Metal complexation chemistry used for phosphate and nucleotide determination: an investigation of the Yb3+–pyrocatechol violet sensor

Metal complexation reactions can be used effectively as sensors to measure concentrations of phosphate and phosphate analogs. Recently, a method was described for the detection of phosphate or ATP in aqueous solution based on the displacement by these ligands of pyrocatechol violet (PV) from a putative 2:1 (Yb³⁺)₂PV complex. We have not been able to reproduce this stoichiometry and report this work in order to correct the coordination chemistry upon which sensor applications are based. In our work, colorimetric and spectrophotometric detection of phosphate was confirmed qualitatively (blue PV + Yb³⁺; yellow + P_i); however, the sequence of visual changes on the titration of PV with 2 equiv. of Yb³⁺ and back titration with ATP as described previously could not be reproduced. In contrast to the linear response to P_i that was reported previously, the absorbance response at 443 or 623 nm was found to be sigmoidal using the recommended 2:1 Yb³⁺:PV solution (100 μM:50 μM, pH 7, HEPES). Furthermore, both continuous variation titration and molar ratio analysis (Job plot) experiments are consistent with 1:1, not 2:1, YbPV complex stoichiometry at pH 7 in HEPES buffer, indicating that the deviation from linearity is produced by excess Yb³⁺. Indeed, using a 1:1 Yb³⁺:PV ratio produces a linear response in ΔAbs at 443 or 623 nm on back titration with analyte (phosphate or ATP). In addition, speciation analysis of the Yb–ATP system demonstrates that a 1:1 complex containing Yb³⁺ and ATP predominates in solution at μM metal ion and ATP concentrations. Paramagnetic ¹H NMR spectroscopy directly establishes the formation of Yb³⁺–solute complexes in dilute aqueous solution. The 1:1 YbPV complex can be used for the colorimetric measurement of phosphate and ATP concentrations from ~2 μM. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Phlebotomus (Legeromyia) multihamatus subg. nov., sp. nov. from Gabon (Diptera: Psychodidae)

During a research project aimed at the study of the Culicinae fauna of Gabon and carried out in the National Park of La Lopé, we captured an unknown sandfly male specimen (genus Phlebotomus) by CDC miniature light trap belonging to a new species for Science. Furthermore, the originality of his genitalia does not allow us to include this species in one of the existing subgenus, thus in this paper we propose the creation of a new subgenus, as Phlebotomus (Legeromyia) multihamatus sp. nov., subg. nov. described from the National Park of La Lopé, through one male captured with CDC miniature light trap. A new species and a new subgenus of sandfly is characterised by a short style with three spines, a paramere wearing a basal hook as well as a basal pouch and the absence of basal lobe on the coxite. The originality of the genitalia of the male gives way to discussion about potential primary homologies between P. multihamatus sp. nov. and Phlebotomus (Abonnencius) fortunatarum, Phlebotomus (Anaphlebotomus) stantoni and Phlebotomus (Euphlebotomus) argentipes, which should be verified for future studies. The discovery of this new species in Gabon must encourage the study of sandflies in this country.     

The impact of roads on the avifauna of páramo grasslands in Cajas National Park,Ecuador

National parks are an important tool for conserving biodiversity, particularly in areas of high biodiversity and endemism such as the tropical Andes. However, national parks often face a variety of stressors related to recreation, road construction and illegal extraction of natural resources. Unfortunately, the influence of these stressors for biodiversity is rarely well documented. Cajas National Park in Ecuador is no exception. Despite being traversed by the Cuenca-Molleturo-Naranjal road, effects of the road construction on biodiversity have not been determined. We therefore assessed the influence of road proximity on bird species richness and abundance as well as composition of bird habitat groups in Cajas National Park using transect walks at 25 m and 250 m distance to the road (overall 18 transects, each 1 km length). In total, we recorded 1110 individuals of 28 páramo bird species. Overall species richness did not differ between transects near and far from the road. Nevertheless, the average abundance of shrubby páramo species was significantly higher far from the road than near the road (Far = 36, Near = 25). Moreover, we found a tendency towards differences in the composition of bird habitat groups between transects near and far from the road. One aspect potentially driving the observed patterns was the increasing proportion of planted non-native woody tree species within páramo grassland near the road, which may have caused reduced abundances of shrubby páramo bird species there. While roads represented a clear impact on the composition of bird species in the páramo, the major effect seems to be driven by the introduction of non-native plant species along the roadside. In order to reduce the impact of roads to a minimum, we suggest that park managers should control the introduction of such plant species.     

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.     

Human Angiogenin Lacks Specific Antimicrobial Activity

The antimicrobial activities of commercially available human angiogenin were studied against two pathogens, namely, Candida albicans and Streptococcus pneumoniae. In contrast to the data published earlier, antimicrobial action of angiogenin was rather limited and comparable to that of bovine serum albumin.     

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.     

Virus variation resources at the National Center for Biotechnology Information: dengue virus

Background  

There is an increasing number of complete and incomplete virus genome sequences available in public databases. This large body of sequence data harbors information about epidemiology, phylogeny, and virulence. Several specialized databases, such as the NCBI Influenza Virus Resource or the Los Alamos HIV database, offer sophisticated query interfaces along with integrated exploratory data analysis tools for individual virus species to facilitate extracting this information. Thus far, there has not been a comprehensive database for dengue virus, a significant public health threat.     

Control of feedback repression of the enzymes of purine biosynthesis in Aerobacter aerogenes

Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes: P-ribosyl-PP amidotransferase (I), P-ribosyl glycinamide synthetase (II), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase (VIII-IIA), adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured.