DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma.

PURPOSE: We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well as the predictive value of quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS: Thirty-three patients with newly diagnosed glioblastoma multiforme (GBM) and five patients with newly diagnosed anaplastic astrocytoma (AA) were treated with Temodal at a starting dose of 200 mg/m2 daily for 5 consecutive days with repeat dosing every 28 days after the first daily dose. Immunochemistry for the detection of the human DNA mismatch repair proteins MSH2 and MLH1 and the DNA repair protein AGT was performed with monoclonal antibodies and characterized with respect to percent positive staining. RESULTS: Of the 33 patients with GBM, complete responses (CRs) occurred in three patients, partial responses (PRs) occurred in 14 patients, stable disease (SD) was seen in four patients, and 12 patients developed progressive disease (PD). Toxicity included infrequent grades 3 and 4 myelosuppression, constipation, nausea, and headache. Thirty tumors showed greater than 60% cells that stained for MSH2 and MLH1, with three CRs, 12 PRs, three SDs, and 12 PDs. Eight tumors showed 60% or less cells that stained with antibodies to MSH2 and/or MLH1, with 3 PRs, 3 SDs, and 2 PDs. Eleven tumors showed 20% or greater cells that stained with an antibody to AGT, with 1 PR, 2 SDs, and 8 PDs. Twenty-five tumors showed less than 20% cells that stained for AGT, with 3 CRs, 12 PRs, 4 SDs, and 6 PDs. CONCLUSION: These results suggest that Temodal has activity against newly diagnosed GBM and AA and warrants continued evaluation of this agent. Furthermore, pretherapy analysis of tumor DNA mismatch repair and, particularly, AGT protein expression may identify patients in whom tumors are resistant to Temodal.

conformation and phasing of dystrophin structural repeats.

The presumptive rod domain of dystrophin contains a series of degenerate repeating sequences with homology to those of spectrin. To determine the relation of the implied structural repeating units to the sequence repeat (the phasing), recombinant fragments of the domain of dystrophin were prepared by expression in Escherichia coli. The phasing was established by identifying the minimum sequence element that would form a stable fold of high (approx. 75%) alpha-helicity: by contrast, incorrectly phased fragments had labile structure with an average alpha-helicity of about 40%. The isolated folded structural repeat showed high stability towards proteolysis and a urea-denaturation profile with a plateau at low denaturant concentration, indicative of a unique folded conformation. The phasing is consistent with a structure inferred from analysis of the amino acid sequence and also found in spectrin, in which each structural repeat comprises a three-stranded coiled-coil, made up of one short helix (approx. 30 residues) and the N and C-terminal halves of two separate long helices, such that each long helix participates in the formation of two contiguous structural units.

Decreased skeletal muscle mitochondrial DNA in patients with statin-induced myopathy.

Statins are widely used to treat hyperlipidemia and lower cardiovascular disease risk. While statins are generally well tolerated, some patients experience statin-induced myopathy (SIM). Statin treatment has been associated with mitochondrial dysfunction and mitochondrial DNA (mtDNA) depletion. In this retrospective study, skeletal muscle biopsies from patients diagnosed with SIM were studied. These were compared with biopsies from patients clinically assessed as having statin-unrelated myopathy but whose biopsy showed no or negligible pathology. For each biopsy sample, mtDNA was quantified relative to nuclear DNA (mtDNA content) by qPCR, mtDNA deletions were investigated by long-template PCR followed by gel densitometry, and mtDNA oxidative damage was quantified using a qPCR-based assay. For a subset of matched samples, mtDNA heteroplasmy and mutations were investigated by cloning/sequencing. Skeletal muscle mtDNA content was significantly lower in SIM patients (N=23, mean±SD, 2036±1146) than in comparators (N=24, 3220±1594), p=0.006. There was no difference in mtDNA deletion score or oxidative mtDNA damage between the two groups, and no evidence of increased mtDNA heteroplasmy or somatic mutations was detected. The significant difference in skeletal muscle mtDNA suggests that SIM or statin treatments are associated with depletion of skeletal muscle mtDNA or that patients with an underlying predisposition to SIM have lower mtDNA levels. If statins induce mtDNA depletion, this would likely reflect decreased mitochondria biogenesis and/or increased mitochondria autophagy. Further work is necessary to distinguish between the lower mtDNA as a predisposition to SIM or an effect of SIM or statin treatment.

Thermal degradation of injectable epinephrine.

