First steps in the standardization of immunoglobulin IgG myeloperoxidase-anti-neutrophil cytoplasmic antibody measurements.

The standardization of immunoassays for immunoglobulin (Ig)G myeloperoxidase-anti-neutrophil cytoplasmic antibodies (MPO-ANCA) could contribute to a more accurate diagnosis and follow-up of small vessels-associated vasculitis, a systemic autoimmune disorder that leads to necrosis of blood vessel walls. Despite significant efforts by different groups, the level of comparability of results from commercially available immunoassays used for IgG MPO-ANCA detection is still poor. Therefore, the potential for improvement using reference materials was assessed. The evaluation of a set of 30 patient samples with 11 assays showed that differences between assays result in different interpretations for individual patients. Only 10 of 30 patient samples had the same clinical interpretation among 11 assays applying the cut-off values provided by each respective manufacturer. The correlation between results from 13 different assays was assessed in a pairwise manner. The correlation between results from patient samples was systematically very good for combinations of seven of those assays. The correlation of results ranged from reasonable to good for combinations with four other assays, therefore it should be possible to improve the comparability of results using a commutable reference material for calibration. Feasibility studies were conducted in order to find a reference material format most suitable for a calibrator. Two sets of candidate reference materials were produced from different raw materials, and assessed according to their suitability. A final format was selected, and a candidate reference material was produced.

Non-myeloablative allogeneic stem cell transplantation: a new treatment option for acquired angioedema?

INTRODUCTION: Acquired angioedema is a rare disorder causing recurrent life-threatening angioedema, due to decreased activity of C1 esterase inhibitor. CASE REPORT: A 57-year-old man presented to our hospital with recurrent swelling of the hands, lips, tongue, scrotum and throat. Lab examination showed the presence of an IgM kappa monoclonal antibody. Additional analysis showed that in the IgM fraction autoantibody activity against C1 esterase inhibitor was present. This confirmed the diagnosis of acquired angioedema in the presence of lymphoplasmacytic lymphoma. Despite standard therapy, there was an increase in the episodes of laryngeal oedema. Therefore it was decided to perform a non-myeloablative allogeneic haematopoietic stem cell transplantation, with his HLA-identical brother as donor. The post-transplantation course was without complications. Five years following alloSCT he is in complete remission without symptoms and with increased C1 esterase inhibitor activity. DISCUSSION: In this case all other known treatment options for severe acquired angioedema failed. This is the first case describing treatment of severe acquired angioedema, caused by lymphoplasmacytic lymphoma, with an alloSCT.

International standards for IgG and IgM anti-ß2glycoprotein antibody measurement.

International standards for anti-beta2 glycoprotein I (anti-ß2GPI) testing are needed. We evaluated the suitability of polyclonal/monoclonal candidate reference materials (RM) for the assay. IgG/IgM anti-ß2GPI were affinity-purified (AP) from high-positive antiphospholipid syndrome sera and IgG from HCAL clone supernatant. Igs were tested for purity by SDS-PAGE, pooled, concentrated, sterile-filtered and the protein concentration determined. One unit was defined as the binding activity of 1 µg/ml of AP anti-ß2GPI Ig. IgG/IgM RM were each assigned a unit value using the respective AP material as a calibrator. Polyclonal/monoclonal RM and 30 samples were evaluated for linearity, unit equivalency and commutability. Polyclonal AP material was assigned a value of 100 U IgG and 15 U IgM anti-ß2GPI, respectively. IgG-RM had a value of 270 IgG and the IgM-RM of 220.3 IgM anti-ß2GPI U. The linearity (R (2)) of each RM curve for the various assays ranged from 0.96 to 0.99. Commutability samples fit very well within 95% prediction intervals and had excellent correlation when comparing assays. IgG and IgM polyclonal and IgG monoclonal RM displayed excellent linearity and commutability, being good candidates for better standardization of anti-ß2GPI immunoassays.

A discussion regarding the contribution of intrapulmonary gas mixing to O2 isotope fractionation by respiration using experimental data for 36Ar and 40Ar.

We determined the argon (Ar) isotope ratio in samples of expired alveolar gas gathered during Ar washout from residual gas relative to this ratio in samples of expired alveolar gas gathered just before the beginning of this washout in 13 young, healthy human subjects at rest. These data were determined for a limited number of breaths in early washout and were used to calculate the relative difference between the alveolar ventilations of (36)Ar and (40)Ar (Delta(rel)V (A)((36)Ar,(40)Ar)). Mean Delta(rel)V (A)((36)Ar,(40)Ar) amounted to 1.6 per thousand (S.D.=1.3 per thousand). This result was then used to discuss the contribution of intrapulmonary gas mixing by diffusion to oxygen isotope fractionation of alveolar gas by respiration. On the basis of our finding for Delta(rel)V (A)((36)Ar,(40)Ar) and further theoretical considerations we arrived at the conclusion that this contribution for subjects at rest is small (about 1 per thousand) and that this contribution is negative irrespective of the level of exercise.

