Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning

GTPases of the Rab5 and Rab7 families were shown to control vacuolar sorting but their specific subcellular localization is controversial in plants. Here, we show that both the canonical as well as the plant-specific Rab5 reside at the newly discovered 'late prevacuolar compartment' (LPVC) while Rab7 partitions to the vacuolar membrane when expressed at low levels. Higher expression levels of wild-type Rab5 GTPases but not Rab7 lead to dose-dependent inhibition of biosynthetic vacuolar transport. In the case of Ara6, this included aberrant co-localization with markers for earlier post-Golgi compartments including the trans-Golgi network. However, nucleotide-free mutants of all three GTPases (Rha1, Ara6 and Rab7) cause stronger dose-dependent inhibition of vacuolar sorting. In addition, nucleotide-free Rha1 led to a later maturation defect and co-localization of markers for the prevacuolar compartment (PVC) and the LPVC. The corresponding Rab7 mutant strongly inhibited vacuolar delivery without merging of PVC and LPVC markers. Evidence for functional differentiation of the Rab5 family members is underlined by the fact that mutant Rha1 expression can be suppressed by increasing wild-type Rha1 levels while mutant Ara6 specifically titrates the nucleotide exchange factor Vps9. A model describing the sequential action of Rab5 and Rab7 GTPases is presented in the light of the current observations.     

Biodiversity impacts from water consumption on a global scale for use in life cycle assessment

Purpose

Agriculture is a major water user worldwide, potentially depriving many ecosystems of water. Comprehensive global impact assessment methodologies are therefore required to assess impacts from water consumption on biodiversity. Since scarcity of water, as well as species richness, varies greatly between different world regions, a spatially differentiated approach is needed. Therefore, our aim is to enhance a previously published methodology in terms of spatial and species coverage.

Methods

We developed characterization factors for lifecycle impact assessment (LCIA) targeting biodiversity loss of various animal taxa (i.e., birds, reptiles, mammals, and amphibians) in wetlands. Data was collected for more than 22,000 wetlands worldwide, distinguishing between surface water- and groundwater-fed wetlands. Additionally, we account for a loss of vascular plant species in terrestrial ecosystems, based on precipitation. The characterization factors are expressed as global fractions of potential species extinctions (PDF) per cubic meter of water consumed annually and are developed with a spatial resolution of 0.05 arc degrees. Based on the geographic range of species, as well as their current threat level, as indicated by the International Union for Conservation of Nature (IUCN), we developed a vulnerability indicator that is included in the characterization factor.

Results and discussion

Characterization factors have maximal values in the order of magnitude of 10^?11 PDF·year/m³ for animal taxa combined and 10^?12 PDF·year/m³ for vascular plants. The application of the developed factors for global cultivation of wheat, maize, cotton, and rice highlights that the amount of water consumption alone is not sufficient to indicate the places of largest impacts but that species richness and vulnerability of species are indeed important factors to consider. Largest impacts are calculated for vascular plants in Madagascar, for maize, and for animal taxa; in Australia and the USA for surface water consumption (cotton); and in Algeria and Tunisia for groundwater consumption (cotton).

Conclusions

We developed a spatially differentiated approach to account for impacts from water consumption on a global level. We demonstrated its functionality with an application to a global case study of four different crops.

     

JUST (Java User Segmentation Tool) for semi-automatic segmentation of tomographic maps

We are presenting a program for interactive segmentation of tomographic maps, based on objective criteria so as to yield reproducible results. The strategy starts with the automatic segmentation of the entire volume with the watershed algorithm in 3D. The watershed regions are clustered successively by supervised classification, allowing the segmentation of known organelles, such as membranes, vesicles and microtubules. These organelles are processed with topological models and input parameters manually derived from the tomograms. After known organelles are extracted from the volume, all other watershed regions can be organized into homogeneous assemblies on the basis of their densities. To complete the process, all voxels in the volume are assigned either to the background or individual structures, which can then be extracted for visualization with any rendering technique. The user interface of the program is written in Java, and computational routines are written in C. For some operations, involving the visualization of the tomogram, we refer to existing software, either open or commercial. While the program runs, a history file is created, that allows all parameters and other data to be saved for the purposes of comparison or exchange. Initially, the program was developed for the segmentation of synapses, and organelles belonging to these structures have thus far been the principal targets modeled with JUST. Since each organelle is clustered independently from the rest of the volume, however, the program can accommodate new models of different organelles as well as tomograms of other types of preparations of tissue, such as citoskeletal components in vitreous ice.     

