Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.     

Fluorescent ligands for the histamine H2 receptor: synthesis and preliminary characterization

3-[3-(Piperidinomethyl)phenoxy]alkyl, N-cyano-N′-[ω-[3-(1-piperidinylmethyl)phenoxy]alkyl]guanidine and 2-(5-methyl-4-imidazolyl)methyl thioethyl derivatives containing fluorescent functionalities were synthesized and the histamine H₂ receptor affinity was evaluated using the H₂ antagonist [¹²⁵I]-aminopotentidine. The compounds exhibited weak to potent H₂ receptor affinity with pK_i values ranging from <4 to 8.85. The highest H₂ receptor affinity was observed for N-cyano-N′-[ω-[3-(1-piperidinylmethyl)phenoxy]alkyl]guanidines substituted with methylanthranilate (13), cyanoindolizine (6) and cyanoisoindole (11) moieties via an ethyl or propyl linker.     

Mussel disturbance dynamics: signatures of oceanographic forcing from local interactions

Local interactions, biotic and abiotic, can have a strong influence on the large-scale properties of ecosystems. However, ecological models often explore the influence of local biotic interactions where physical disturbance is included as a large-scale and imposed source of variability but is not allowed to interact with biotic processes at the local scale. In marine intertidal communities dominated by mussels, wave disturbances create gaps in the mussel bed that recover through a successional sequence. We present a lattice model of mussel disturbance dynamics that allows local interactions between wave disturbance and mussel recolonization, in which each cell of the lattice can be empty, occupied by a mussel bed element, or disturbed (which corresponds to a newly disturbed cell that has unstable edges). As in natural ecosystems, wave disturbance can also spread from disturbed to adjacent occupied cells, and recolonization can also spread from occupied to adjacent empty cells. We first validate the local rules from artificial gap experiments and from natural gap monitoring along the Oregon coast. We analyze the properties of the model system as a function of different oceanographic forcings of productivity and disturbance. We show that the mussel bed can go through phase transitions characterized by a large sensitivity of mussel cover and patterns to oceanographic forcings but also that criticality (scale invariance) is observed over wide ranges of parameters, which suggests self-organization. We also show that spatial patterns in the intertidal can provide a robust signature of local processes and can inform about oceanographic regimes. We do so by comparing the large-scale patterns of the simulation (scaling exponents) with field data, which suggest that some experimental sites are close to criticality. Our results suggest that regional patterns in disturbed populations can be explained by local biotic and abiotic processes submitted to oceanographic forcing.     

NMR studies of the interaction of tryparedoxin with redox-inactive substrate homologues

Tryparedoxins (TXNs) are trypanothione-dependent peroxiredoxin oxidoreductases involved in hydroperoxide detoxification that have been shown to determine virulence in trypanosomatids. The structure of (15)N,(13)C-doubly-labeled, C-terminally-His-tagged tryparedoxin 1 from Crithidia fasciculata (Cf TXN1) was elucidated by three-dimensional NMR spectroscopy. Global folding was found to be similar to the crystal structure, but regions near the active site, especially the onset of helix alpha1 with the redox-active Cys 43 and helix alpha2 relevant to substrate binding, were less well defined in solution. The redox-inactive inhibitory substrate analogue N(1),N(8)-bis(ophthalmyl)spermidine was used to study the substrate/TXN interaction by two-dimensional (1)H,(15)N NMR spectroscopy. The NMR data complemented by molecular modeling revealed several alternative modes of ligand binding. The results confirm and extend the concept of TXN action and specificity derived from X-ray analysis and site-directed mutagenesis and thus improve the rational basis for inhibitor design.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

Protein recovery using two-phase systems

The technology and the main economic aspects of extractive recovery of intracellular enzymes and other biological active proteins are reviewed briefly. It will be seen that the method has high potential and is already used industrially.     

Chaetomium elatum (Kunze: Chaetomiaceae) as a root-colonizing fungus in avocado: is it a mutualist, cheater, commensalistic associate, or pathogen?

Plants support numerous root colonists that may share morphological characteristics with mycorrhizal fungi but may play different roles in the rhizosphere. To determine the function of one such root-colonizing fungus, Chaetomium elatum, the infectivity and composition of inoculum containing C. elatum were varied independently of and in association with the known mutualist Glomus intraradices under two light intensities. Maximum plant benefit occurred with mixtures of both G. intraradices and C. elatum and under high light intensity. Under low light intensity and in monoculture, C. elatum functioned as a weak pathogen that was able to kill host plants. Here, maximum plant mortality was associated with the highest levels of C. elatum infectivity. When G. intraradices was present, no negative impact of C. elatum was detected. Intraspecific interactions were important in predicting sporulation rates for both fungi, whereas no interspecific fungal interactions were detected. In the presence of G. intraradices, C. elatum appears to function as a "commensalistic associate," neither impacting plant growth nor sporulation by G. intraradices. Overall, C. elatum appears to be multifunctional, serving as both a rhizoplane and rhizophere fungus, opportunistically colonizing plant roots and only becoming pathogenic when resources are severely limited and intraspecific competition is high. This multifunctional strategy may be shared with other fungi that form similar structures in roots.     

Heavy metals and thiol pool in three strains of <Emphasis Type="Italic">Tetracladium marchalianum</Emphasis>

Growth of three strains of Tetracladium marchalianum was inhibited by Cd-, and, to a lesser extent, by Cu-and Zn-chloride. In the presence of 50 μM Cd(II), all strains increased total thiol and glutathione production to 6, 11, and 21 μmoles · mg⁻¹ dry mass, respectively. Cd(II) also induced the synthesis of one to several compounds reacting with 5,5′-dithio-bis-(2-nitrobenzoic acid). In order to identify buffer-soluble thiolic compounds other than cysteine, γ-EC and γ-ECG (glutathione) were analyzed and confirmed by mass spectrometry. No water soluble sulfides were detectable in any of the culture filtrates, but Cd(II) exposure at a concentration of 50 μM raised sulfide levels in the mycelia of two of the strains between 3 and 7-fold, Cu(II) and Zn(II) had no effect. Energy Dispersive X-ray-analysis (EDX) and Electron Spectrometry-Images (ES-I) of one strain revealed increased levels of Cu and Zn in the cytoplasm and even higher levels in vacuolar precipitates. Zn and Cu are accumulated in the vacuoles as polyphosphates, identified by Electron Energy Loss-Spectrometry (EELS). Cd was found only in the vacuoles.     

Trypanothione synthesis in crithidia revisited

In Crithidia fasciculata the biosynthesis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBanktrade mark accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg(2+)-ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K(m) values for GSH, Mg(2+)-ATP, and glutathionylspermidine were 407, 222, and 480 microm, respectively, and the k(cat) was 8.7 s(-1) for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.