Phase I and pharmacokinetic study of the novel chemoprotector BNP7787 in combination with cisplatin and attempt to eliminate the hydration schedule.

BNP7787 (disodium 2,2'-dithio-bis-ethane sulphonate; Tavocept) is a novel agent developed to protect against cisplatin (cis-diammine-dichloroplatinum(II))-associated chronic toxicities. In this study, we determined the recommended dose of BNP7787 when preceding a fixed dose of cisplatin, the pharmacokinetics (PKs) and the possible reduction of saline hydration. Patients with advanced solid tumours received BNP7787 in escalating doses of 4.1-41 g m(-2) as a 15-min intravenous (i.v.) infusion followed by cisplatin 75 mg m(-2) as a 60-min i.v. infusion together with pre- and postcisplatin saline hydration in a volume of 2200 ml; cycles were repeated every 3 weeks. PK was carried out using BNP7787, cisplatin and the combination. Twenty-five patients were enrolled in stage I of the study to determine the recommended dose of BNP7787. No dose-limiting toxicity was reached. The highest dose level of 41 g m(-2) resulted in a low incidence of grade 2 toxicities, being nausea and vomiting, dry mouth or bad taste and i.v. injection site discomfort. Doses of BNP7787 > or = 18.4 g m(-2) did not show a drug interaction between BNP7787 and cisplatin. In stage II of the study, patients received a fixed dose of BNP7787 of 18.4 g m(-2) preceding cisplatin and were entered in prespecified reduced saline hydration steps. A total of 21 patients in cohorts of six to nine patients received reduced saline hydration of 1600 ml (step A), 1000 ml (step B) and 500 ml (step C). In step C, two out of six evaluable patients experienced grade 1 nephrotoxicity. Cisplatin acute toxicities in all 46 patients were as expected. Only five patients complained of paresthesias grade 1 and six developed slight audiometric changes. Partial tumour response was observed in four patients and stable disease in 15 patients. In conclusion, BNP7787 was tolerated well up to doses of 41 g m(-2). The recommended dose of 18.4 g m(-2) enabled safe reduction of the saline hydration schedule for cisplatin to 1000 ml. Further studies will assess whether BNP7787 offers protection against platinum-related late side effects.

Structural study of the type II 3-dehydroquinate dehydratase from Actinobacillus pleuropneumoniae.

The structure of the type II dehydroquinate dehydratase (DHQase) from Actinobacillus pleuropneumoniae, the third enzyme of the shikimate pathway, has been determined. Crystals diffracting to 1.7 A were obtained in space and on earth using the counter-diffusion technique. The structure was solved using molecular replacement and refined to high resolution. The overall structure of the dodecameric enzyme is described and compared with structures of DHQases from other bacteria. DHQases contain a flexible loop that presumably closes over the active site upon substrate binding. The enzyme can exist in an open or closed conformation. The present structure displays the open conformation, with a sulfate anion bound in the active site. The availability of this structure opens a route to structure-based antibiotics targetting this pathogenic bacterium.

Arsenate reductase from S. aureus plasmid pI258 is a phosphatase drafted for redox duty.

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 plays a role in bacterial heavy metal resistance and catalyzes the reduction of arsenate to arsenite. The structures of the oxidized and reduced forms of ArsC were solved. ArsC has the PTPase I fold typical for low molecular weight tyrosine phosphatases (LMW PTPases). Remarkably, kinetic experiments show that pI258 ArsC also catalyzes the tyrosine phosphatase reaction in addition to arsenate reduction. These results provide evidence that ArsC from pI258 evolved from LMW PTPase by the grafting of a redox function onto a pre-existing catalytic site and that its evolutionary origin is different from those of arsenate reductases from Escherichia coli plasmid R773 and from Saccharomyces cerevisiae. The mechanism proposed here for the catalysis of arsenate reduction by pI258 ArsC involves a nucleophilic attack by Cys 10 on arsenate, the formation of a covalent intermediate and the transport of oxidative equivalents by a disulfide cascade. The reaction is associated with major structural changes in the ArsC.

Trimeric domain-swapped barnase.

The structure of a trimeric domain-swapped form of barnase (EC 3.1. 27.3) was determined by x-ray crystallography at a resolution of 2.2 A from crystals of space group R32. Residues 1-36 of one molecule associate with residues 41-110 from another molecule related through threefold symmetry. The resulting cyclic trimer contains three protein folds that are very similar to those in monomeric barnase. Both swapped domains contain a nucleation site for folding. The formation of a domain-swapped trimer is consistent with the description of the folding process of monomeric barnase as the formation and subsequent association of two foldons.

Hydrolysis of a slow cyclic thiophosphate substrate of RNase T1 analyzed by time-resolved crystallography.

Here we present a time-resolved crystallographic analysis of the hydrolysis of exo (Sp) guanosine 2',3'-cyclophosphorothioate by RNase T1. The use of a slow substrate and fast crystallization methods made it possible to perform the study with conventional data-collection techniques. The results support the idea that the hydrolysis reaction proceeds through a mechanism that is the inverse of the transesterification reaction. In addition, the structures provide an explanation for the differential behavior of RNase T1 towards exo- and endo-cyclic thiophosphates.

