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Biomedical subjects

J M McDonnell

Publications and source records attributed to J M McDonnell.

At least 19 recordsLinked to original sources
PubMed2001

Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition.

With the introduction of new instruments and improved sensor chip chemistries, surface plasmon resonance (SPR) is finding new applications for molecular interaction studies. Easy access to high-quality kinetic and thermodynamic data for macromolecular binding events is providing insights into the fundamental mechanisms of molecular recognition. Progress is being made to allow larger-scale interaction studies. In addition, combining SPR with other analytical methods is enabling SPR-based analysis of interaction proteomics.

Animals↗
PubMed2001

The structure of the IgE Cepsilon2 domain and its role in stabilizing the complex with its high-affinity receptor FcepsilonRIalpha.

The stability of the complex between IgE and its high-affinity receptor, FcepsilonRI, on mast cells is a critical factor in the allergic response. The long half-life of the complex of IgE bound to this receptor in situ ( approximately 2 weeks, compared with only hours for the comparable IgG complex) contributes to the permanent sensitization of these cells and, hence, to the immediate response to allergens. Here we show that the second constant domain of IgE, Cepsilon2, which takes the place of the flexible hinge in IgG, contributes to this long half-life. When the Cepsilon2 domain is deleted from the IgE Fc fragment, leaving only the Cepsilon3 and Cepsilon4 domains (Cepsilon3-4 fragment), the rate of dissociation from the receptor is increased by greater than 1 order of magnitude. We report the structure of the Cepsilon2 domain by heteronuclear NMR spectroscopy and show by chemical shift perturbation that it interacts with FcepsilonRIalpha. By sedimentation equilibrium we show that the Cepsilon2 domain binds to the Cepsilon3-4 fragment of IgE. These interactions of Cepsilon2 with both FcepsilonRIalpha and Cepsilon3-4 provide a structural explanation for the exceptionally slow dissociation of the IgE-FcepsilonRIalpha complex.

Base Sequence↗
PubMed2000

Conformation of the isolated cepsilon3 domain of IgE and its complex with the high-affinity receptor, FcepsilonRI.

Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcepsilonRI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (Cepsilon3) in binding to FcepsilonRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cepsilon3 and Cepsilon4 domains. We have expressed the isolated Cepsilon3 in Escherichia coli, measured its affinity for FcepsilonRI, and examined its conformation alone and in the complex with FcepsilonRI. Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer. The kinetics of binding to an immobilized fragment of the FcepsilonRI alpha-chain, measured by surface plasmon resonance, yields an affinity constant K(a) = 5 x 10(6) M(-)(1), as compared with 4 x 10(9) M(-)(1) for IgE. The circular dichroism spectrum and measurements of fluorescence as a function of the concentration of a denaturant do not reveal any recognizable secondary structure or hydrophobic core. On binding to the FcepsilonRI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cepsilon3 is unchanged in the complex. Thus the isolated Cepsilon3 domain is sufficient for binding to FcepsilonRI, but with lower affinity than IgE. This may be due to the loss of its native immunoglobulin domain structure or to the requirement for two Cepsilon3 domains to constitute the complete binding site for FcepsilonRI or to a combination of these factors.

Cell Line↗
PubMed2000

Cross-reactivity of antiidiotypic antibodies with DNA in systemic lupus erythematosus.

OBJECTIVE: To assess the functional relationship between antibodies reactive with DNA and antibodies reactive with the idiotypes (idiopeptides) of anti-DNA antibodies that are associated with systemic lupus erythematosus (SLE) in mice. METHODS: Antiidiotypic antibodies that appeared spontaneously in lupus mice, and others that were induced by immunization of normal, non-lupus mice, were analyzed for their reactivity by a range of direct binding, competition enzyme-linked immunosorbent assay (ELISA), and surface plasmon resonance (SPR) methods. Their reactions were assessed against synthetic peptides representing sequences of the V(H) region of anti-DNA monoclonal antibody (mAb) V-88, against the native mAb itself, and against mammalian DNA. RESULTS: In lupus mice, only sera with the highest reactivity against double-stranded DNA (dsDNA) also reacted with idiopeptides in ELISA, and this showed a strong statistical correlation. However, there was no significant relationship between antiidiotypic antibodies and anti-single-stranded DNA antibodies. Immunization of (BALB/c x NZW)F1 mice with idiopeptides p64 (V(H) residues 64-80) or p92 (V(H) residues 92-105) induced antibodies that reacted not only against the respective peptides, but also against the native parent anti-DNA mAb V-88. Furthermore, the immune antiidiopeptide antibodies cross-reacted with dsDNA. Competition SPR experiments with the BIAcore system supported this observation. The binding reaction of V(H) peptide p64 (representing the CDR-H2/FR-H3 region of V-88) with antiidiopeptide antibodies was inhibited by dsDNA. CONCLUSION: This study identified a unique set of autoantibodies in SLE. They react with both autoantibody idiotopes and with dsDNA, thus having a dual specificity for 2 autoantigens. Because these antiidiotope antibodies arise naturally during the development of lupus disease, and because they bind also to dsDNA, this provides a mechanism whereby the production of anti-dsDNA antibodies is stimulated. These idiotopes on autoantibodies in lupus act as natural mimotopes for inducing anti-dsDNA antibodies, which, due to their dual specificity, may significantly contribute to the pathology of nephritis in SLE.

