Shoreline algal wash as a factor in reed decline in Lake Constance-Untersee

This paper assesses the chemical and mechanical impact of algal wash (Cladophora, Spirogyra, Chara) upon the lakeside reed belt (Phragmites australis) using field mapping methods, bioassays with Scenedesmus acutus in batch culture, and field experiments. Heavy mats of filamentous algae are correlated with a reduction in number of the outermost reed stalks. The water pressed from decaying heaps of Cladophora and Spirogyra reduced the growth rate of Scenedesmus significantly, but mats from Chara did not. It is assumed that the toxic substance is an organic compound. In field experiments the detrimental effect could not be clearly evidenced. The reasons for this are discussed. It is concluded that mechanical impact is of major importance.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

Effects of a retaining wall and an artificial embankment on nearshore littoral habitats and biota in a large Alpine lake

Hydrobiologia - The littoral zones of many Central European lakes are severely altered by lake-side retaining walls. These are suspected to impair littoral biota due to the reflection of incoming...     

Effects of mooring management on submerged vegetation, sediments and macro-invertebrates in Lake Constance, Germany

Mooring areas are a common form of berthing in many of Germany’s inland waters for the growing number of sporting boats. A circular swinging zone around the anchor zone is formed, in which the submerged macrophyte vegetation is destroyed to a large extent, and the sediment surface is eroded. We investigated the effects of two types of buoy (conventional and so-called hook-buoys) in comparison with undisturbed reference sites nearby. The aim of the study was to identify possible harmful consequences of mooring sites to lake littoral habitats in Lake Constance-Untersee, and to provide information to managers to aid them in the formulation of mooring management plans with the least ecological impact. The study focused on submerged vegetation, surface sediments and macro-invertebrate colonisation. In the swinging circle of conventional buoys (87 m²) we observed a significant sediment matter erosion of 0.9 tonnes and a reduction of organic matter amount by 4.5% of the undisturbed reference. The vegetation free area increased by 122%, and the phytomass (mainly Chara div. spp.) was reduced by 14.6% per berth. The psammophilous macro-invertebrate numbers were not significantly affected (−3%) in contrast to the phytophilous taxa which were reduced by 12.7% per berth. The mayfly larvae were the most negatively affected taxon. Oligochaetes profited from the clearing of the sediment surface in the swinging circles. The ecological effects of the hook-buoys were more minor, mainly due to the smaller swinging circle (6 m²). We concluded that the detrimental effects of mooring can be significantly reduced by mooring systems, e. g. the hook-buoy system as it was used in this study, which reduce the area disturbed and cleared by the anchor chain. We argue that these results can be generalised to mooring areas with soft bottom and dense macrophyte vegetation in Lake Constance and other large lakes.     

Disturbance and rehabilitation of lakeside Phragmites reeds following an extreme flood in Lake Constance (Germany)

Hydrobiologia - After an extreme flood in Lake Constance in 1999 the Phragmites australisbelt showed a severe decline in area and vitality. A three year monitoring project was installed in 2000 to...     

Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.     

Phosphoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex.

Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and identical N-terminal sequence was also found. Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T. maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far. The corresponding pgk and tpi genes are part of the apparent gap operon of T. maritima. This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene. On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.     

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.