Natural and artificial cystine knots for assembly of homo- and heterotrimeric collagen models

Native collagens are molecules that are difficult to handle because of their high tendency towards aggregation and denaturation. It was discovered early on that synthetic collagenous peptides are more amenable to conformational characterization and thus can serve as useful models for structural and functional studies. Single-stranded collagenous peptides of high propensity to self-associate into triple-helical trimers were used for this purpose as well as interchain-crosslinked homotrimers assembled on synthetic scaffolds. With the growing knowledge of the biosynthetic pathways of natural collagens and the importance of their interchain disulfide crosslinks, which stabilize the triple-helical structure, native as well as de novo designed cystine knots have gained increasing attention in the assembly of triple-stranded collagen peptides. In addition, natural sequences of collagens were incorporated in order to biophysically characterize their functional epitopes. This review is focused on the methods developed over the years, and future perspectives for the production of collagen-mimicking synthetic and recombinant triple-helical homo- and heterotrimers.     

Interference in immunoassays by human IgM with specificity for the carbohydrate moiety of animal proteins

In allergen characterization common immunoassays such as ELISA and immunoblotting are often used to evaluate human IgE binding of sera from allergic individuals. In an attempt to find and characterize new cat allergens, heavily stained bands were found at 67 and 90 kDa on blotted cat dander extract. Further characterization confirmed these bands to be cat IgA and IgM heavy chains. Analyses in ELISA of purified cat immunoglobulins linked the human IgE binding to cat IgA and IgM and also revealed complete mutual cross-reactivity between the two isotypes. IgE reactivity was found to be directed to carbohydrates of the immunoglobulins and to IgM from 7 out of 9 animal species, but not to human immunoglobulins. Further investigations revealed that the reaction was not mediated by human IgE but by a factor in the serum cross-linking animal immunoglobulins and alkaline phosphatase. The factor was further studied and shown to be human IgM. This IgM fits into the heterophile antibody classification and is directed against carbohydrates on animal immunoglobulins and on calf intestine alkaline phosphatase.     

Proteinaceous bacterial adhesins and their receptors

The adhesion of bacteria to surfaces is an ecologically important property which enables them to colonize their natural habitats. Adhesion between bacteria mediated by sex pili and aggregation substances may also promote gene transfers. In this review, we describe the adhesive properties of bacteria (to eukaryotic and prokaryotic cells, and inert surfaces) and emphasize the characteristics of adhesins (structure, function, genetics, and morphology) and their cognate receptors on target surfaces. The physiochemical interactions between bacteria and surfaces can be described by the DLVO theory, but the interaction between bacterial adhesins and their receptor is better described as a ligand receptor interaction. The DLVO theory predicts that no physical contact can occur between bacteria and surface and, hence, predicts that adhesins must be filamentous in order to bridge the space between the two bodies and allow attachment of the bacteria. Adhesins are primarily proteinaceous, although adhesins of streptococci may involve dextrans or lipoteichoic acids. The cognate receptors for adhesins all appear to contain carbohydrates and as such as likely to be glycoconjugates with carbohydrate moieties acting as the receptor sites.     

Application of carbohydrate array technology to antigen discovery and vaccine development

Carbohydrate arrays are a new technology developed for high-throughput evaluation of interactions between carbohydrates and proteins, cells or viruses. Carbohydrate arrays contain many different carbohydrate structures on a solid support. The format allows one to probe hundreds or thousands of potential receptor-ligand interactions while using only tiny amounts of material. Recently, carbohydrate arrays have been applied to vaccine development in several ways. First, carbohydrate arrays have been utilized for the discovery and characterization of carbohydrate antigens. Second, they have been used to evaluate immune responses to vaccine candidates. Third, carbohydrate arrays have been used to identify and characterize reagents necessary for vaccine development. Although still at an early stage, carbohydrate array technology has tremendous potential for accelerating vaccine development.     

