Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

Effectiveness of autochthonous bacterium and mycorrhizal fungus on Trifolium growth, symbiotic development and soil enzymatic activities in Zn contaminated soil

AIMS: This study investigates how autochthonous micro-organisms [bacterium and/or arbuscular mycorrhizal (AM) fungi] affected plant tolerance to Zn contamination. METHODS AND RESULTS: Zinc-adapted and -nonadapted Glomus mosseae strains protected the host plant against the detrimental effect of Zn (600 microg g(-1)). Zn-adapted bacteria increased root growth and N, P nutrition in plants colonized by adapted G. mosseae and decreased the specific absorption rate (SAR) of Cd, Cu, Mo or Fe in plants colonized by Zn-nonadapted G. mosseae. Symbiotic structures (nodule number and extraradical mycelium) were best developed in plants colonized by those Zn-adapted isolates that were the most effective in increasing plant Zn tolerance. The bacterium also increased the quantity and quality (metabolic characteristics) of mycorrhizal colonization, with the highest improvement for arbuscular vitality and activity. Inocula also enhanced soil enzymatic activities (dehydrogenase, beta-glucosidase and phosphatase) and indol acetic acid (IAA) accumulation, particularly in the rhizosphere of plants inoculated with Zn-adapted isolates. CONCLUSIONS: Glomus mosseae strains have a different inherent potential for improving plant growth and nutrition in Zn-contaminated soil. The bacterium increased the potential of mycorrhizal mycelium as inoculum. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycorrhizal performance, particularly that of the autochthonous strain, was increased by the bacterium and both contributed to better plant growth and establishment in Zn-contaminated soils.     

Spatial and temporal expressions of prune reveal a role in Müller gliogenesis during Xenopus retinal development

The development of stratified retinal cell architecture is highly conserved in all vertebrates, implying that a common fundamental molecular mechanism is involved in the generation of the organized retina. However, the detailed molecular mechanisms of retinal development are not fully understood. Here we have identified the Xenopus ortholog of prune and show that it is expressed in both differentiating and differentiated retinal domains during development. Interestingly, these spatial and temporal expression patterns coincide with the expression of prune binding partners, the NM23 family members. Overexpression of prune in retinal precursor cells significantly increases the ratio of Müller glial cells as observed by modulation of NM23 activity (Mochizuki et al., 2009). However, a mutated form of prune that has replacement of four aspartate (D) residues (D'Angelo et al., 2004), essential for phosphodiesterase activity, does not exhibit gliogenic activity. Our observations suggest that Xenopus prune may regulate Müller gliogenesis through phosphodiesterase-mediated regulation of NM23 family members.     

Infectious Agents and Neurodegeneration

A growing body of epidemiologic and experimental data point to chronic bacterial and viral infections as possible risk factors for neurodegenerative diseases, including Alzheimer??s disease, Parkinson??s disease and amyotrophic lateral sclerosis. Infections of the central nervous system, especially those characterized by a chronic progressive course, may produce multiple damage in infected and neighbouring cells. The activation of inflammatory processes and host immune responses cause chronic damage resulting in alterations of neuronal function and viability, but different pathogens can also directly trigger neurotoxic pathways. Indeed, viral and microbial agents have been reported to produce molecular hallmarks of neurodegeneration, such as the production and deposit of misfolded protein aggregates, oxidative stress, deficient autophagic processes, synaptopathies and neuronal death. These effects may act in synergy with other recognized risk factors, such as aging, concomitant metabolic diseases and the host??s specific genetic signature. This review will focus on the contribution given to neurodegeneration by herpes simplex type-1, human immunodeficiency and influenza viruses, and by Chlamydia pneumoniae.     

Galactose recognition by the apicomplexan parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.     

The population genetics of Quechuas, the largest native South American group: autosomal sequences, SNPs, and microsatellites evidence high level of diversity

Elucidating the pattern of genetic diversity for non-European populations is necessary to make the benefits of human genetics research available to individuals from these groups. In the era of large human genomic initiatives, Native American populations have been neglected, in particular, the Quechua, the largest South Amerindian group settled along the Andes. We characterized the genetic diversity of a Quechua population in a global setting, using autosomal noncoding sequences (nine unlinked loci for a total of 16 kb), 351 unlinked SNPs and 678 microsatellites and tested predictions of the model of the evolution of Native Americans proposed by (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496). European admixture is <5% and African ancestry is barely detectable in the studied population. The largest genetic distances were between African versus Quechua or Melanesian populations, which is concordant with the African origin of modern humans and the fact that South America was the last part of the world to be peopled. The diversity in the Quechua population is comparable with that of Eurasian populations, and the allele frequency spectrum based on resequencing data does not reflect a reduction in the proportion of rare alleles. Thus, the Quechua population is a large reservoir of common and rare genetic variants of South Amerindians. These results are consistent with and complement our evolutionary model of South Amerindians (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496), proposed based on Y-chromosome data, which predicts high genomic diversity due to the high level of gene flow between Andean populations and their long-term effective population size.     

TLR3 but not TLR7/8 ligand induces allergic sensitization to inhaled allergen

Epidemiological studies suggest that viral infections during childhood are a risk factor for the development of asthma. However, the role of virus-specific pattern recognition receptors in this process is not well defined. In the current study, we compare the effects of the inhaled viral TLR ligands polyinosinic-polycytidylic acid (TLR3) and resiquimod (TLR7/8) on sensitization to a model allergen (OVA) in a murine model. Both compounds enhance the migration, activation, and Ag-processing of myeloid dendritic cells from the lung to the draining lymph nodes comparable to the effects of LPS. Application of polyinosinic-polycytidylic acid [poly(I:C)] or LPS induces production of allergen-specific IgE and IgG1, whereas resiquimod (R848) had no effect. In addition, rechallenge of mice with OVA resulted in airway inflammation and mucus production in animals that received either poly(I:C) or LPS but not after application of R848. In summary, these results show that activation of TLR3 in combination with inhaled allergen results in induction of dendritic cell activation and migration similar to the effects of LPS. This leads to the development of allergic airway disease after allergen rechallenge, whereas mice treated with R848 did not develop allergic airway disease. These findings give further insight into the effects of stimulation of different TLRs on the development of asthma.     

In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species

Efficient micropropagation and cryopreservation of Hypericum richeri ssp. transsilvanicum, an endemic species in Romania, and Hypericum umbellatum, a rare and endangered Daco-Balkan species, was achieved. The effects of type of explant and cytokinin on in vitro plant regeneration were investigated. Shoot organogenesis was achieved in all explants, but stem nodes regenerated best. Organogenesis from nodal segments was promoted by incubating these explants on Murashige and Skoog (MS) medium in the presence of cytokinins (6-benzyladenine, thidiazuron, kinetin or 6-??,??-dimethylallylaminopurine), each tested at four concentrations. The best morphogenic response for both Hypericum species (number of shoots per explant, shoot length, axillary branching of shoot, and frequency of shoot organogenesis) was observed when explants were incubated on MS medium containing 0.44 or 1.11???M 6-benzyladenine. Root induction was achieved only when regenerated shoots were transferred to fresh medium with or without auxin. Maximum rooting was recorded on MS medium supplemented with 2.45???M indole-3-butyric acid. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development. Shoot tips and axillary buds excised from the in vitro regenerated plants were successfully cryopreserved in liquid nitrogen by the droplet-vitrification method. Following preculture in 0.25?M sucrose, dehydration and cryopreservation, the highest regeneration rates were obtained in both species by using axillary buds (68?% for H. richeri ssp. transsilvanicum and 71?% for H. umbellatum).     

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.