New Insights into the Structure and Mode of Action of Mo-CBP3, an Antifungal Chitin-Binding Protein of Moringa oleifera Seeds

Mo-CBP₃ is a chitin-binding protein purified from Moringa oleifera Lam. seeds that displays inhibitory activity against phytopathogenic fungi. This study investigated the structural properties and the antifungal mode of action of this protein. To this end, circular dichroism spectroscopy, antifungal assays, measurements of the production of reactive oxygen species and microscopic analyses were utilized. Mo-CBP₃ is composed of 30.3% α-helices, 16.3% β-sheets, 22.3% turns and 30.4% unordered forms. The Mo-CBP₃ structure is highly stable and retains its antifungal activity regardless of temperature and pH. Fusarium solani was used as a model organism for studying the mechanisms by which this protein acts as an antifungal agent. Mo-CBP₃ significantly inhibited spore germination and mycelial growth at 0.05 mg.mL⁻¹. Mo-CBP₃ has both fungistatic and fungicidal effects, depending on the concentration used. Binding of Mo-CBP₃ to the fungal cell surface is achieved, at least in part, via electrostatic interactions, as salt was able to reduce its inhibitory effect. Mo-CBP₃ induced the production of ROS and caused disorganization of both the cytoplasm and the plasma membrane in F. solani cells. Based on its high stability and specific toxicity, with broad-spectrum efficacy against important phytopathogenic fungi at low inhibitory concentrations but not to human cells, Mo-CBP₃ has great potential for the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogens.     

Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.     

Biochemical Evidence for a Timing Mechanism in Prochlorococcus

Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.Cyanobacteria are photosynthetic prokaryotes that are known to possess a true circadian clock. Gene expression and other biological activities follow rhythmic cycles with a circa 24-h period. Rhythmic behavior is maintained even in the absence of environmental stimuli such as light and temperature. The underlying core oscillator consisting of the clock proteins KaiA, KaiB, and KaiC is the only characterized prokaryotic circadian oscillator. It was previously demonstrated that these three proteins, together with ATP, can produce 24-h oscillations of KaiC phosphorylation in vitro (17). The essential roles of KaiA and KaiB in oppositely influencing KaiC phosphorylation are well documented for the oscillator of “Synechococcus elongatus” PCC 7942 (hereafter S. elongatus), the species for which most bacterial circadian research has been conducted. Thus, it is puzzling that marine cyanobacteria of the genus Prochlorococcus, probably the most abundant photosynthetic organisms on Earth (5, 28), contain homologs of only two of these clock proteins, KaiC and KaiB (3, 11, 20). Laboratory cultures (10) as well as natural Prochlorococcus populations (24) display a rhythmic cell cycle together with a daily periodicity of gene expression that can be explained by the functioning of a circadian clock. Alternatively, these rhythms could be controlled directly by the daylight (10). The functional role of the Kai proteins from Prochlorococcus has remained entirely unclear and has not been experimentally addressed thus far.In the well-studied protein clock of S. elongatus, KaiC hexamers are at the center of the circadian oscillator, combining three intrinsic enzymatic activities: autokinase, autophosphatase, and ATPase. KaiA and KaiB modulate KaiC''s activities in opposite manners. KaiA seems to be essential for the shift between autophosphatase and autokinase, and for generating KaiC phosphorylation rhythms, by stabilizing C-terminal residues of KaiC, the A-loops (12). Thus, the absence of KaiA should have consequences for the enzymatic activities of the remaining Kai proteins of Prochlorococcus. In this study, the previously unknown functions of the Prochlorococcus sp. strain MED4 protein KaiB (ProKaiB) and ProKaiC are examined. In our in vitro experiments, we analyzed the recombinant proteins ProKaiB and ProKaiC in direct comparison to the core oscillator of S. elongatus, which consists of S. elongatus KaiA (SynKaiA), SynKaiB, and SynKaiC. We show here that both clock proteins from Prochlorococcus sp. strain MED4 independently exhibit their known biochemical functions, although the influence of ProKaiB on ProKaiC dephosphorylation is different certainly due to the absence of KaiA, the third protein of the oscillator. For ProKaiC, we demonstrate ATPase activity as well as the phosphorylation of serine 427 (S427) and threonine 428 (T428) using mass spectrometry and high-resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Moreover, we suggest that the deletion of kaiA is compensated by the enhanced autophosphorylation activity of ProKaiC. Our results might have further implications for the analysis of a possible timing mechanism in other bacterial species, such as purple bacteria that encode KaiB and KaiC homologs but that lack the KaiA component.     

Blue native PAGE

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein-protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.     