The degradation of epinephrine in USP injectable cartridges was investigated under different heating conditions. Epinephrine (EPI) and EPI sulfonic acid (EPI-SA) levels in 1:10,000 (0.1 mg/mL) EPI injectable solutions subjected to either cyclical (65 degrees C for 8 hr/d for 4 to 12 weeks) or constant (65 degrees C for 7 days) heating were determined using high-pressure liquid chromatography with diode array and electrochemical detection. Constant (169 total hours of heat exposure) heating resulted in complete degradation of both compounds and dark brown discoloration of the solution. Cyclical heating (672 total hours of heat exposure) resulted in a 31% reduction in EPI concentration and a 225% increase in EPI-SA concentration with no discoloration of the solution. In laboratory-prepared solutions, the degradation of EPI and the formation of EPI-SA was found to be dependent on sodium metabisulfite concentration and the duration of cyclical heating. These results indicate that the thermal stability of EPI and the formation of EPI-SA depends on the method of heat exposure and the amount of bisulfite present in the solution.

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.

Thermodynamics of the interaction of human immunoglobulin E with its high-affinity receptor Fc epsilon RI.

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.

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.

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.

Upregulation of FcepsilonRI on human basophils by IgE antibody is mediated by interaction of IgE with FcepsilonRI.

BACKGROUND: IgE is now known to upregulate the expression of FcepsilonRI on human basophils. It is not known which receptor on basophils mediates this process of upregulation. OBJECTIVE: We sought to determine whether galectin-3, FcepsilonRII (CD23), or FcepsilonRI were involved in the upregulation of FcepsilonRI by IgE. METHODS: The role of galectin-3 was examined by measuring the influence of alpha-lactose on upregulation. Basophils were examined for expression of FcepsilonRII (CD23) by flow cytometry and messenger (m)RNA expression. Functional discrimination between binding to FcepsilonRII or FcepsilonRI was examined through the use of mutant IgE-Fc fragments or anti-FcepsilonRII antibody. RESULTS: Upregulation of FcepsilonRI on basophils in the presence of IgE was not altered by coincubation with alpha-lactose, eliminating a role for galectin-3. Basophils were not found to express FcepsilonRII, as determined by flow cytometry with enriched basophil preparations or RT-PCR with highly purified basophil preparations. A mutant of the Fc fragment of IgE (IgE-Fc), which binds to FcepsilonRI with a greater than 10-fold lower affinity than IgE or wild-type IgE-Fc but exhibits no change in affinity for FcepsilonRII, allowed us to distinguish between the functions of the two Fc receptors. The mutant (R334S; Henry et al 1997) was required at about 30-fold higher concentration than the wild-type IgE-Fc for the same stimulation of FcepsilonRI expression on basophils, thus excluding a role for FcepsilonRII in the response. In addition, treatment of basophils with anti-FcepsilonRII antibody (MHM6), which is known to be competitive with IgE, had no effect on the expression of FcepsilonRI or the ability of IgE to upregulate expression of FcepsilonRI. CONCLUSION: Collectively, these data indicate that IgE interacts with FcepsilonRI to upregulate its expression on human basophils.

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.

Expression and relationships of seven public idiotypes of DNA-binding autoantibodies on monoclonal antibodies and serum immunoglobulins.

Many studies have shown that DNA-reactive autoantibodies share cross-reactive public idiotypes that are defined, usually, by single anti-idiotype reagents. Because anti-idiotype antibodies or antisera will be limited in their ability to detect all the idiotopes of a particular antibody, their use will tend to underestimate the full extent of idiotype sharing between different antibodies. In order to define more comprehensively the extent of idiotype sharing in DNA autoantibodies, a panel of DNA-binding monoclonal autoantibodies from lupus mice was examined with a range of anti-idiotype antisera prepared in rabbits (five sera), guinea pigs (four sera) and a sheep. Each idiotype was detected on more antibodies than its original reference monoclonal antibody, and idiotopes of each were also present on serum immunoglobulins from lupus mice. Of 23 monoclonal antibodies 65% reacted with one or more of the anti-idiotype reagents. On these criteria, all the idiotypes were public; none was private in its expression. In about half the cases the idiotypes were located in or near the antigen-binding sites of the antibodies, but a direct relationship to specificity was not obvious except in the case of Id.228 present on antibodies with a relatively high affinity for single-stranded DNA. In other cases there was no obvious relationship between idiotype and specificity. Antibodies from the same mouse did not each express the same array of idiotopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants.

We have constructed an expression vector that leads to secretion of the whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more homogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosylated in Chinese hamster ovary cells. Both the double mutant and wild-type hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (Ka in the range 10(10)-10(11) M-1), and were able to sensitize isolated human basophils for anti-IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity receptor, Fc epsilon RII (Ka = 7.3 x 10(7) M-1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1 x 10(6) M-1).