Low avidity antibody: a reliable method to diagnose a recent HIV-1 infection

Standard serological tests (both EIA and Immunoblotting) have reached high levels of sensitivity and reproducibility, but do not indicate whether infection is recent or longstanding. Since many patients with HIV-1 infection are not usually diagnosed until symptom presentation, the possibility to distinguish between acute and chronic infection has become increasingly important for the purposes of therapeutic decision-making, partner notification and epidemiological surveillance. We evaluated a guanidine-based-antibody-avidity assay in a selected group of recent (within six months from seroconversion) and chronic (more than forty eight months) HIV-1 infections in an attempt to shed more light on the significance of the avidity index in establishing the time of infection. Sera from newly infected individuals showed a low mean avidity index (ranging from 0.35 to 0.60 with a standard deviation 0.09) at baseline and a clear increasing value at the following times of observation. Our data showed that an avidity index <0.70 might be presumptive of infection occurring within 9 months. Avidity index levels might distinguish between acute and chronic infection. The method is semi-automated, inexpensive and easy to perform, and estimates the time elapsed from seroconversion, thereby identifying a recent infection.     

A role for <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis> Cu,ZnSOD in resistance to heme toxicity

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.     

Primate cathelicidin orthologues display different structures and membrane interactions

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.     

Species identity, diversity and microbial carbon flow in reassembling macrobenthic communities

Intense disturbance may locally destroy patches of habitat and shape the landscape into a mosaic of reassembling communities. The development of ecosystem properties during such community reassembly is poorly understood. In intertidal bare sediments, trophic relations between microphytobenthos or heterotrophic bacteria and macrofauna invertebrates may guarantee fundamental ecosystem properties such as carbon flow through the food web. We studied the dynamic relation between reassembling macrofauna communities and such microbial carbon flow during recovery after severe disturbance. We deliberately induced prolonged hypoxia in winter and early summer and allowed recolonisation for periods of two and five months. Carbon flow was quantified from basal resources (microphytobenthos and bacteria) to intermediate consumers using ¹³C as a tracer. Within the period of study (5 months), microbial carbon flow fully recovered, although macrofauna diversity was still very low compared to the natural communities (ranging from 6 to 17 species). More than 90% of microbial carbon flow to macrofauna was due to the consumers that recolonised within two months. Two of these species were dominant contributors to microphytobenthos carbon transfer to fauna. Furthermore, at an early stage of reassembly, this ecosystem property was remarkably similar when disturbance took place at different times of the year (winter or early summer), although there were differences in assemblage composition and functional diversity. We conclude that species assemblages and ecosystem function developed relatively independently in this benthic system. We discuss which ecological factors may have caused such non-parallel development of macrofaunal communities and carbon flow.     

Inhibition of net nitrification activity in a Mediterranean woodland: possible role of chemicals produced by Arbutus unedo

Nitrification is a key biological process for the control of soil NO₃ ^? availability and N losses from terrestrial ecosystems. The study investigates the causes for the absence of net nitrification activity in the soil of a Mediterranean monospecific woodland of Arbutus unedo, focusing in particular on the possible role of chemicals produced by this plant. The mineral N pool, net rates of mineralization and nitrification were measured in the soil top 10 cm over 18 months. Raw extracts of leaves and roots of Arbutus unedo and soil underneath Arbutus plant canopy were purified using chromatographic techniques and the structure of chemicals was defined using spectroscopic and spectrometric methods. Leaf extracts (raw, aqueous and organic fractions) were tested for their toxicity on net nitrification, using a test soil. Field and laboratory incubations showed soil NO₃ ^? concentration below the detection limit over the whole study period, despite the significant NH₄ ⁺ availability. Toxicity tests indicated that more than 400 μg of extract g^?1 dry soil were needed to have more than 50% reduction of net NO₃ ^? production. Gallocatechin and catechin were among the most abundant chemicals in the extracts of leaves, roots and soil. Their soil concentration was significantly higher than the annual calculated input via leaf litter, and it was in the range of toxic concentrations, as deduced from the dose-response curve of the toxicity test. Data support the hypothesis that plant produced chemicals might be involved in the limited net nitrate production in this Mediterranean woodland.