Conditional expression of Spry1 in neural crest causes craniofacial and cardiac defects

Background  

Growth factors and their receptors are mediators of organogenesis and must be tightly regulated in a temporal and spatial manner for proper tissue morphogenesis. Intracellular regulators of growth factor signaling pathways provide an additional level of control. Members of the Sprouty family negatively regulate receptor tyrosine kinase pathways in several developmental contexts. To gain insight into the role of Spry1 in neural crest development, we analyzed the developmental effects of conditional expression of Spry1 in neural crest-derived tissues.     

First selective agonist of the neuropeptide Y1‐receptor with reduced size

Selective NPY analogues are potent tools for tumour targeting. Their Y₁‐receptors are significantly over‐expressed in human breast tumours, whereas normal breast tissue only expresses Y₂‐receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y₁‐receptor agonists enhance the tumour to background ratio compared to Y₁‐receptor antagonists, we were interested in the development of Y₁‐receptor selective agonists. We designed 19 peptides containing the C‐terminus of NPY (28–36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y₁‐receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N‐terminally 5(6)‐carboxyfluorescein (CF) labelled peptides resulted in half‐lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y₁‐receptor internalization. For the first time, here we show the design and characterization of a small Y₁‐receptor selective agonist. This agonist might be a useful novel ligand for NPY‐mediated tumour diagnostics and therapeutics. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

β-(1,3)-Glucan Exposure Assessment by Passive Airborne Dust Sampling and New Sensitive Immunoassays

Associations between house dust-associated β-(1,3)-glucan exposure and airway inflammatory reactions have been reported, while such exposures in early childhood have been suggested to protect against asthma and wheezing. Most epidemiological studies have used reservoir dust samples and an inhibition enzyme immunoassay (EIA) for β-(1,3)-glucan exposure assessment. The objective of this study was to develop inexpensive but highly sensitive enzyme immunoassays to measure airborne β-(1,3)-glucans in low-exposure environments, like homes. Specificities of available anti-β-(1,3)-glucan antibodies were defined by direct and inhibition experiments. Three suitable antibody combinations were selected for sandwich EIAs. β-(1,3)-Glucans in passive airborne dust collected with an electrostatic dust fall collector (EDC) and floor dust from seven homes were measured with the three EIAs. Floor dust samples were additionally analyzed in the inhibition EIA. The sandwich EIAs were sensitive enough for airborne glucan measurement and showed different specificities for commercial glucans, while the β-(1,3)-glucan levels in house dust samples correlated strongly. The feasibility of measuring glucans in airborne dust with the recently introduced EDC method was further investigated by selecting the most suitable of the three EIAs to measure and compare β-(1,3)-glucan levels in the EDC and in floor and actively collected airborne dust samples of the previously performed EDC validation study. The EDC β-(1,3)-glucan levels correlated moderately with β-(1,3)-glucans in actively collected airborne dust and floor dust samples, while the glucan levels in the airborne dust and floor dust samples did not correlate. The combination of the newly developed β-(1,3)-glucan sandwich EIA with EDC sampling now allows assessment in large-scale population studies of exposure to airborne β-(1,3)-glucans in homes or other low-exposure environments.β-(1,3)-Glucans are polysaccharides produced by plants, bacteria, and fungi. Their chain lengths, their degrees of branching, and the numbers and positions of their other glycosidic linkages, like β-(1,4)- and/or β-(1,6)-linkages, may vary largely. While β-(1,3)-(1,4)-glucan structures are typically found in plant material, β-(1,3)-(1,6)-chains are more prevalent in fungi and bacteria (31). Because they are typical microbe-associated molecular patterns (MAMPs), β-(1,3)-glucans activate cells of the innate immune system by binding to glucan-specific receptors like dectin-1 (1, 4, 6) and other cellular membrane receptors (5, 21). Associations between indoor β-(1,3)-glucan exposure and inflammatory reactions of the respiratory system have been reported (3, 10, 25, 33, 34, 40), but protective effects of glucan exposure in early childhood against the development of asthma and allergy have also been suggested (9, 13, 15, 29). β-(1,3)-Glucans are less potent inducers of inflammatory reactions than bacterial endotoxins (16, 30, 35), but since their total amounts in our environment may be much higher—glucans are measured in micrograms per milligram of house dust, whereas endotoxins are measured in nanograms per milligram of house dust (10, 14, 29, 37)—their proinflammatory impact may be similar to that of endotoxin exposure.An inexpensive and relatively simple β-(1,3)-glucan-specific inhibition immunoassay was introduced in the mid-1990s by Douwes et al. (8). This assay has found wide application in large-scale population studies in which glucans have been routinely measured in dust from mattresses and living room and/or bedroom floors (9, 10, 12, 13, 29). However, while useful for quantification of β-(1,3)-glucans in extracts with >1 to 2% (wt/vol) floor or mattress dust, the sensitivity of the assay is usually too low for airborne measurements. Even in environments with high microbial contaminations, like the household waste recycling industry (36), β-(1,3)-glucan levels in airborne dust samples may often remain under the limit of detection. Until recently, the only published methods sensitive enough to measure β-(1,3)-glucans in airborne dust samples were the modified Limulus amebocyte lysate (LAL) assay (a modification of the endotoxin assay with which glucans can be specifically detected [11]) and two sandwich enzyme immunoassays (EIAs) (2, 23, 27). Due to its high cost, which is at least 5-fold higher than that of the inhibition EIA, the LAL assay has thus far hardly been used in epidemiological studies. The assay developed by Sander et al. (27) has been applied to only a limited number of samples from the work environment, and the EIA described by Blanc et al. (2) and Rao et al. (23) has been used only to analyze reservoir and airborne dust samples from heavily mold-contaminated houses in New Orleans after the hurricanes Katrina and Rita. A third sensitive EIA makes use of galactosyl ceramide, a receptor specific for β-(1,3)-glucans (41), as the capture reagent and of a monoclonal antibody specific for β-(1,3)-(1,6)-glucans as the detecting antibody (20). Application of this EIA in population studies has, however, not yet been reported.Apart from the low sensitivity of the inhibition EIA and/or high cost of the modified LAL assay, the time, equipment, and budget needed for active sampling of airborne dust are reasons why epidemiological studies have relied mainly on β-(1,3)-glucan analyses of reservoir dust samples from floors or mattresses. β-(1,3)-Glucan levels in airborne dust samples may, however, be more representative of real inhalatory exposures.The aim of this study was to develop new sensitive but inexpensive assays for β-(1,3)-glucans in airborne dust from homes or other locations with low exposure levels. We combined methods and reagents from three laboratories that previously developed and applied β-glucan EIAs (2, 8, 23, 27). The specificities of available antibodies to a panel of 13 different glucans were determined to assess whether it is possible to develop sandwich assays that would show clear differences in specificities toward glucans from different taxonomic sources—bacterial, fungal, or plant derived—and/or between glucans with different chemical structures.Another objective of the present study was to explore the feasibility of using our recently developed passive airborne dust sampling method, the electrostatic dust fall collector (EDC) (22), for assessing exposure to glucans in airborne dust in the home environment, when combined with the new sensitive immunoassays.     

