Hidden in the host – Unexpected vegetative hibernation of the holoparasite Cuscuta epithymum (L.) L. and its implications for population persistence

Several reports have referred to the possible perennial character of holoparasitic Cuscuta species, but the frequency and ecological importance of perennation have not been studied yet. We determined that Cuscuta epithymum is capable of overwintering vegetatively, especially on its most common perennial host Calluna vulgaris. To examine the impact of successional stages on the capability of C. epithymum to perennate vegetatively, ten C. epithymum populations in Calluna-dominated sites in a successional gradient were studied. Although the number of overwintering haustoria varied between different populations, on average 85% of each C. epithymum population was the result of resprouting haustoria. Thus, the pseudoannual growth habit is an important, but overlooked life-strategy for long-term survival of this species. Furthermore, the stage of heath succession significantly determined the overwintering probabilities of C. epithymum and therefore strongly influenced its annual growth. Most sprouted haustoria were found in 1- or 2-year-old vegetation followed by a gradual decline at older successional stages. The parasite was more likely to overwinter on unlignified (and nutritious) than on lignified parts of C. vulgaris. The number of sprouted haustoria positively affects C. epithymum population size and thus flower abundance. Hence, vegetative perennation and subsequent increased flowering success are two aspects that may increase the chance for these populations to cope with environmental influences and survive in the long run.     

A new development of triterpene acid-containing extracts from Viscum album L. displays synergistic induction of apoptosis in acute lymphoblastic leukaemia

Objectives: Aqueous Viscum album L. extracts are widely used for anti‐cancer therapies. Due to their low solubility, triterpenes (which are known to act on cancers), do not occur in aqueous extracts in significant amounts. Using cyclodextrins, we have found it possible to solubilize mistletoe triterpene acids and to determine their effects on acute lymphoblastic leukaemia (ALL) in vitro and in vivo. Materials and methods: A C.B‐17/SCID model of pre‐B ALL (NALM‐6) was used to test efficacy and mechanisms of treatment with lectin‐ and triterpene acid containing preparations in vivo. Cytotoxicity of increasing concentrations of V. album L. preparations was assessed in vitro. Apoptosis was determined using mitochondrial membrane potential measurements, annexin V/PI, western blot analyses and caspase inhibitor assays. Results: Solubilized triterpene acid‐ or lectin‐containing V. album L. extracts inhibited cell proliferation and demonstrated cytotoxic properties in vitro. Annexin V/PI and mitochondrial membrane potential assays indicated that dose‐dependent induction of apoptosis was the main mechanism. Combination (viscumTT) of lectin‐ (viscum) and triterpene‐containing (TT) extracts resulted in greatest induction of apoptosis. Furthermore, caspase activity demonstrated that these extracts were able to induce apoptosis through both caspase‐8 and ‐9 dependent pathways. In vivo experimentation showed that treatment of mice with viscumTT combination prolonged mean survival to 50.5 days compared to 39.3 days in the phosphate‐buffered saline group. Conclusion: Here for the first time, we have demonstrated that either solubilized triterpene acids or lectins and combinations thereof, induce dose‐dependent apoptosis in the ALL cell line NALM‐6 via caspase‐8 and ‐9 dependent pathways.     

Crystal structure of the intrinsically flexible addiction antidote MazE

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.     

Recognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment. Structure,thermodynamics of binding,stability, and influence on interactions with DNA

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.     

Chromosomal evolution in Pleurothallidinae (Orchidaceae: Epidendroideae) with an emphasis on the genus Acianthera: chromosome numbers and heterochromatin

In this study, we analysed chromosome number variation and chromomycin A₃/4′,6‐diamidino‐2‐phenylindole (CMA/DAPI) banding patterns in 48 species belonging to 12 genera of subtribe Pleurothallidinae (Orchidaceae) in order to understand the chromosome evolution based on recent phylogenetic hypotheses and taxonomic treatments. All species had small chromosomes, with numbers ranging from 2n = 20 in two Specklinia spp. to 2n = 80 in an unidentified Octomeria sp. In Acianthera, the most highly represented genus in this study, a great diversity of chromosome number and pattern of fluorescent bands was observed, showing heterochromatin accumulation in Acianthera section Sicariae subsection Pectinatae. Interspecific ascending and, mainly, descending dysploidy were the main mechanisms of chromosome number evolution in subtribe Pleurothallidinae. For Pleurothallidinae, x = 20 is suggested as the basic chromosome number, the same suggested for the related subtribe Laeliinae and for the whole tribe Epidendreae. The Brazilian species of the mega‐genus Stelis had chromosomes with small amounts of heterochromatin and chromosome numbers based on x₂ = 16. These are generally divergent from those reported for Andean and Meso‐American species, but in agreement with the monophyletic hypothesis proposed for Stelis spp. with a Brazilian Atlantic distribution. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 102–120.     