Animals↗
PubMed2000

Specificity and binding kinetics of murine lupus anti-DNA monoclonal antibodies implicate different stimuli for their production.

The origin and relative biological importance of the many different DNA-reactive antibodies that appear in systemic lupus erythematosus are not well understood. A detailed analysis of their fine specificity and binding characteristics with DNA is a necessary step in understanding their biology. We have examined here two monoclonal antibodies (mAb) IV-228 and V-88 that are, respectively, characteristic of antibodies, which bind exclusively to single-stranded (ss) DNA and to both double-stranded (ds) DNA and ssDNA. By surface plasmon resonance (SPR) on BIAcore, we characterized the kinetics of binding of each antibody to synthetic ss and ds oligonucleotides. Antibody V-88 and IV-228 showed different patterns of reactivity for both ss and ds oligonucleotides, characterized by distinctly different kinetic parameters. Analysis of their binding kinetics indicates the importance of base composition in defining DNA epitopes, and shows that some epitopes, such as that recognized by mAb V-88, are expressed on dsDNA and ssDNA, whereas others, as recognized by IV-228, are not. The base preferences of V-88 for ds GC-rich structures over AT-rich, and of IV-228 for ss T-rich structures, also reveal distinct differences between these antibodies. We conclude that the different binding properties of the antibodies will relate to their biological activities. The base preferences of the antibodies suggest that they might be induced by different immunological stimuli, such as those that could be provided by the various DNA fragments and structures released during programmed cell death.

Animals↗
PubMed1999

Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists.

Members of the BCL2 family of proteins are key regulators of programmed cell death, acting either as apoptotic agonists or antagonists. Here we describe the solution structure of BID, presenting the structure of a proapoptotic BCL2 family member. An analysis of sequence/structure of BCL2 family members allows us to define a structural superfamily, which has implications for general mechanisms for regulating proapoptotic activity. It appears two criteria must be met for proapoptotic function within the BCL2 family: targeting of molecules to intracellular membranes, and exposure of the BH3 death domain. BID's activity is regulated by a Caspase 8-mediated cleavage event, exposing the BH3 domain and significantly changing the surface charge and hydrophobicity, resulting in a change of cellular localization.

Amino Acid Sequence↗
PubMed1998

Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus.

A biologically active construct of the retroviral M domain from the avian Rous sarcoma virus is defined and its solution structure described. This M domain is fully active in budding and infectivity without myristylation. In spite of a sequence homology level that suggests no relationship among M domains and the family of matrix proteins in mammalian retroviruses, the conserved structural elements of a central core allow an M domain sequence motif to be described for all retroviruses. The surface of the M domain has a highly clustered positive patch comprised of sequentially distant residues. An analysis of the backbone dynamics, incorporating rotational anisotropy, is used to estimate the thermodynamics of proposed domain oligomerization.

Amino Acid Sequence↗
PubMed1997

Participation of the N-terminal region of Cepsilon3 in the binding of human IgE to its high-affinity receptor FcepsilonRI.

The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.

Animals↗
PubMed1997

Identification of contact residues in the IgE binding site of human FcepsilonRIalpha.