Protein kinase C isotypes in signal transduction for the 1,25D3-MARRS receptor (ERp57/PDIA3) in steroid hormone-stimulated phosphate uptake

We undertook studies to determine which isotype(s) of protein kinase C (PKC) is/are activated by ligand binding to the 1,25D₃-MARRS receptor (ERp57/PDIA3) and subsequent stimulation of phosphate uptake. Isolated intestinal epithelial cells from vitamin D-replete chicks were exposed to 1,25(OH)₂D₃ for 1, 3, or 5 min, thoroughly chilled, homogenized, and P₂ fractions (20,000 × g post-nuclear pellet) prepared. Western analyses with anti-pan PKC revealed steroid-stimulated redistribution to P₂ membranes 1 min after hormone. Using this time point, cells were treated with vehicle, 130-, 300- or 650-pM hormone. Western blots with anti-PKCα exhibited redistribution to membranes in a biphasic dose–response curve: slightly stimulated at the lowest dose, maximal at 300 pM 1,25(OH)₂D₃, and equivalent to control levels at the highest dose, paralleling hormone-mediated phosphate uptake. Westerns with anti PKCβ also revealed hormone-mediated differences, while those with anti PKCγ did not. RNAi studies were then performed with siRNA against PKCα or PKCβ. Untransfected cells treated with hormone for 7 min exhibited enhanced ³²P uptake relative to vehicle controls. Cells transfected with either active siRNA revealed decreased ³²P uptake in both controls (relative to untransfected controls), and hormone treated cells. However, control and transfected cells treated with hormone had equivalent levels of uptake. Western blot analyses confirmed decreased immunoreactivity in transfected cells. Chemical PKCα (safingol) and PKCβ ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] blockers also confirmed the results from siRNA and demonstrated decreased ³²P uptake in cells treated with 1,25(OH)₂D₃ plus blockers in comparison with cells treated with 1,25(OH)₂D₃ alone. Thus, PKCα and PKCβ are both involved in steroid-stimulated phosphate uptake.     

Die Rückkehr der Wölfe. Das erste Jahrzehnt

The first decade: the return of the wolves Wolves returned to Saxony in the year 2000 since then they have been regularly rearing pups. Nowadays at least 60‐80 wolves are living in Germany. To face its attendant conflicts a wolf management has been installed including wolf monitoring, public relation work and damage compensation. According to the monitoring wolves feed almost completely on wild ungulates, whereas livestock does not play a major role. The wolves' natural origin from north‐eastern Poland could be proven by genetic analyses. By the use of radiotelemetry important information could be gained on the adaption of the wolves to the anthropogenic landscape.