Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.     

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

Plasmolysis effects and osmotic potential of two phylogenetically distinct alpine strains of Klebsormidium (Streptophyta)

The osmotic potential and effects of plasmolysis were investigated in two different Klebsormidium strains from alpine habitats by incubation in 300–2,000 (3,000) mM sorbitol. Several members of this genus were previously found to tolerate desiccation in the vegetative state yet information was lacking on the osmotic potentials of these algae. The strains were morphologically determined as Klebsormidium crenulatum and Klebsormidium nitens. These species belong to distinct clades, as verified by phylogenetic analysis of the rbcL gene. K. crenulatum is part of to the K. crenulatum/mucosum (‘F’ clade) and K. nitens of the ‘E2’ clade. Plasmolysis occurred in K. crenulatum at 800 mM sorbitol (961 mOsmol kg^?1, Ψ?=??2.09 MPa) and in K. nitens at 600 mM sorbitol (720 mOsmol kg^?1, Ψ?=??1.67 MPa). These are extraordinarily high osmotic values (very negative osmotic potentials) compared with values reported for other green algae. In K. crenulatum, the maximum photosynthetic rate (Pmax) in the light-saturated range was 116 μmol O₂ h^?1 mg^?1 chl a. Incubation in 1,000 mM sorbitol decreased Pmax to 74.1% of the initial value, whereas 2,000 mM sorbitol (Ψ?=??5.87 MPa) lead to an almost complete loss of oxygen production. In K. nitens, Pmax was 91 μmol O₂ h^?1 mg^?1 chl a under control conditions and incubation in 800 mM sorbitol did not decrease Pmax, 2,000 mM sorbitol decreased Pmax only to about 62.6% of the initial value whereas 3,000 mM sorbitol stopped oxygen evolution. This indicated a broader amplitude for photosynthesis in the examined strain of K. nitens. Control samples and samples plasmolysed for 3 h in 800 mM sorbitol (K. nitens), 1,000 mM sorbitol (K. crenulatum), or 2,000 mM sorbitol were investigated by transmission electron microscopy after chemical or high-pressure freeze fixation. In cells undergoing plasmolysis the protoplasts were retracted from the cell wall, the cytoplasm appeared dense, vacuoles were small and fragmented, and the cytoplasm was filled with ribosomes. Thin cytoplasmic strands were connected to the cell wall; 2,000 mM sorbitol increased the effect. The content of soluble carbohydrates in these two strains was investigated by HPLC, as this is one known mechanism for cells to maintain high osmotic pressure of the cytosol. Both Klebsormidium species contained diverse soluble carbohydrates, including a dominant mixed peak of unidentified oligosaccharides, and more minor amounts of raffinose, sucrose, glucose, xylose, galactose, mannose, inositol, fructose, glycerol, mannitol, and sorbitol. The total content of soluble carbohydrates was approximately 1.2% of the dry weight, indicating that this is not a major factor contributing to the high osmotic potential in these strains of Klebsormidium.     

Breeding habitat and nest‐site selection by an obligatory “nest‐cleptoparasite”, the Amur Falcon Falco amurensis

The selection of a nest site is crucial for successful reproduction of birds. Animals which re‐use or occupy nest sites constructed by other species often have limited choice. Little is known about the criteria of nest‐stealing species to choose suitable nesting sites and habitats. Here, we analyze breeding‐site selection of an obligatory “nest‐cleptoparasite”, the Amur Falcon Falco amurensis. We collected data on nest sites at Muraviovka Park in the Russian Far East, where the species breeds exclusively in nests of the Eurasian Magpie Pica pica. We sampled 117 Eurasian Magpie nests, 38 of which were occupied by Amur Falcons. Nest‐specific variables were assessed, and a recently developed habitat classification map was used to derive landscape metrics. We found that Amur Falcons chose a wide range of nesting sites, but significantly preferred nests with a domed roof. Breeding pairs of Eurasian Hobby Falco subbuteo and Eurasian Magpie were often found to breed near the nest in about the same distance as neighboring Amur Falcon pairs. Additionally, the occurrence of the species was positively associated with bare soil cover, forest cover, and shrub patches within their home range and negatively with the distance to wetlands. Areas of wetlands and fallow land might be used for foraging since Amur Falcons mostly depend on an insect diet. Additionally, we found that rarely burned habitats were preferred. Overall, the effect of landscape variables on the choice of actual nest sites appeared to be rather small. We used different classification methods to predict the probability of occurrence, of which the Random forest method showed the highest accuracy. The areas determined as suitable habitat showed a high concordance with the actual nest locations. We conclude that Amur Falcons prefer to occupy newly built (domed) nests to ensure high nest quality, as well as nests surrounded by available feeding habitats.     

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.     

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein''s neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.     

Vegetation controls methane emissions in a coastal brackish fen

Climate change and associated sea level rise will likely affect coastal ecosystems and lead to more frequent inundations. Plants are an important control for methane (CH₄) emissions in peatlands because the metabolism of the living plant can either enhance or attenuate CH₄ emissions and plant litter supplies an easily available carbon source for methanogenesis. Here we compare the contribution of various dominant plant species to methane emissions in a degraded, rewetted coastal brackish fen at the southern Baltic Sea coast in Northeast Germany. We analyse one year of bi-weekly static closed chamber data gathered at measurement spots that were located in different mono-dominant vegetation stands (Bolboschoenus maritimus (L.) Palla, Schoenoplectus tabernaemontani (C.C.Gmel.) Palla, Carex acutiformis Ehrh.). Furthermore, data on water level, water temperature, conductivity (sulphate), and several peat characteristics were recorded. Generally, the annual methane emissions were low with an average across vegetation stands of 14 kg CH₄ ha^?1 a^?1, which we related to high decomposition of peat after drainage and to relatively low water levels in summer. Nevertheless, methane emissions varied between different vegetation types with significantly higher methane fluxes (31.8 ± 5.7 kg CH₄ ha^?1 a^?1) from Bolboschoenus maritimus stands compared to Carex acutiformis and Schoenoplectus tabernaemontani stands (4.3 ± 1.2 and 5.7 ± 2.4 kg CH₄ ha^?1 a^?1, respectively). None of the environmental variables that have been recorded can explain this difference. Thus, vegetation composition seems to be an important driver for methane emissions in coastal brackish fens and may therefore be crucial with regard to recreation measures.     

Three-step purification of a fragment of the large immunophilin FKBP52

PPIases catalyze the interconversion of cis and trans isomers of peptidyl–prolyl (Xaa–Pro) bonds in peptide and protein substrates. The PPIase family comprises three subfamilies, two of which interact with immunosuppressant drugs and are therefore termed immunophilins. One subgroup of the immunophilins are the FK506 binding proteins (FKBPs). FKBPs of a relative molecular mass higher than 40 000 also display chaperone activity and are part of the multichaperone complex that Hsp90 forms with substrate proteins. Their function in this chaperone complex is still enigmatic. To further characterize the function of FKBP52 we want to analyze constructs of FKBP52-fragments. Here we describe a fast and effective three-step purification procedure for a fragment of FKBP52 with a relative molecular mass of 48 000, termed FKBP52–123, consisting of affinity chromatography, anion-exchange column and gel-permeation chromatography. A yield of 1 mg pure protein per gram of cells was achieved.     

Novel Humanized and Highly Efficient Bispecific Antibodies Mediate Killing of Prostate Stem Cell Antigen-Expressing Tumor Cells by CD8+ and CD4+ T Cells

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8(+) and CD4(+) T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4(+) T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.     

Transduction of peptides and proteins into live cells by cell penetrating peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.