Chemical basis for the affinity maturation of a camel single domain antibody

Affinity maturation of classic antibodies supposedly proceeds through the pre-organization of the reactive germ line conformational isomer. It is less evident to foresee how this can be accomplished by camelid heavy-chain antibodies lacking light chains. Although these antibodies are subjected to somatic hypermutation, their antigen-binding fragment consists of a single domain with restricted flexibility in favor of binding energy. An antigen-binding domain derived from a dromedary heavy-chain antibody, cAb-Lys3, accumulated five amino acid substitutions in CDR1 and CDR2 upon maturation against lysozyme. Three of these residues have hydrophobic side chains, replacing serines, and participate in the hydrophobic core of the CDR1 in the mature antibody, suggesting that conformational rearrangements might occur in this loop during maturation. However, transition state analysis of the binding kinetics of mature cAb-Lys3 and germ line variants show that the maturation of this antibody relies on events late in the reaction pathway. This is reflected by a limited perturbation of k(a) and a significantly decreased k(d) upon maturation. In addition, binding reactions and the maturation event are predominantly enthalpically driven. Therefore, maturation proceeds through the increase of favorable binding interactions, or by the reduction of the enthalpic penalty for desolvation, as opposed to large entropic penalties associated with conformational changes and structural plasticity. Furthermore, the crystal structure of the mutant with a restored germ line CDR2 sequence illustrates that the matured hydrophobic core of CDR1 in cAb-Lys3 might be compensated in the germ line precursor by burying solvent molecules engaged in a stable hydrogen-bonding network with CDR1 and CDR2.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.     

Activity Pattern of Polecats Mustela putorius L. in Relation to Food Habits and Prey Activity

The activity pattern of polecats Mustela putorius L. was studied by radiotracking five males and four females in the wetlands of western France from Jul. 1988 to Mar. 1992. Polecats were nocturnal throughout the year with no differences between males and females. The diel activity remained moderate (31 %). Seasonal food habits were characterized by the exploitation of anurans in spring, while rodents were preyed upon from summer to winter. Seasonal changes in the activity rhythm of polecats appeared synchronized with the activity of the main prey. It is suggested that the activity patterns exhibited in different periods of the year are a result of changes in feeding tactics.     

Synthesis and characterization of a catalytic antibody-HPMA copolymer-Conjugate as a tool for tumor selective prodrug activation

Selective chemotherapy remains a key issue for successful treatment in cancer therapy. The use of targeting approaches like the enhanced permeability and retention (EPR) effect of macromolecules, is consequently needed. Here, we report the preparation of a novel catalytic antibody-polymer conjugate for selective prodrug activation. HPMA copolymer was conjugated to catalytic antibody 38C2 through an amide bond formation between epsilon-amino group of lysine residue from the antibody molecule and a p-nitrophenyl ester of the polymer. The conjugate was purified over a size exclusion column using FPLC. In the isolated fraction, one or two molecules of polymer were conjugated to one molecule of antibody based on gel analysis. The resulting conjugate retained most of its catalytic activity (75-81%) in comparison to the free antibody. The activity was monitored with a fluorogenic substrate and a prodrug activation assay using HPLC. Furthermore, the conjugate was evaluated in vitro for its ability to activate an etoposide prodrug using two different cancer cell lines. Cells growth inhibition using the prodrug and the conjugate was almost identical to inhibition by the free antibody and the prodrug. For the first time, a catalytic antibody was conjugated to a passive targeting moiety while retaining its catalytic ability to activate a prodrug. The conjugate described in this work can be used for selective activation of prodrug in the PDEPT (polymer directed enzyme prodrug therapy) approach by replacing the enzyme component with catalytic antibody 38C2.