Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding

BACKGROUND: Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2. METHODOLOGY/PRINCIPAL FINDINGS: Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab' fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients. CONCLUSIONS/SIGNIFICANCE: Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates

Reproductive failure, possible maternal infanticide, and cannibalism in wild moustached tamarins, Saguinus mystax

Maternal infanticide in wild non-human primates has only been reported twice. In this paper, we report a possible new case of infanticide and cannibalism within a series of four successive reproductive failures in wild moustached tamarins, Saguinus mystax. Necropsy and genetic analyses of the corpses enabled us to rule out any pathology, and to determine paternity. The mother was seen biting and then eating the head of its own infant during a period when another female was pregnant and gave birth just 1 month later. Before that, the perpetrator had given birth to twins three times successfully when four to five adult and subadult males were present in the group. Although we do not know for certain that the infant was alive when the mother started biting it, our field observations preceding the event suggest it probably was. The possible infanticide case and the two cases of births and early death of the infants occurred while only two to three adult males were present in the group. This could be the second case of maternal infanticide reported in the genus Saguinus and the similar circumstances suggest a common pattern. We discuss these events in the light of the different functional explanations of infanticide and conclude that parental manipulation was the most likely: the mother could have terminated the investment in offspring that had low chances of survival in a group with low availability of helpers

Actin binding domains direct actin-binding proteins to different cytoskeletal locations

<p>Abstract</p> <p>Background</p> <p>Filamin (FLN) and non-muscle α-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell.</p> <p>Results</p> <p>We have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD's complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and α-actinin (GFP-αA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-αA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and α-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant.</p> <p>Conclusion</p> <p>These observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton.</p

Non-invasive cardiac imaging techniques and vascular tools for the assessment of cardiovascular disease in type 2 diabetes mellitus

Cardiovascular disease is the major cause of mortality in type 2 diabetes mellitus. The criteria for the selection of those asymptomatic patients with type 2 diabetes who should undergo cardiac screening and the therapeutic consequences of screening remain controversial. Non-invasive techniques as markers of atherosclerosis and myocardial ischaemia may aid risk stratification and the implementation of tailored therapy for the patient with type 2 diabetes. In the present article we review the literature on the implementation of non-invasive vascular tools and cardiac imaging techniques in this patient group. The value of these techniques as endpoints in clinical trials and as risk estimators in asymptomatic diabetic patients is discussed. Carotid intima–media thickness, arterial stiffness and flow-mediated dilation are abnormal long before the onset of type 2 diabetes. These vascular tools are therefore most likely to be useful for the identification of ‘at risk’ patients during the early stages of atherosclerotic disease. The additional value of these tools in risk stratification and tailored therapy in type 2 diabetes remains to be proven. Cardiac imaging techniques are more justified in individuals with a strong clinical suspicion of advanced coronary heart disease (CHD). Asymptomatic myocardial ischaemia can be detected by stress echocardiography and myocardial perfusion imaging. The more recently developed non-invasive multi-slice computed tomography angiography is recommended for exclusion of CHD, and can therefore be used to screen asymptomatic patients with type 2 diabetes, but has the associated disadvantages of high radiation exposure and costs. Therefore, we propose an algorithm for the screening of asymptomatic diabetic patients, the first step of which consists of coronary artery calcium score assessment and exercise ECG