Application of a kinetic gelation simulation to the characterization of in situ cross-linking biomaterials

Multifunctional monomers that are polymerized in situ to form highly cross-linked biomaterials have been an area of recent interest for medical applications. From a biomaterial application standpoint, the relationship between the reaction conditions, polymer structure, and final physical properties is important, particularly for in situ formed materials, as they must have optimum network properties immediately after Formation. However, multifunctional monomer reaction mechanisms are complicated by mobility restrictions on the reacting species, cyclization of pendant groups, unequal reactivity of functional groups, and structural heterogeneities. Also, experimental characterization of these complexities is difficult and limited by the insolubility of the resulting cross-linked structure. Thus, in this contribution, a kinetic gelation simulation was used to understand and characterize the evolution of in situ forming, three-dimensional polymer structures. Specifically, the reaction of tetrafunctional monomers (i.e. divinyl monomers) to form high strength networks with degradable cross-links was modeled. This work focuses on using a lattice-based model to characterize network properties important for biomaterial applications and compare them with an experimental system (i.e. cross-linked polyanhydrides) where appropriate. Simulated results for pendant group reactivity, kinetic chain lengths, and radical concentrations (trapped and free) are presented herein.     

Homing receptors and addressins

Molecular cloning of several homing receptors and their placement within unique families of adhesion receptors over the past 2 years will now permit detailed analyses of structure, function and regulation. Novel tools have significantly contributed to the characterization of carbohydrates as essential parts of the recognition site in addressins whose molecular structures remain to be elucidated.     

Introducing allergists to food chemistry

A report of common analytical methods for the identification and characterization of food allergens is presented. The different electrophoretic and immunologic methods describe the present analytical tools to characterize allergens. With respect to the lack of knowledge on the structure and the properties of food allergens, additional chemical techniques such as chromatography, NMR and MALDI-TOF are of particular importance.     

Sociological differences between women and men: implications for autoimmunity

There are an enormous number of incorrect stereotypes that characterize the differences between women and men. Indeed, nearly all of these stereotypes are based on cultural inaccuracies and faulty data without consideration of biology and the distinct sociological differences between genders. Sociological differences are those that relate to the development, structure, interaction and behavior of organized groups of human beings, or societies, and their values and beliefs. Gender is a social construct referring "to the culturally and historically based differences in the roles, attitudes and behaviors of men and women" ([1], p.1) as shaped by norms and stereotypes. Sex, on the other hand, serves to classify living things according to their reproductive organs and functions assigned by chromosomal complement (according to the US Institute of Medicine) and the physical and biological characteristics arising from these organs and functions. The two terms are generally viewed as dichotomous; however, they are closely intertwined in as yet hardly understood ways, and it is frequently difficult to distinguish between them since gendered life experiences can have profound effects on body structure and function [2]. In this review, we will examine to what extent gender roles and stereotypes shape the daily lives of women in their roles as students, employees, wives, and mothers and their health. These data have implications for the etiology of autoimmunity and also for differences in the natural history of disease.     

Overlap between molecular markers expressed by naturally occurring CD4+CD25+ regulatory T cells and antigen specific CD4+CD25+ and CD8+CD28- T suppressor cells

Alloantigen specific CD8+CD28- T suppressor (TS) cells differ from naturally occurring CD4+CD25+ T-regulatory (natural TR) cells not only by their phenotype but also by their mechanism of action. Natural TR have been extensively studied, leading to the identification of characteristic "molecular markers" such as Forkhead box P3 (FOXP3), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). We have investigated the expression of these genes in alloantigen specific TS and CD4+CD25+ T regulatory (TR) cells and found that they are expressed at levels similar to those observed in natural TR. Furthermore, similar to natural CD4+CD25+ TR, antigen-specific CD8+CD28-CD62L+ TS cells have more suppressive capacity than CD8+CD28-CD62L- TS cells. In spite of these similarities, natural TR are not antigen-specific and inhibit other T cells by T cell-to-T cell interaction, whereas TS are antigen-specific and exert their inhibitory function by interacting with antigen-presenting cells and render them tolerogenic to other T cells. The molecular characterization of TS cells may contribute to a better understanding of mechanisms involved in inhibition of immune responses in autoimmunity, transplantation, and chronic viral infection.     

Secretory products of helminth parasites as immunomodulators

Parasitic helminths release molecules into their environment, which are generally referred to as excretory-secretory products or ES. ES derived from a wide range of nematodes, trematodes and cestodes have been studied during the past 30–40 years, their characterization evolving from simple biochemical procedures such as SDS-PAGE in the early days to sophisticated proteomics in the 21st century. Study has incorporated investigation of ES structure, potential as vaccines, immunodiagnostic utility, functional activities and immunomodulatory properties. Immunomodulation by ES is increasingly the area of most intensive research with a number of defined helminth products extensively analyzed with respect to the nature of their selective effects on cells of the immune system as well as the molecular mechanisms, which underlie these immunomodulatory effects. As a consequence, we are now beginning to learn the identities of the receptors that ES employ and are increasingly acquiring detailed knowledge of the signalling pathways that they interact with and subvert. Such information is contributing to the growing idea that the anti-inflammatory properties of a number of ES products makes them suitable starting points for the development of novel drugs for treating human inflammatory disease.     

