Mimicry of human IgE epitopes by anti-idiotypic antibodies

According to Jerne's network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunization, and confirm Jerne's hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.     

Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.     

MECHANICAL PROPERTIES WITHIN THE GROWTH ZONE OF CORN ROOTS INVESTIGATED BY BENDING EXPERIMENTS II. DISTRIBUTIONS OF MODULUS AND COMPLIANCE IN BENDING

A bending technique was used to infer the spatial distributions of rheological properties within the growth zone of the root of corn, Zea mays. “Bending modulus” (ratio of stress to strain, calculated from engineering theory of bending) falls from 20 MPa near the root tip (3 mm from the tip) to 6 MPa at the location 6 mm from the tip and then remains uniform through the basal region of the growth zone. Where growth stops, at 11–12 mm, there is a sharp rise in bending modulus. The profile of bending moduli is not changed by root incubation temperature during the growth period prior to bending, but it is shifted to the left in roots growing more slowly than the average at either of two temperatures (19 and 29 C). The spatial distribution of “compliance” (reciprocal of bending modulus and a measure of tissue extensibility) resembles the distribution of swelling in response to osmotic perturbation. The distribution of compliance does not parallel that of growth rate. Attempts to explain the discrepancy between compliance and growth rate lead us to examine the theoretical basis for the calculations and to suggest that the dependence of compliance on rate of stretching is physiologically important.     

Effects of acute tryptophan depletion on brain serotonin function and concentrations of dopamine and norepinephrine in C57BL/6J and BALB/cJ mice

Acute tryptophan depletion (ATD) is a method of lowering brain serotonin (5-HT). Administration of large neutral amino acids (LNAA) limits the transport of endogenous tryptophan (TRP) across the blood brain barrier by competition with other LNAAs and subsequently decreases serotonergic neurotransmission. A recent discussion on the specificity and efficacy of the ATD paradigm for inhibition of central nervous 5-HT has arisen. Moreover, side effects such as vomiting and nausea after intake of amino acids (AA) still limit its use. ATD Moja-De is a revised mixture of AAs which is less nauseating than conventional protocols. It has been used in preliminary clinical studies but its effects on central 5-HT mechanisms and other neurotransmitter systems have not been validated in an animal model. We tested ATD Moja-De (TRP-) in two strains of mice: C57BL/6J, and BALB/cJ, which are reported to have impaired 5-HT synthesis and a more anxious phenotype relative to other strains of mice. ATD Moja-De lowered brain TRP, significantly decreased 5-HT synthesis as indexed by 5-HTP levels after decarboxlyase inhibition, and lowered 5-HT and 5-HIAA in both strains of mice, however more so in C57BL/6J than in BALB/cJ. Dopamine and its metabolites as well as norepinephrine were not affected. A balanced (TRP+) control mixture did not raise 5-HT or 5-HIAA. The present findings suggest that ATD Moja-De effectively and specifically suppresses central serotonergic function. These results also demonstrate a strain-specific effect of ATD Moja-De on anxiety-like behavior.     

Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.     

Biochemical changes in human cervical connective tissue after intracervical application of prostaglandin E2

Cervical biopsies were taken during the first trimester from primigravidae and plurigravidae at different time points after intracervical application of prostaglandin E₂-gel. Collagenase activity was determined by a highly specific technique using native, triple helical collagen as substrate. Elastase-_α1-proteinase-inhibitor complex (elastase) was measured by a commercially available assay, and glycosaminoglycan (GAG) analyses were performed as described by Greiling et al. (5, 6). The maximum activity of collagenase was found 2 hours after PGE₂ application in plurigravidae and 4 hours after application in primigravidae. Elastase activity rose nearly 7-fold to maximum values 4 hours after PGE₂ application. The total GAG concentrations and the dermatan sulfate concentrations increased by approximately 10 %, while the hyaluronic acid concentrations were found to be elevated significantly by nearly 50 % in the PGE₂-primed cervices.We conclude that a time-dependent enzymatic collagen degradation by collagenases and other proteinases and an increase in hyaluronic acid concentrations are the significant biochemical events underlying PG-induced cervical ripening.