A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1.

The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.

The structures of RNase A complexed with 3'-CMP and d(CpA): active site conformation and conserved water molecules.

The interactions of RNase A with cytidine 3'-monophosphate (3'-CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.

Crystallographic study of Glu58Ala RNase T1 x 2'-guanosine monophosphate at 1.9-A resolution.

Glu58 is known to participate in phosphodiester transesterification catalyzed by the enzyme RNase T1. For Glu58 RNase T1, an altered mechanism has been proposed in which His40 replaces Glu58 as the base catalyst [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. Glu58Ala Rnase T1 has been cocrystallized with guanosine 2'-monophosphate (2'-GMP). The crystals are of space group P2(1), with one molecule per asymmetric unit (a = 32.44 A, b = 49.64 A, c = 26.09 A, beta = 99.17 degrees). The three-dimensional structure of the enzyme was determined to a nominal resolution of 1.9 A, yielding a crystallographic R factor of 0.178 for all X-ray data. Comparison of this structure with wild-type structures leads to the following conclusions. The minor changes apparent in the tertiary structure can be explained by either the mutation of Glu58 or by the change in the space group. In the active site, the extra space available through the mutation of Glu58 is occupied by the phosphate group (after a reorientation) and by a solvent molecule replacing a carboxylate oxygen of Glu58. This solvent molecule is a candidate for participation in the altered mechanism of this mutant enzyme. Following up on a study of conserved water sites in RNase T1 crystallized in space group P2(1)2(1)2(1) [Malin, R., Zielenkiewicz, P., & Saenger, W. (1991) J. Mol. Biol. 266, 4848-4852], we investigated the hydration structure for four different packing modes of RNase T1.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of RNase T1 with 3'-guanylic acid and guanosine.

A modified method for the synthesis and separation of endo and exo guanosine 2',3'-cyclophosphorothioate (cGPS) has been developed. The exo diastereoisomer has been co-crystallized with RNase T1. cGPS is known to be a RNase T1 inhibitor but is also a very slow substrate. It was hydrolyzed during the crystallization, leaving 3'-guanylic acid (3'-GMP) in the active site. As a guanosine was also found to be bound in a subsite, the enzyme contains the products of the reaction of guanylyl-3',5'-guanosine. The structure was refined to a resolution of 1.7 A and yielded a final R value of 14.5%. In contrast to previous 3'-GMP complexes of RNase T1, the ribose phosphate moiety of the inhibitor is in contact with all the active site residues. The phosphate forms hydrogen bonds with Asn36, Tyr38, Arg77, His92, and with Asn49 from a symmetry-related molecule. The ribose 2'-OH is hydrogen-bonded to both Glu58 and His40. The interactions in the active site of the present structure are compared to those found in the 2'-GMP complex of RNase T1.

Structure of the crystalline complex of cytidylic acid (2'-CMP) with ribonuclease at 1.6 A resolution. Conservation of solvent sites in RNase-A high-resolution structures.

The X-ray structure of the inhibitor complex of bovine ribonuclease A with cytidylic acid (2'-CMP) has been determined at 1.6 A resolution and refined by restrained least squares to R = 0.17 for 11 945 reflections. Binding of the inhibitor molecule to the protein is confirmed to be in the productive mode associated with enzyme activity. A study of conserved solvent sites amongst high-resolution structures in the same crystal form reveals a stabilizing water cluster between the N and C termini.

Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate.

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.

Role of histidine-40 in ribonuclease T1 catalysis: three-dimensionalstructures of the partially active His40Lys mutant.

Histidine-40 is known to participate in phosphodiester transesterification catalyzed by the enzyme ribonuclease T1. A mutant enzyme with a lysine replacing the histidine-40 (His40Lys RNase T1) retains considerable catalytic activity [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. We report on the crystal structures of His40Lys RNase T1 containing a phosphate anion and a guanosine 2'-phosphate inhibitor in the active site, respectively. Similar to previously described structures, the phosphate-containing crystals are of space group P2(1)2(1)2(1), with one molecule per asymmetric unit (a = 48.27 A, b = 46.50 A, c = 41.14 A). The complex with 2'-GMP crystallized in the lower symmetry space group P2(1), with two molecules per asymmetric unit (a = 49.20 A, b = 48.19 A, c = 40.16 A, beta = 90.26). The crystal structures have been solved at 1.8- and 2.0-A resolution yielding R values of 14.5% and 16.0%, respectively. Comparison of these His40Lys structures with the corresponding wild-type structures, containing 2'-GMP [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368] and vanadate [Kostrewa, D., Hui-Woog Choe, Heinemann, U., & Saenger, W. (1989) Biochemistry 28, 7692-7600] in the active site, respectively, leads to the following conclusions. First, the His40Lys mutation causes no significant changes in the overall structure of RNase T1; second, the Lys40 side chains in the mutant structures occupy roughly the same space as His40 in the corresponding wild-type RNase T1 structures.(ABSTRACT TRUNCATED AT 250 WORDS)