Glycobiology of the neuromuscular junction

Most molecules that are present at synapses are glycosylated with carbohydrates, and some carbohydrate structures are themselves uniquely synaptic in their localization. Thus, proteins or lipids at the synapse may bear distinct carbohydrates that alter their localization or function. Here, I will review the evidence that there are unique synaptic carbohydrates at the neuromuscular junction. Then, I will review the evidence that such carbohydrates can affect the function of synaptic proteins, with particular attention to agrin, dystroglycan, and the neural cell adhesion molecule (NCAM). Finally, I will review recent data that demonstrates a role for one carbohydrate structure, the cytotoxic T cell (CT) antigen, in neuromuscular development. These studies suggest that glycosylation is an important modification to consider in studies of synapse formation and function.     

Molecular and phenotypic characterization of Acinetobacter strains able to degrade diesel fuel

Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1(T) and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.     

Automated detection of qualitative spatio-temporal features in electrocardiac activation maps

OBJECTIVE: This paper describes a piece of work aiming at the realization of a tool for the automated interpretation of electrocardiac maps. Such maps can capture a number of electrical conduction pathologies, such as arrhytmia, that can be missed by the analysis of traditional electrocardiograms. But, their introduction into the clinical practice is still far away as their interpretation requires skills that belongs to very few experts. Then, an automated interpretation tool would bridge the gap between the established research outcome and clinical practice with a consequent great impact on health care. METHODS AND MATERIAL: Qualitative spatial reasoning can play a crucial role in the identification of spatio-temporal patterns and salient features that characterize the heart electrical activity. We adopted the spatial aggregation (SA) conceptual framework and an interplay of numerical and qualitative information to extract features from epicardial maps, and to make them available for reasoning tasks. RESULTS: Our focus is on epicardial activation isochrone maps as they are a synthetic representation of spatio-temporal aspects of the propagation of the electrical excitation. We provide a computational SA-based methodology to extract, from 3D epicardial data gathered over time, (1) the excitation wavefront structure, and (2) the salient features that characterize wavefront propagation and visually correspond to specific geometric objects. CONCLUSION: The proposed methodology provides a robust and efficient way to identify salient pieces of information in activation time maps. The hierarchical structure of the abstracted geometric objects, crucial in capturing the prominent information, facilitates the definition of general rules necessary to infer the correlation between pathophysiological patterns and wavefront structure and propagation.     

Treating natural disaster victims is dealing with shortages: an orthopaedics perspective

During natural disasters such as earthquakes or tsunamis, most of the casualties are known to suffer from musculoskeletal injuries. This leads to an enormous need of orthopaedic (surgical) implants such as osteosynthesis plates, which are difficult to provide in developing countries that rely on imported ones. One of the alternatives is utilization of local resources, but only after they have been proven safe to use, and meet the international standards set. Through this paper we would like to urge the international community to include locally produced biomedical products, like osteosynthesis plates in their scientific evaluations and communications. When the quality of local products is proven, the reluctance to use local products also by surgeons from developing countries will disappear and larger scale production can be initiated. This in its turn solves many problems that come after natural disasters and stimulates the national economy in an efficient and effective way.     

Isolation and characterization of major banana allergens: identification as fruit class I chitinases