The high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is an alphabetagamma2 tetramer found on mast cells, basophils, and several other types of immune effector cells. The interaction of IgE with the alpha-subunit of FcepsilonRI is central to the pathogenesis of allergy. Detailed knowledge of the mode of interaction of FcepsilonRI with IgE may facilitate the development of inhibitors for general use in the treatment of allergic disease. To this end we have performed site-directed mutagenesis on a soluble form of the FcepsilonRI alpha-chain (sFcepsilonRIalpha). The effects of four mutations in the second immunoglobulin-like domain of sFcepsilonRIalpha upon the kinetics of binding to IgE and fragments of IgE have been analyzed using surface plasmon resonance. As described in the preceding paper of this issue [Henry, A. J., et al. (1997) Biochemistry 36, 15568-15578], biphasic binding kinetics was observed. Two of the mutations had significant effects on binding: K117D reduced the affinity of sFcepsilonRIalpha for IgE by a factor of 30, while D159K increased the affinity for IgE by a factor of 7, both principally through changes in the rates of dissociation of the slower phase of the interaction. Circular dichroism spectra of sFcepsilonRIalpha incorporating either of these mutations were indistinguishable from those of wild-type sFcepsilonRIalpha, demonstrating that the native conformation had not been disrupted. Our results, together with those from site-directed mutagenesis on fragments of IgE presented in the accompanying paper, define the contact surfaces in the IgE:sFcepsilonRIalpha complex.

Binding Sites↗
PubMed1997

Peptide analogs that inhibit IgE-Fc epsilon RI alpha interactions ameliorate the development of lethal graft-versus-host disease.

Significant increases in serum levels of IgE have often been observed in allogeneic bone marrow transplantation patients and have generally been thought to be diagnostic of graft-versus-host disease (GVHD), rather than an agent involved in the pathogenesis of the disease. Experimental murine GVHD models have also indicated associations of hyper-IgE activity, yet the role of IgE in GVHD pathogenesis has never been tested directly. In the current study, we have tried to address this issue by using recently developed peptide analog antagonists for the interaction of IgE with the Fc epsilon RI receptor, which is necessary for triggering mast cells and other cell types when cross-linked by antigens. A synthetic cyclized 13-amino acid peptide was previously designed from the modeled C-C' loop region of the Fc epsilon RI alpha-chain and was found to act as a competitive inhibitor of IgE-Fc epsilon RI alpha binding. The peptide was generated in two forms, a cyclic L-(L-IgEtide) and retro D-amino acid composition (rDIgEtide), the latter to increase resistance to protease degradation for in vivo applications. These two inhibitor peptides were then used to test the hypothesis that IgE could be involved in the pathogenesis of acute GVHD, in the B10.D2-->DBA/2 (900 cGy) strain combination, with GVHD directed to minor histocompatibility antigens. Both peptides demonstrated significant inhibition of the development of lethal GVHD, supporting the involvement of IgE at some level of disease pathogenesis.

Animals↗
PubMed1996

Calreticulin binding affinity for glycosylated laminin.

Several lines of evidence indicate that calreticulin has lectin-like properties. As a molecular chaperone, calreticulin binds preferentially to nascent glycoproteins via their immature carbohydrates; this property closely resembles that seen for calnexin, a chaperone with extensive molecular identity to calreticulin. A cell surface form of calreticulin also exhibits lectin-like properties, binding specific oligomannosides including those covalently linked to laminin. In the present study we examined the interaction between calreticulin and laminin by means of surface plasmon resonance. The results show that calreticulin specifically binds to glycosylated laminin but fails to specifically bind tunicamycin-derived unglycosylated laminin or bovine serum albumin. Calreticulin binding to glycosylated laminin requires calcium and is abolished in the presence of EDTA. Scatchard analysis of binding yields an apparent association constant, Ka, of 2.1 +/- 0.9 x 10(6) m-1 while kinetic analysis yields an estimate of the association on rate, (Kassoc), as 2 x 10(5) m-1 s-1. The composite results support calreticulin's lectin-like properties as well as its proposed role in laminin recognition, both in the cell interior and on the cell surface.

Animals↗
PubMed1996

Structure based design and characterization of peptides that inhibit IgE binding to its high-affinity receptor.

We have designed synthetic peptide inhibitors of the interaction between IgE and its high affinity receptor, Fc epsilon RI. The structure of the second domain of CD2 was used as a modelling template for the second alpha-chain domain of Fc epsilon RI, the C-C' loop of which has been implicated in the interaction with IgE. An L-amino acid peptide and a retro-enantiomeric D-amino acid peptide were designed to mimic the conformation of the C-C' region. Both peptides were cyclized by disulphide bond formation between terminal cysteine residues, and show mirror image symmetry by circular dichroism analysis. The C-C' peptide mimics act as competitive inhibitors of IgE binding. The cyclic L- and retro D-peptides exhibited KDs of approximately 3 microM and 11 microM, respectively, for IgE. Further, the peptides inhibit IgE-mediated mast cell degranulation, an in vitro model of an allergic response.

