Arbuscular mycorrhizal fungal community structures differ between co-occurring tree species of dry Afromontane tropical forest, and their seedlings exhibit potential to trap isolates suited for reforestation

The diversity of arbuscular mycorrhizal (AM) fungi and their broad or narrow association with distinct plant species in natural environments are crucial information in the understanding of the ecological role of AM fungi on plant co-existence. This knowledge is also needed for appropriate mycorrhization of nursery-grown seedlings for forestation efforts. Here, we report results from comparative studies on three co-occurring indigenous tree species of the dry Afromontane forests of Ethiopia and their seedlings grown under controlled conditions in soil collected from the sites. AM fungal SSU rDNA fragment was amplified and sequenced from mycorrhizas of adult plants and seedlings of Olea europaea subsp. cuspidata and Prunus africana, and from Podocarpus falcatus seedlings. AM fungal identity, diversity and community structure were analyzed based on sequence types defined by the NS31-AM1 SSU rDNA fragment similarity in order to compare with data from other habitats. A total of 409 sequences, grouped in 32 sequence types, belonging to Glomeraceae, Diversisporaceae and Gigasporaceae were found. Some sequence types are close to the widespread Glomus intraradices, G. hoi, G. etunicatum, G. cf. etunicatum and Gigaspora margarita. However, the majority (59%) of sequence types are so far specific for the sites including 11 new types when compared with previous data from the same area. The AM fungal community associated with adult plants, including data previously obtained from adult Podocarpus falcatus seedlings, and seedlings of a host species differed significantly, where seedlings trapping a surprising large number of native fungi. AM fungal community structure also differed significantly between host species and sites, respectively. The results confirm previous results from the same area indicating distinct fungal communities associated with the diverse tree species and suggests the potential of these indigenous tree seedlings to trap a wide range of AM fungi appropriate for successful afforestation.     

Activation loop phosphorylation regulates B‐Raf in vivo and transformation by B‐Raf mutants

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B‐Raf signaling in vivo. Here, we generated a conditional knock‐in mouse allowing the expression of the B‐Raf^AVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase‐impaired protein, the Braf^AVKA allele does not phenocopy the lethality of Braf‐knockout or paradoxically acting knock‐in alleles. However, Braf^AVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B‐Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B‐Raf^AVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non‐V600E B‐Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP‐competitive inhibitors.     

Waterborne compounds from the green seaweed Ulva reticulata as inhibitive cues for larval attachment and metamorphosis in the Polychaete Hydroides elegans

Field observations in Hong Kong waters have shown the marine macroalga Ulva reticulata Forsskal (Chlorophyta) to be free of fouling. In this study, the presumptive antifouling mechanism in this alga was investigated and attempts were made to distinguish between possible mechanisms. Waterborne algal compounds were analyzed in larval behavioral bioassays with the marine polychaete Hydroides elegans, a major fouling organism in tropical waters around the world. Larval attachment and metamorphosis in this sessile species is induced by specific bacterial strains in natural bio‐organic films. In bioassays with alga‐conditioned seawater, levels of larval metamorphosis were significantly reduced in comparison to controls. The results demonstrate that the inhibitive effect was caused by direct larval deterrence. Following a bio‐assay‐guided isolation procedure, an inhibitive fraction for larval metamorphosis was purified from U. reticulata‐conditioned seawater. Preliminary chemical analysis of the biologically active fraction pointed to polysaccharides, proteins or glycoconjugates with a molecular weight > 100 kD.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.     

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg²⁺, cdG·Ca²⁺ complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.     

Synthesis of hydroxy derivatives of highly potent non-steroidal CYP 17 inhibitors as potential metabolites and evaluation of their activity by a non cellular assay using recombinant human enzyme.

Inhibition of CYP 17 is a promising strategy for the treatment of prostate cancer. Recently two non-steroidal compounds with high in vitro activity were synthesized in our group (BW19 and BW95). However, after a few hours they showed in vivo a strong decrease in their activity. This might be due to a fast biodegradation. Potential hydroxy and epoxy metabolites were synthesized and their inhibitory activities were tested by a new non-cellular assay using recombinant enzyme. As source, membrane fractions of E. coli pJL17/OR coexpressing human CYP 17 and rat NADPH-P450-reductase were, used. Showing a high and constant CYP 17 activity and a fast and easy isolation procedure the new method was advantageous compared with the microsomal assay. Interestingly, all the new synthesized hydroxy and epoxy compounds except one showed a lower inhibition of CYP 17 than the parent compounds. Thus, the loss of in vivo activity may be partly explained.