BACKGROUND: Banana allergy has been associated with the latex-fruit syndrome. Several IgE-binding components, the relevant ones being proteins of 30-37-kDa, have been detected in banana fruit, but none of them have been isolated and characterized yet. Objective To purify and characterize the 30-37 kDa banana allergens. METHODS: Fifteen patients allergic to banana were selected on the grounds of a latex-allergic population. Prick by prick tests to this fruit were performed. Total and specific IgE to banana were determined. Banana allergens were isolated by affinity chromatography, followed by cation-exchange chromatography. Their characterization includes N-terminal sequencing, enzymatic activity assays, immunodetection with sera from allergic patients and with antichitinase antibodies, and CAP and immunoblot inhibition tests. Skin prick tests with banana extracts and with the purified allergens were also carried out. RESULTS: Two major IgE-binding proteins of 34 and 32 kDa, also recognized by polyclonal antibodies against chestnut chitinases, were immunodetected in crude banana extracts. Purification and characterization of both proteins have allowed their identification as class I chitinases with an hevein-like domain. Each isolated allergen reached inhibition values higher than 90% in CAP inhibition assays, and fully inhibited the IgE-binding by the crude banana extract when tested by an immunoblot inhibition method. The two purified allergens provoked positive skin prick test responses in more than 50% of the banana-allergic patients. CONCLUSIONS: Class I chitinases with an hevein-like domain are major allergens in banana fruit. Their presence in other fruits and nuts, such as avocado and chestnut, could explain the cross-sensitization among these foods.     

Immunological identification and characterization of individual food allergens.

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Finding their voice: developing emotional, cognitive, and behavioral congruence in female abuse survivors through equine facilitated therapy

The use of equine therapy for abused women requires hard work and the confluence of significant resources. First, there must be a therapist with the knowledge and skill necessary to use the gifts horses offer us and who is comfortable and experienced working through the aftermath of abuse. It also requires well-trained and socialized horses able to stay connected in the company of clients as they process their abuse and find their voice in a natural environment conducive to the work. Finally, it takes clients willing to pursue nontraditional options for treatment. Facing ones past abuse and its lingering effects is always a difficult journey. Having the courage to take that journey in the company of horses is particularly challenging for women without voice. When it all comes together, the results are synergistic and profound and the relationships gained are often long maintained. It is not uncommon for the women who have done this work to desire continued connection with the horses that took the journey with them. The congruence in the ongoing relationship seems to keep them centered. More than once, I have heard their voices ring out loud and clear across the pasture calling Waggoner, Ready, or Mystic, and the hoofbeats of a nearing horse in response.     

Identification and characterization of novel mammalian spermatogenic genes conserved from fly to human

Spermatogenesis is a complex and highly regulated developmental process by which round spermatogonial stem cells undergo mitotic proliferation and meiosis, followed by extraordinary differentiation into highly specialized elongated mature sperm. Extensive differences in terms of sperm production such as testicular structure and organization, hormonal regulation are reported between humans and insects, yet it is not known to what extent components of the process could be conserved and furthermore to what extent the underlying genetic regulators could be shared from insects to mammals. We hence take a genomic approach to identify genes which are expressed in the testes of both fly and mouse through in silico analysis and are phylogenetically conserved across metazoans. Fifty eight testis-enriched, phylogenetically conserved from fly to mouse genes were identified. Among them, 12 genes are novel. Detailed characterization of their murine and human homologs indicate most of them are testis-restricted or enriched and developmentally regulated, thus suggesting that they are important regulators of sperm development in mammals and potential human fertility factors. Our results reveal the existence of spermatogenic homologs with similar testicular expression across a large evolutionary distance, further functional study will be needed to explore the functional conservation among those spermatogenic orthologs.     

P-glycoprotein in autoimmune diseases

Multidrug resistance-1 (MDR-1) is characterized by overfunction of P-glycoprotein (P-gp), a pump molecule that decreases intracellular drug concentration by effluxing them from the intracellular space. Broad ranges of structurally unrelated compounds are transported by P-gp, including antineoplastic agents, HIV protease inhibitors, prednisone, gold salts, methotrexate, colchicine as well as several antibiotics. In contrast, many other compounds such as calcium channel blockers (verapamil) and immunosupressors (cyclosporine-A) are able to inhibit P-gp function. The P-gp role in therapeutic failures has been extensively studied in cancer; however, there is little information regarding MDR-1 phenotype in autoimmune disorders. It has been reported that an increased number of lymphocytes are able to extrude P-gp substrates in rheumatoid arthritis, immune thrombocytopenic purpura and systemic lupus erythematosus, the patients with poor response to treatment being the ones that exhibit the highest values. This may be due, at least in part, to a simultaneous long-term usage of several drugs that induce P-gp function. Since abnormally activated cell compartments characterize autoimmune diseases, it is possible that those cells are the ones that exhibit drug resistance. The study of drug resistance mechanisms in autoimmunity may be helpful for the optimization of the current therapeutic schemes through their combination with low doses of P-gp inhibitors.