Amino Acid Sequence↗
PubMed1994

A rationally designed CD4 analogue inhibits experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an acute inflammatory autoimmune disease of the central nervous system that can be elicited in rodents and is the major animal model for the study of multiple sclerosis (MS). The pathogenesis of both EAE and MS directly involves the CD4+ helper T-cell subset. Anti-CD4 monoclonal antibodies inhibit the development of EAE in rodents, and are currently being used in human clinical trials for MS. We report here that similar therapeutic effects can be achieved in mice using a small (rationally designed) synthetic analogue of the CD4 protein surface. It greatly inhibits both clinical incidence and severity of EAE with a single injection, but does so without depletion of the CD4+ subset and without the inherent immunogenicity of antibody. Furthermore, this analogue is capable of exerting its effects on disease even after the onset of symptoms.

Amino Acid Sequence↗
PubMed1994

Keratitis as a complication of bilateral, simultaneous radial keratotomy.

During a one-month period, we examined four patients referred for evaluation of probable microbial keratitis after bilateral, simultaneous radial keratotomy. Each patient had midstromal infiltrates compatible with microbial keratitis that involved one or more of the radial incisions. In two patients the keratitis was bilateral. All patients had been treated empirically with antibiotic agents; superficial cultures with cotton-tipped applicators and corneal scraping by inserting a platinum spatula into the radial incisions were negative. Corneal biopsy of one patient disclosed gram-positive rods and culture of the biopsy specimen grew diphtheroids. The infiltrates gradually resolved over a period of several months with intensive antibiotic therapy. Sight-threatening infectious keratitis can occur after radial keratotomy, and we believe that simultaneous bilateral ocular surgery of any kind should be discouraged.

Adult↗
PubMed1994

Keratocyte loss after corneal deepithelialization in primates and rabbits.

PURPOSE: To evaluate the response of stromal keratocytes to central corneal deepithelialization. METHODS: Rabbits and monkeys underwent unilateral mechanical deepithelialization with a blunt instrument and were killed at intervals ranging from 15 minutes to 24 hours after surgery. Two rabbits underwent unilateral deepithelialization under a fluid bath containing corneal preservation medium. Two rabbits were treated unilaterally with corneal preservation medium topically applied every 15 minutes for 16 hours after epithelial removal. Four rabbits underwent linear keratotomy immediately after deepithelialization of the cornea or on normal unoperated corneas and were killed 1 day (two animals) and 14 days (two animals) after surgery. RESULTS: Deepithelialization resulted in severe ultrastructural changes in keratocytes within 30 minutes after surgery. After 24 hours, the number of keratocytes in the anterior stroma underneath the deepithelialized area had decreased significantly in rabbits (P = .0001) and in monkeys (P = .0007) compared with controls. The wound healing was altered and delayed when the epithelium was not present after keratotomy. The use of storage media during and after deepithelialization minimized the early keratocyte changes and appeared to stimulate reepithelialization. CONCLUSIONS: Removal of corneal epithelium causes loss of superficial stromal keratocytes in rabbits and monkeys. Keratocyte death may results from osmotic changes that alter the corneal wound healing response.

Animals↗
PubMed1994

Histopathologic changes following irradiation for uveal tract melanoma.

Histopathologic studies were performed on 38 eyes in patients with uveal melanoma who had enucleation. Of the 38 eyes examined, enucleation was required in 11 (29%) following episcleral radioactive plaque therapy (RPT), which was performed in 83 patients. The reasons for enucleation in the 11 patients who had RPT were progressive tumor in 5 and treatment complications in 6 eyes. The histologic findings in these 11 patients were compared to those seen in 7 patients (18%) who received a planned course of preoperative external beam radiotherapy (RT) prior to enucleation and with 20 uveal melanoma patients (53%) who were treated with enucleation alone. Tumor necrosis was found in the eyes of patients from all three groups examined. It was, however, seen more frequently and to a greater extent in the 11 RPT patients as compared to the 7 preoperative RT and 20 enucleation alone patients, p = .01. There was no difference in the incidence or extent of tumor necrosis in the 7 preoperative RT patients as compared to the 20 primary enucleation patients, p = .18. In all 3 study groups, no correlation was found between tumor size and necrosis. In the 11 RPT patients, necrosis was independent of cell type and the radiation dose. As expected, the RPT patients had a greater incidence of neovascularization on the iris and scleral necrosis than those of the other two study groups (70 vs. 12.5% and 33 vs 0%, respectively), p = .004. A major effort needs to be made to optimize episcleral RPT in order to reduce treatment complications and increase the incidence of primary tumor control.

Brachytherapy↗