Mechanisms preventing a decrease in phytoplankton biomass after phosphorus reductions in a German drinking water reservoir—results from more than 50 years of observation

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Synthesis and characterization of natural and modified antifreeze glycopeptides: glycosylated foldamers

In Arctic and Antarctic marine regions, where the temperature declines below the colligative freezing point of physiological fluids, efficient biological antifreeze agents are crucial for the survival of polar fish. One group of such agents is classified as antifreeze glycoproteins (AFGP) that usually consist of a varying number (n = 4–55) of [AAT] _n -repeating units. The threonine side chain of each unit is glycosidically linked to β-d-galactosyl-(1 → 3)-α-N-acetyl-d-galactosamine. These biopolymers can be considered as biological antifreeze foldamers. A preparative route for stepwise synthesis of AFGP allows for efficient synthesis. The diglycosylated threonine building block was introduced into the peptide using microwave-enhanced solid phase synthesis. By this versatile solid phase approach, glycosylated peptides of varying sequences and lengths could be obtained. Conformational studies of the synthetic AFGP analogs were performed by circular dichroism experiments (CD). Furthermore, the foldamers were analysed microphysically according to their inhibiting effect on ice recrystallization and influence on the crystal habit.     

Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli

Applied Microbiology and Biotechnology - Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid...     

Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.     

Fe- but not Mg-protophorphyrin IX binds to a transmembrane b-type cytochrome

Transmembrane b-type cytochromes, which are crucially involved in electron transfer chains, bind one or more heme (Fe-protoporphyrin IX) molecules non-covalently. Similarly, chlorophylls are typically also non-covalently bound by several membrane integral polypeptides involved in photosynthesis. While both, chlorophyll and heme, are tetrapyrrole macrocycles, they have different substituents at the tetrapyrrole ring moiety. Furthermore, the central metal ion is Mg²⁺ in chlorophyll and Fe^2+/3+ in heme. As heme and chlorophyll a have similar structures and might both be ligated by two histidine residues of a polypeptide chain, and as the local concentration of chlorophyll a might be up to 100-times higher than the concentration of heme, the question arises, as to how an organism ensures specific binding of heme, but not of chlorophyll, to transmembrane apo-cytochromes involved in photosynthetic electron transfer reactions. As shown here, Fe-protoporphyrin IX derivatives with modified substituents at the tetrapyrrole ring moiety still bind to an apo-cytochrome; however, association appears to be reduced. This indicates that hydrophobic and polar interactions of the ring substituents with the protein moiety stabilize the protein/heme-complex but are not essential per se. However, removal or replacement of the central Fe-ion completely abolishes formation of a holo-protein complex, and thus the central iron ion appears to determine heme binding to apo-cytochrome b₆.     

Pinning down a polymorphic parasite: new genetic and morphological descriptions of Eimeria macropodis from the Tammar wallaby (Macropus eugenii)

Identification of the protozoan parasite, Eimeria has traditionally relied on oocyst morphology, host range and life-cycle attributes. However, it is increasingly recognized that Eimeria species can vary in size and shape across their host range, an attribute known as 'polymorphism' that presents a unique challenge for identification. Advances in molecular tools hold promise for characterising Eimeria that may otherwise be misclassified based on morphology. Our study used morphologic and molecular traits of the oocyst life stage to identify a polymorphic parasite, Eimeria macropodis in a captive Tammar wallaby (Macropus eugenii) population in Australia. Molecular characterization highlighted the need to use multiple genetic markers (18S SSU and cytochrome c oxidase subunit I) to accurately identify E. macropodis owing to heterozygous alleles at the 18S SSU locus. This study provided an opportunity to assess the utility and shortcomings of morphologic and molecular techniques for 'pinning down' a polymorphic species. Moreover, our study was able to place E. macropodis in an evolutionary context and enhance resolution of the under-studied marsupial clade.     

High-throughput screening for small-molecule inhibitors of plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase

Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites' pentose phosphate pathway. In this study, we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries-namely, LOPAC (Sigma-Aldrich, 1280 compounds), Spectrum (MicroSource Discovery Systems, 1969 compounds), and DIVERSet (ChemBridge, 49 971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC(50) values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.     

The rotation-coupled sliding of EcoRV

It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE-DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D(1)), revealing a significant size dependency. To account for the dependence of D(1) on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.