The solution structure of the adhesion protein Bd37 from Babesia divergens reveals structural homology with eukaryotic proteins involved in membrane trafficking

Babesia divergens is the Apicomplexa agent of the bovine babesiosis in Europe: this infection leads to growth and lactation decrease, so that economical losses due to this parasite are sufficient to require the development of a vaccine. The major surface antigen of B. divergens has been described as a 37 kDa protein glycosyl phosphatidyl inositol (GPI)-anchored at the surface of the merozoite. The immuno-prophylactic potential of Bd37 has been demonstrated, and we present here the high-resolution solution structure of the 27 kDa structured core of Bd37 (Δ-Bd37) using NMR spectroscopy. A model for the whole protein has been obtained using additional small angle X-ray scattering (SAXS) data. The knowledge of the 3D structure of Bd37 allowed the precise epitope mapping of antibodies on its surface. Interestingly, the geometry of Δ-Bd37 reveals an intriguing similarity with the exocyst subunit Exo84p C-terminal region, an eukaryotic protein that has a direct implication in vesicle trafficking. This strongly suggests that Apicomplexa have developed in parallel molecular machines similar in structure and function to the ones used for endo- and exocytosis in eukaryotic cells.     

An entirely cell-based system to generate single-chain antibodies against cell surface receptors

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.     

Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells

The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Antibody-based approaches in Alzheimer's research: safety, pharmacokinetics, metabolism, and analytical tools

High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease. The efficacy of anti-amyloid immunization strategies has been demonstrated preclinically, prompting the design of clinical studies. However, the detailed mechanisms of action of therapeutic antibodies, especially their influence on the complex amyloid β peptide (Aβ) metabolism and various Aβ-equilibria present both within and outside the CNS, are far from being clear. Furthermore, physiological Aβ metabolism is poorly understood and the analytical tools to characterize and quantify treatment effects on Aβ metabolism are suboptimal. Thus, the design of immunization strategies with optimized benefit-to-risk ratios for patients is subjected to significant obstacles. Indeed, an active immunization trial with Aβ was discontinued because of severe adverse effects. Anti-Aβ immunization protocols designed to attain high blood levels of antibodies bear the potential to induce brain inflammation and/or hemorrhage, thus directing the biomedical research towards development of more predictable therapies for minimizing the risk of adverse effects. The focus of this review is to summarize current knowledge of Aβ metabolism under physiological and antibody-based therapeutic conditions and to introduce a promising approach, namely the passive immunization using antibody fragments, which are characterized by entirely different pharmacokinetic and pharmacodynamic properties compared with conventional monoclonal antibodies.     

Photochemistry, remotely sensed physiological reflectance index and de-epoxidation state of the xanthophyll cycle in Quercus coccifera under intense drought

Quercus coccifera L. is a Mediterranean sclerophyllous shrub with a high capacity to resist intense drought stress. Therefore, it could be used in the study of physiological changes suffered by plants at very low water potentials. A remote sensing sensor was used to measure continuously the physiological reflectance index (PRI; defined as the changes in reflectance at 531 nm with respect to those at 570 nm; PRI = [(R531 − R570)/(R531 + R570)] at canopy level and under field conditions in an artificial carpet of seedlings of Q. coccifera during a drought cycle. Correlations between leaf level-measured chlorophyll fluorescence parameters as well as the de-epoxidation state of the xanthophyll cycle [(A + Z)/(V + A + Z)] and canopy level-measured PRI were reasonably good (R ² = 0.57–0.63, P < 0.01), and quite interesting for water stress remote sensing purposes. The instrument’s temporal resolution allowed us to follow the rapid response of PRI to changing photosynthetic active radiation, and to resolve, in response to cloud-induced changes in light intensity, a fast and a slow PRI component. We report the disappearance of the rapid one under conditions of intense drought in response to a sudden increase in light intensity. The underlying photoprotection mechanisms that Q. coccifera shows in response to intense drought stress periods seem to be related to the existence of a low intrathylakoid lumenal pH at the end of the drought cycle. Under intense drought, these mechanisms allow this species to avoid oxidative damage, which was evidenced by the maintenance of an unaltered photosynthetic pigment composition and constant photosystem II efficiency in the mornings. It is concluded that, contrary to early reports, PRI is a sensible, indirect, non-destructive water stress indicator, even in plants experiencing intense drought. Preliminary results of this work were presented at the 3rd International Workshop on Remote Sensing of Vegetation Fluorescence (February 2007, Florence, Italy).     

