vitro method to study cell migration after mechanical damage is the scratch assay, however, there is no established model for thermomechanical damage to characterise cellular reactions. In this study, we established a new in vitro model to investigate exposure to high temperature in a carefully controlled cell culture system. Heatable thermostat-controlled aluminium stamps were developed to induce local damage in primary human umbilical vein endothelial cells (HUVEC). The thermomechanical damage invoked is reproducibly locally confined, therefore allowing studies, under the same experimental conditions, of cells affected to various degrees as well as of unaffected cells. We show that the unaffected cells surrounding the thermomechanical damage zone are able to migrate into the damaged area, resulting in a complete closure of the ‘wound’ within 48 h. Initial studies have shown that there are significant morphological and biological differences in endothelial cells after thermomechanical damage compared to the mechanical damage inflicted by using the unheated stamp as a control. Accordingly, after thermomechanical damage, cell death as well as cell protection programs were activated. Mononuclear cells adhered in the area adjacent to thermomechanical damage, but not to the zone of mechanical damage. Therefore, our model can help to understand the differences in wound healing during the early phase of regeneration after thermomechanical vs. mechanical damage. Furthermore, this model lends itself to study the response of other cells, thus broadening the range of thermal injuries that can be analysed.     

The Social System of Lariang Tarsiers <Emphasis Type="Italic">(Tarsius lariang)</Emphasis> as Revealed by Genetic Analyses

Previous sociobiological studies of tarsiers were invariably based on field observations. Sulawesi tarsiers are known for monogamous or facultative polygynous social mating systems, but, to date, no data exist to describe the genetic mating system. We here bring together behavioral studies and molecular tools for the first time to elucidate mating behavior and kinship within a tarsier population. We investigated the social system of the recently described Tarsius lariang, which researchers have never studied before. Between September and November 2005, we conducted field observations and sampling in central Sulawesi, Indonesia, where this species is endemic. Ten of 11 social groups of the focal population were composed of 1 adult male, 1 adult female, and putative offspring. To enlighten genetic relationships, we used 12 microsatellite loci and mitochondrial DNA sequences of 26 captured and sampled Lariang tarsiers for parentage and relatedness analyses. A significant number of young were the offspring of the sampled group adults, suggesting a predominantly monogamous social and genetic mating system. There is evidence for extrapair young in groups in which adult pairs exhibit close relationships, leading to the assumption that extrapair mating is solicited to avoid inbreeding. Ten of 11 social groups lived in monogamous social systems, indicating monogamy to be the rule in Tarsius lariang.     

Localization of iron-reducing activity in paddy soilby profile studies

Profiles of iron speciations (porewaterFe(II) and Fe(III), solid-phase Fe(II) andFe(III)) have been studied to localize both ironreduction and oxidation in flooded paddy soil. Sulfateand nitrate were determined to analyze interactions ofredox reactions involved in the iron cycle with thoseof the sulfur and nitrogen cycle. The development ofthe iron(II) and iron(III) profiles was observed inmicroscale over a time period of 11 weeks. After 11weeks the profiles were stable and showed lowestconcentrations of solid-phase iron(II) on the soilsurface with increasing concentrations to a soil depthof 10 mm ( 100 µmol/cm³). Profilesof iron(III) showed a maximum of iron(III) at a depthof 2 to 4 mm ( 100--200 µmol/cm³).Porewater iron(II) concentrations were three orders ofmagnitude lower than extracted iron(II) and indicatedthat most iron(II) was adsorbed to the solid-phase orimmobilized as siderite and vivianite. Diffusive lossof iron from the soil was indicated by iron recovery(0.3 µmol gdw^–1) in the flooding water after12 weeks. The organic content of the soil influencedthe concentrations of solid-phase iron(II) in deepersoil layers (> 6 mm); higher Fe(II) concentrationsin soil with limiting amounts of electron donors mayindicate lower consumption of CO₂ by methanogenicbacteria and therefore a higher sideriteprecipitation. Soil planted with rice showed similariron(II) profiles of fresh paddy soil cores. However,maximal iron(III) concentrations ( 350µmol/cm³) were present in planted soil at adepth of 1 to 2.5 mm where oxygen is provided by a matof fine roots. Sulfate and nitrate concentrations inthe porewater were highest on the soil surface (10µM NO₃ ^–, 40 µM SO₄ ^2–) anddecreased with depth. Similar profiles were detectedfor malate, acetate, lactate, and propionate, theconcentrations decreased gradually from the surface toa depth of 4 mm. Profiles of oxygen showed highestconcentrations at the surface due to photosyntheticproduction and a depletion of oxygen below 3 mm depth.Methane production rates measured from soil layersincubated separately in closed vessels were zero atthe soil surface and increased with depth. In soildepths below 4 mm where iron(III) concentrationsdecreased higher methane production rates werefound.     

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Purpose

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods

We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results

The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions

We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.