Estimating plant responses to climate by direct gradient analysis and geographic distribution analysis

We characterised the climatic behaviour of 53 woody species in terms of the climatic factors that play the main role in controlling species distribution in the study area. Floristic and climatic data were obtained from 150 stands in sites under climatic control (i.e. eu-climatopes). The sampling strategy used allowed a reliable match between floristic and climatic observations. Different methods of frequency analysis and goodness-of-fit tests were used to identify associations between species occurrence and climatic characteristics. The species' responses were summarised by statistics describing ecological preferences and amplitudes, and species were grouped accordingly. A Gaussian response model was fitted to the abundance data along the main climatic gradients for selected species and response surfaces were derived by spatial analysis for a set of indicator species. Frequency analysis methods detected 42 indicator taxa for the Baudiere's Qe drought index, and lower numbers, 34 and 22, respectively, for the mean minimum coldest-month temperature and the daily temperature range in the coldest month. Goodness-of-fit tests revealed a lower number of ecological profiles with statistically significant deviations from equidistribution. We discuss the relative performance of the different methods and suggest that the combined use of statistical tests and frequency analyses may improve estimation of the environmental requirements of species. We also recommend using the species' responses to key environmental factors as reliable criteria in the definition of plant functional types. This revised version was published online in August 2006 with corrections to the Cover Date.     

Herbivory and climatic warming: a Mediterranean outbreaking caterpillar attacks a relict, boreal pine species

Climate change can harm many species by disrupting existing interactions or by favouring new ones. This study analyses the foreseeable consequences of climatic warming in the distribution and dynamics of a Mediterranean pest that causes severe defoliation, the pine processionary caterpillar Thaumetopoea pityocampa, and the effects upon the relict Andalusian Scots pine Pinus sylvestris nevadensis in the Sierra Nevada mountains (southeastern Spain). We correlated a set of regional data of infestation by T. pityocampa upon Scots pine, from a broad ecological gradient, with climatic data for the period 1991–2001, characterized by alternating warm and cold winters. Defoliation intensity shows a significant association with previous warm winters, implying that climatic warming will intensify the interaction between the pest and the Scots pine. The homogeneous structure of the afforested pine woodlands favours the outbreak capacity of the newcomer, promoting this new interaction between a Mediterranean caterpillar pest and a boreal tree at its southern distribution limit.     

传染性法氏囊病病毒野毒株的致病性及其vp2基因比较

对4个IBDV野毒株的致病性和它们的vp2基因高变区序列同时做了比较分析。结果表明,4个IBDV毒株在致病性程度上存在较大差异。其中有一个是真正的超强毒,即GX8／99,其他几个虽然达不到真正超强毒的毒力,但也比经典的标准毒的致死性高得多。在vp2基因高变区,这4个IBDV野毒株与超强毒参考株HK46同源性很高,在DNA水平为96．8％～99．5％,在氨基酸(aa)水平为96．6％～100％o而与疫苗毒D78有很大差异,分别只有91．7％～93．6％和91．8％～93．2％。说明IBDV毒株的vp2基因高变区确实与其致病性有一定关系。特别是SD-1／97、SD-3／98、JS-30／99株之间及其与HK46在DNA和aa水平的同源性高达98．4％和98．6％以上,SD-3／99、JS-30／99株之间及其与HK46的氨基酸同源性为100％。然而,致病性特别高的GX8／99株病毒与其他3个野毒株及HK46在DNA和氨基酸水平的同源性相对较低,在DNA和aa水平的同源性只有96．8％～9712％和96．6％～97．9％。相对于国内的流行毒株和香港超强毒参考株HK46,GX8／99株在致病性和VP2高变区都已发生了一定的变异。     

Reversible Redox-Dependent Inactivation of Photosystem II as Photosynthesis Regulation in Chlorella

Inactivation of photosystem II (PSII) in the alga Chlorella pyrenoidosa Chick induced by photoinhibition (high light illumination at an intensity 10 times higher than photosynthesis-saturating light) or by incubation at a supraoptimum temperature (41°C) in darkness, resulted in a decrease in the relative yield of variable fluorescence due to a selective suppression of the slow phase of its rise. This indicates that low-activity PSII complexes, with a low efficiency of Q_A ^– formation are inactivated first. We suppose that the transition of normal PSII complexes to a low-activity state precedes the complete loss of their photochemical activity. The existence of some common stages of PSII inactivation, when induced by photoinhibition or incubation at supraoptimum temperature in darkness, is discussed. We suggest a scheme of the sequential stages in the regulation of photosynthetic light reactions involving a reversible redox-dependent PSII inactivation.